genetic and molecular analyses of nodulation in chickpea...

GENETIC AND MOLECULAR ANALYSES OF NODULATION

IN CHICKPEA (Cicer arietinum L.)

BY

ROZINA GUL

DEPARTMENT OF PLANT BREEDING & GENETICS FACULTY OF CROP PRODUCTION SCIENCES KHYBER PUKHTUNKHWA AGRICULTURAL

UNIVERSITY PESHAWAR, PAKISTAN JUNE, 2010

GENETIC AND MOLECULAR ANALYSES OF NODULATION

IN CHICKPEA (Cicer arietinum L.)

BY

ROZINA GUL

A dissertation submitted to the KP Agricultural University, Peshawar in partial fulfillment of the requirement for the degree of

DOCTOR OF PHILOSoPHY IN AGRICULTURE

(PLANT BREEDING AND GENETICS)

DEPARTMENT OF PLANT BREEDING & GENETICS FACULTY OF CROP PRODUCTION SCIENCES KHYBER PUKHTUNKHWA AGRICULTURAL

UNIVERSITY PESHAWAR, PAKISTAN JUNE, 2010

GENETIC AND MOLECULAR ANALYSES OF NODULATION IN

CHICKPEA (Cicer arietinum L.)

BY

ROZINA GUL

A dissertation submitted to the KPK Agricultural University, Peshawar in partial fulfillment of

the requirement for the degree of

DOCTOR OF PHILOSoPHY IN PLANT BREEDING AND GENETICS APPROVED BY: _________________________ Prof. Dr. Farhatullah Chairman, Supervisory Committee _________________________ Prof. Dr. Ko Harda Co-Supervisor for Research Forest Resource Biology Laboratory Faculty of Agriculture Ehime University Matsuyama Japan ______________________________ Prof. Dr.Iftikhar Hussain Khalil Member _________________________ Prof. Dr. Zahir Shah Member Deptt. of Soil and Envir. Science _________________________ Dr. Gul Sanat Shah Addl. Member Principle Scientist Nuclear Institute for Food and Agriculture Peshawar _________________________ Prof. Dr. Farhatullah Chairman/Convener Board of Studies _________________________ Prof. Dr. Zahoor A. Swati Dean, Faculty of Crop Production Sciences _________________________ Prof. Dr. Farhatullah Director, Advanced Studies and Research

DEPARTMENT OF PLANT BREEDING & GENETICS FACULTY OF CROP PRODUCTION SCIENCES

KHYBER PAKHTUNKHWA AGRICULTURAL UNIVERSITY PESHAWAR, PAKISTAN

JUNE, 2010

I dedicate this humble effort to

my loving Parents, caring husband and my sweet kids

i

TABLE OF CONTENTS _____________________________________________________________ Chapter No. Title Page

Table of contents i List of tables iv List of figures vi List of appendix vii List of abbreviations viii Acknowledgements ix Abstract xi

1 General Introduction and Background Information ........... 1

1.1 Origin and botanical classification of chickpea ................................ 1 1.2 Mode of reproduction ....................................................................... 1 1.3 Importance and utilization ................................................................ 2 1.4 Medicinal use of chickpea ................................................................ 2 1.5 Taxonomy, morphology and types ................................................... 3

1.6 Chemistry of chickpea ...................................................................... 3 1.7 Area and production ......................................................................... 3 1.8 Genetic variability in chickpea .......................................................... 4 1.9 Nodulation and nitrogen fixation ....................................................... 5 1.10 Nodulation mutants in chickpea ....................................................... 6 1.11 Inoculation of chickpea with rhizobium ............................................ 7 1.12 Linkage study in chickpea ............................................................... 7 1.13 Molecular study in chickpea ............................................................. 8 1.14 Aims and objectives of the study ...................................................... 9

2 Review of Literature ......................................................... 1

2.1 Characterization of chickpea for morphological markers and other quantitative traits ............................................................................ 10

2.3 Characterization of genotypes for nodulation and effect of rhizobil inoculation on nodulation ............................................................... 12

2.4 Molecular characterization of chickpea germplasm using microsatellite (SSR) markers……………………………………………………………………………16

2.6 Inheritance and linkage study of genes of nodulation in chickpea .. 19

ii

3 General Materials and Methods………………………………………………… 22

3.1 Procurement of seed ...................................................................... 22 3.2 Experimental sites .......................................................................... 23 3.3 Characterization of germplasm for various morphological markers and Quantitative traits .................................................................... 26 3.4 Characterization of genotypes for nodulation and effect of rhizobial

Inoculation on nodules number and seed yield plant-1 .................. 28 3.5 Inheritance and linkage study of nodulation in chickpea ............... 32 3.6 Molecular characterization of germplasm using microsatellite markers …. ............................................................................... 35

4 RESULTS AND DISCUSSIONS ......................................... 41

4.1 Expt.1. Characterization for morphological markers and

quantitative traits. ................................................. 41

4.1.1 Introduction ………………………………………………………………………………………………………….41 4.1.2 Materials and methods ................................................................... 42 4.1.3 Results . ................................................................................. 43 4.1.3.1 Characterization for genetic marker .............................................. 43 4.1.3.2 Characterization for quantitative characters ................................. 43 4.1.4 Discussion . .............................................................................. ….52

4.2 Expt.2. Characterization of genotypes for nodulation and

effect of rhizobial inoculation on nodules number

and seed yield plant-1 . ........................................... 55

4.2.1 Introduction .................................................................................. 55 4.2.2 Materials and Methods ................................................................. 57 4.2.3 Results . ................................................................................. 57 4.2.3.1 Characterization of accessions for presence or absence of Nodules . ................................................................................. 57 4.2.3.2 Number of Nodules Plant-1 genotype-1 ................................................................ 58 4.2.3.3 Effect of rhizobial inoculation on number of nodules plant-1 ........ 59

iii

4.2.3.3 Effect of rhizobial inoculation on seed yield plant-1 ................................... 60 4.2.4 Discussion .................................................................................. 66

4.3 Expt.3. Inheritance and linkage studies of nodulation in

chickpea. ............................................................... 71

4.3.1 Introduction ................................................................................. .71 4.3.2 Materials and Methods .................................................................. 73 4.3.3 Results…………………………………………………………………………………………………………………… 75 4.3.3.1 Inheritance of nodulation ............................................................... 75 4.3.3.2 Inheritance of leaf color ................................................................. 76 4.3.3.3 Nodulation Vs leaf color ................................................................ 76 4.3.4 Discussion . ................................................................................. 80

4.4 Expt.4. Molecular characterization of chickpea germplasm using microsatellite markers...................................85

4.4.1 Introduction ................................................................................. 85 4.4.2 Materials and methods ............................................................... 87 4.4.3 Results………………………………………………………………………………………………………………….87 4.4.3.1 Genetic variation ......................................................................... 87

4.4.3.2 Genetic relationship among accessions ..................................... 89

4.4.3.3 Grouping of chickpea genotypes based on morphological traits 91

4.4.4 Discussion ................................................................................ 101

Summary.. .................................................................... 105 Conclusions .................................................................. 109 Recommendations ........................................................ 110 Bibliography ................................................................... 111 Appendices ................................................................... 127

iv

List of Tables

No Title Page _____________________________________________________________

3.1 Pedigree and origin of genotypes/accessions used in the study ...... 25

3.2 Details of primers sequencing ........................................................... 39

4.1.1 Flower color, stem color and seed coat color of evaluated chickpea germplasm ........................................................................ 46 4.1.2 Mean square for days to 50% flowering, days to maturity, plant height and number of leaflets leaf-1 of chickpea germplasm ........... 47 4.1.3 Mean square for leaf area, seed yield plant-1, 100 seed weight and biological yield plant-1 of chickpea germplasm .......................... 47 4.1.4 Mean values of days to 50% flowering, days to maturity, plant height and number of leaflets leaf-1 in chickpea germplasm ........... 48

4.1.5 Mean values of leaf area, seed yield plant-1, 100 seed weight and biological yield plant-1 in chickpea germplasm ......................... 49

4.1.6 Estimates of genotypic variance (Vg), phenotypic variance (vp), genotypic coefficient of variability (PCV), phenotypic coefficient of variability (GCV) and heritability ( h2

(bs) ) for various agronomic traits .................................................................................. 50 4.1.7 Description of chickpea accessions used in present study. ............. 51 4.2.1 Mean square for number of nodules plant-1 of chickpea germplasm .................................................................................. 61

4.2.2 Presence or absence of nodules and number of nodules plant-1 in

chickpea accession ........................................................................ 62

4.2.3 Estimates of variability parameters for number of nodules plant-1 in

chickpea germplasm ........................................................................ 63

4.2.4 Mean square for inoculation effect on number of nodules plant-1

and seed yield plant-1............................................................................................................ 63

4.2.5 Effect of control and rhizobial inoculation on nodules plant-1 in. ...... 64

v

No Title Page __________________________________________________________ 4.2.6 Effect of control and rhizobial inoculation on seed yield plant-1

in chickpea genotypes ...................................................................... 65 4.3.1 Morphological markers of parents and their F1 used in linkage studies of chickpea .......................................................................... 77 4.3.2 Nodulation response of non-nodulated (Nod-) and nodulated

(Nod+) parents, F1, F2, and backcross Progenies of

ICC 19181 X NDC 5-S10 cross to rhizobial Inoculation .................. 77 4.3.3 Nodulation response of non-nodulated (Nod-) and nodulated

(Nod+) parents, F1, F2, and backcross Progenies of

ICC 19181 X NDC 4-20-4 cross to rhizobial Inoculation ................. 78

4.3.4 Inheritance of leaf color in light green and dark green leaf colored parents, F1, F2, and backcross progenies of cross ICC 19181 X NDC 5-S10 ................................................................. 78

4.3.5 Inheritance of leaf color in light green and dark green leaf colored parents, F1, F2, and backcross progenies of cross ICC 19181 X NDC 4-20-4 ................................................................. 79 4.3.6 Joint segregation for markers of chickpea F2 population for leaf color and nodulation .................................................................. 79 4.4.1 Description of chickpea accessions used in Molecular study .......... 96 4.4.2 Primers used for amplifying chickpea microsatellite regions ........... 97 4.4.3 Genetic diversity statistics of 47 chickpea accessions .................... 98 4.4.4 AMOVA analysis of microsatellite data of 47 chickpea accessions 99 4.4.5 Summary of ANOVA for the 4 morphological traits of 47 chickpea

accessionsA ........................................................................................................................... 99 4.4.6 Correlation among four morphological traits of 47 chickpea accessions ................................................................................ 100 4.4.7 Principal component analysis on four morphological traits of 47

chickpea genotypes ....................................................................... 100

vi

List of Figures

No Title Page

3.1 Washing Procedure of Roots to check Nodules .............................. 30

5.1 Nodulated Root ............................................................................... 58

5.2 Non-nodulated Roots ...................................................................... 58

4.3.1 Chickpea Crossed Flower ................................................................ 74

4.3.1 Roots of nodulated (L) and non- nodulated plants (R) .................... 74

4.4.1 (a) UPGMA tree (b) ME tree............................................................. 93

4.4.2 Scatter plot of principal component analysis based on

morphological data ........................................................................... 94

4.4.3 Scatter plot of principal coordinate analysis based on

microsatellite allele frequencies ...................................................... 95

vii

List of Appendices ____________________________________________________________________ No Title Page

1 Analysis of variance for days to 50% flowering ............................... 127

2 Analysis of variance for days to maturity ......................................... 127

3 Analysis of variance for plant height ................................................ 127

4 Analysis of variance for leaflets leaf-1 ........................................................................ 28

5 Analysis of variance for leaf area .................................................... 128

6 Analysis of variance for seed yield plant-1 ........................................................... 128

7 Analysis of variance for 100 seed weight ........................................ 129

8 Analysis of variance for biological yield plant-1 ................................................ 129

9 Analysis of variance for number of nodules plant-1 ....................................... 129

10 Analysis of variance for rhizobium effect on number

of nodules plant-1 .................................................................................................................... 130

11 Analysis of variance for rhizobium effect on seed

yield plant-1 ................................................................................................................................................ 130

viii

List of Abbreviations

AFLP Amplified fragment length polymorphism

AMOVA Analysis of molecular variance

ARS Ahmadwala research station

AUP Agricultural university Peshawar

CIB Cold isolation buffer

cM Centi Morgan

CRD Completely randomized design

CTAB Cetytriammoniumbromide

CV Coefficient of variation

GCV Genotypic co-efficient of variation

GRS Gram research station

h2 Heritability

IBGE Institute of biotechnology and genetic engineering

ICARDA International center for agricultural research in the dry areas, Syria

ICRISAT International crops research Institute for the semi-arid tropics, India

KP Khyber Pakhtunkhwa

LSD Least significant difference

ME Minimum-evolution method

NIFA Nuclear Institute for food and agriculture, Peshawar Pakistan

Nod- Non-nodulated

Nod+ Nodulated

PCA Principal component analysis

PCV Phenotypic co-efficient of variation

PCoA Principal coordinate analysis

PIC polymorphic information content

RAPD Random amplified polymorphic DNA

RCBD Randomized complete block design

RFLP Restriction fragment length polymorphism

SSR Simple sequence repeat

UPGMA: Unweighted pair-group method using arithmetic averages

ix

ACKNOWLEDGEMENTS

Today I am immensely pleased and feel elated on successful completion of

my dissertation-the project assigned to me during the last leg of my PhD

studies. I bow my head to Almighty Allah. The Omnipotent and Omnipresent,

for his blessing, especially the faculties that he endowed upon me and I was

able and capable enough to undertake the task and complete it in a befitting

manner.

It was an honor for me to have Prof. Dr Farhatullah as my major supervisor.

His sincere and continuous efforts, constant supervision, constructive

criticism, critical insight, judicious guidance, excellent editorial suggestions

and patience during these four years were invaluable for the development of

my study and research program.

Special thanks to my co-supervisor, Dr Ko Harada who supervised me in the

molecular part of my research study. His kind and excellent guidance, stable

supervision and incessant efforts were priceless for the completion of my

research project. His outstanding supervision made my study at Japan

memorable.

I am grateful to Dr Gul Sanat Shah, Dr NaquibulIa and Dr Iftikhar for their

help and support. I would extend my appreciation to Sahir, Sajjid, Nazma

and Sabra for helping in the field work. I am also thankful to Mr. Shaheen,

Mr. Naveed and Mr. Akram for providing their services whenever needed.

I would like to present my heartfelt thanks to my great father (Haji Gul Faraz

Khan) and mother for their care, love and life long support.

Finally I am extremely thankful to my considerate husband Dr. Hamayoon

Khan Yousafzai, whose uninterrupted support in various aspects of my life

and studies, kept me going with grace and honor, and the courage and

motivation that he gave me, made me able to complete this project. I am also

much more thankful to my sweet daughter Amina Khan, my precious sons

Mohsin Khan and Abdullah Khan who always remained a source of

refreshment for me and due to their love I remained successful.

Rozina Gul

x

GENETIC AND MOLECULAR ANALYSES OF NODULATION IN

CHICKPEA (Cicer arietinum L.)

Rozina Gul and Farhatullah

Department of Plant Breeding and Genetics

Faculty of Crop Production Sciences

Khyber Pakhtunkhwa Agricultural university Peshawar, Pakistan.

May, 2010

ABSTRACT

A hallmark trait of chickpea (cicer arietinum L.) is its ability to form root nodules

and to fix atmospheric nitrogen in symbiosis with compatible rhizobia.

Chickpea plays a vital role in natural ecosystems, agriculture, and agro-

forestry, where its ability to fix nitrogen in symbiosis makes it an excellent

settler of low-N2 environments, and economic and environmentally friendly

crop. Forty seven chickpea genotypes were procured from Nuclear Institute for

Food and Agriculture (NIFA), Peshawar, Gram Research Station (GRS),

Karak, Pakistan and International Crops Research Institute for the Semi-Arid

Tropics (ICRISAT), India. Entire experiments of the reported project were

carried out from 2006 to 2009 at Agricultural University, and NIFA, Peshawar

except molecular characterization, which was accomplished at Ehime

University Matsuyama, Japan.

All genotypes were characterized for marker traits, quantitative parameters,

nodulation and molecular markers (SSR). Highly nodulated and non-nodulated

parents were picked and hybridized to study mode of inheritance of nodulation

and its linkage with marker trait loci. The germplasm was also grouped as desi

(pink flower, green with purplish tings stem and colored seed coat) and kabuli

(white flower, green stem and white seed coat) types. Highly significant

differences and high heritability estimates were recorded for days to 50%

flowering, days to maturity, leaf area, number of leaflets leaf-1, plant height,

100 seed weight, biological yield plant-1 and grain yield plant-1 in all the

genotypes. Genotypes from NIFA and GRS were nodulated while genotypes

from ICRISAT were Nod-. All genotypes also differed highly and significantly

for number of nodules plant-1. The genotypes NDC 5-S10 and NDC 4-20-4

xi

produced the maximum nodules plant-1. Highly significant response of

rhizobium inoculation was recorded for nodules plant-1 and seed yield plant-1.

Interaction of genotypes with treatments classified NDC 4-20-1(16.66) as

highly Nod+ and Karak 3 (33 g) as high seed yielder plant-1. The maximum

genotypic mean for nodulation and seed yield plant-1 was recorded for

accession NDC 5-S10 (14.83) and Karak 3 (30.20) respectively. Inoculated

genotypes exceeded control in treatment means both for nodules plant-1 (10.33

& 7.22) and seed yield plant-1 (14.40 g & 10.59 g).

Molecular characterization of 47 genotypes was performed using 10 simple

sequence repeat (SSR) markers. Eight of the 10 SSR markers were

polymorphic. Number of alleles ranged from 2 to 16, with an average of 7.4

locus-1. Polymorphic information content (PIC) values ranged from 0.227 to

0.876, with an average of 0.636. The average PIC was 0.582 in desi and 0.577

in kabuli genotypes, shows that both groups are distinct. Significant genetic

differentiation was found between desi and kabuli genotypes by using Analysis

of molecular variance (AMOVA) under stepwise mutation assumption (RST =

0.239, P ≤ 0.001). Unweighted pair-group method using arithmetic averages

(UPGMA) and Minimum-evolution method (ME) trees as well as Principal

Coordinate analysis (PCoA) classified the accessions into 6 groups and all the

6 accessions could be clearly separated. Grouping was mostly the same in

both the phylogenetic trees and PCoA, but the branching order differed

greatly. Inheritance of nodulation was studied in two cross combinations i.e.,

ICC 19181(non-nodulated and dark green leaves) x NDC 5-S10 (nodulated

and light green leaves) - Hybrid A and ICC 19181 x NDC 4-20-4 (Nod+ and

light green leaves) - Hybrid B. Hybrid A, showed monogenic dominant

inheritance, while hybrid B showed duplicate gene action for nodulation

confirming that both Nod+ genotypes are from different clusters. Both hybrids

revealed monogenic dominant inheritance of light green leaf color. Linkage

study revealed that loci for nod and leaf color resides on the same

chromosome at the distance of 15 centi Morgan (cM) in genotype NDC 5-S10

while in genotype NDC 4-20-4 the two loci for nodulation exists at the distance

of 26 cM and 15 cM from the locus of leaf color. The current research findings

show significant diversity both at morphological and molecular levels, and

valuable results regarding rhizobial inoculation, inheritance and linkage study

of nodulation, which could play a vital role in future chickpea breeding

programs.

1

CHAPTER 1

INTRODUCTION

1.1 Origin and botanical classification of chickpea

Chickpea (Cicer arietinum L., 2x = 2n = 16) belongs to genus Cicer,

tribe Cicereae, family Fabaceae, and subfamily Papilionaceae. Cicer is Latin

name originated and derived from the Greek word 'kikus' that means force or

strength. The origin of the word is traced to the Hebrew 'kikar'', where 'kikar'

means round (Duschak, 1871). The word arietinum is also Latin, translated

from the Greek “krios", another name for both ram and chickpea, an allusion

to the shape of the seed which resembles the head of a ram (Aries) (Van der

Maesen, 1987). The oldest report concerning this species was 5450 BC

(Helbaek, 1959) and it has been cultivated for at least 7000 years (Van der

Maesen, 1972).

1.2 Mode of reproduction

Chickpea is highly self pollinated specie because of its cleistogamous

flower (Smithson et al., 1985). Self pollination takes place 12-24 hour before

the flower is fully opened. At the time of pollination the keel is closed and

foreign pollen can’t reach the stigma. Fertilization takes place about 24 hr

after flower is fully expanded. Due to fragility of flower artificial hybridization

are rather poor, overall seed setting is estimated as 5 – 25%. Emasculation

and pollination can be done at any time of the day, but simultaneous

emasculation and pollination gave higher percentage of seed set than

consecutive day operation (Singh and Ackuland, 1975).

2

1.3 Importance and utilization

Chickpea is one of the world's most important but less-studied

leguminous food crop with 740-Mb genome size. Chickpea ranks third

among pulses, fifth among grain legumes, and 15th among grain crops of the

world. It accounts for 12% of the world pulses production, with a 1.9% annual

growth rate of chickpea production during the last 20 years (Upadhyaya,

2003). Chickpea is one of the major pulse crops in West Asian and North

African regions. It has great importance as food, feed and fodder. Due to the

increasing need for legumes, chickpea is no longer considered a subsistence

crop. The rising trend in its trade suggests that the crop is grown increasingly

for the market (Saxena et al., 1996). In the developed world it represents a

valuable crop for export. It provides a protein-rich supplement to cereal-

based diets. Chickpea is valued for its nutritive seeds with high protein

content, 25.3-28.9 %, after de-hulling (Hulse, 1991).

1.4 Medicinal uses of chickpea

Among the food legumes, chickpea is the most hypocholesteremic

agent; germinated chickpea was reported to be effective in controlling

cholesterol level in rats (Geervani, 19991). Glandular secretion of leaves,

stem and pods consist of malic and oxalic acids, giving a sour taste. Acids

are supposed to lower the blood cholesterol levels. Medicinal applications

include use for bronchitis, aphrodisiac, catarrh, cholera, cutamenia,

constipation, diarrhea, flatulence, dyspepsia, snakebite, sunstroke, and

warts. Seeds are considered antibilious (Duke, 1981).

3

1.5 Morphology and types

There are two types of chickpea, depending on seed color, shape,

and size. Kabuli type, the seeds of this type are large, round or ram head,

and cream-colored. The plants are medium to tall (up to one m) in height,

with large leaflets and white flowers, and contain no anthocyanin

pigmentation. The second type is Desi type; this type has small seeds

angular in shape. The seed color varies from cream, black, brown, yellow to

green. There are 2-3 ovules per pod but on an average 1-2 seeds per pod

are produced. The plants are short, more prostrate with small leaflets and

purplish flowers, and contain anthocyanin pigmentation (Muehlbauer et al.,

1982; Cubero, 1975). Chickpea is grown in tropical, sub-tropical and

temperate regions. Kabuli type is grown in temperate regions while the desi

type chickpea is grown in the semi-arid tropics (Muehlbauer and Singh,

1987; Malhotra et al., 1987).

1.6 Chemistry of chickpea

Chickpea is important due to its nutritive seeds which is a rich and

cheap source of protein in human diet as compared to the animal protein.

Chickpea seed has 38-59% carbohydrate, 4.8-5.5% oil, 3% fiber, 3% ash,

0.3% phosphorus and 0.2% calcium,. Digestibility of protein varies from 76-

78% and its carbohydrate from 57-60% (Hulse, 1991).

1.7 Area and Production

Pakistan is the second big producer of chickpea after India in the

world. In Pakistan total cropped area is 22.51 million hectare. Out of this

4

area, 1.492 m.ha is under pulse cultivation which is 7% of the total cropped

area, and 73% of the area under pulse production is occupied by chickpea

cultivation. Total area under chickpea cultivation in our country is 1052.3

thousand hectares with a production of 823 thousand tones and an average

yield of 795.4 Kg ha-1. In Khyber Pakhtunkhwa (KPK) it was cultivated on an

area of 49.0 thousand hectares with a production of 21.0 thousand tones

with an average yield of 427.5 kg ha-1 (MINFAL, 2007-08). In KP about 75%

chickpea is grown on rainfed lands and its cultivation is concentrated in the

southern districts of the province including D.I. Khan, Tank, Lakki Marwat,

Bannu and Karak.

1.8 Genetic variability

Genetic diversity plays a vital role in survival and development of

many natural populations and wild species. The crops genetic variability not

only helps varieties to adopt to diverse environments, to enhance the

tolerance of unfavorable conditions and resistance to pest and diseases, but

also to produce the diversity and to get better yield and quality of product to

serve needs of people. Utilization of exotic and diverse germplasm is needed

to enhance the genetic diversity of cultivars. Genetically diverse lines provide

sufficient opportunity to create favorable gene combinations, and the

probability of producing a unique genotype, increase in proportion to the

number of genes by which the parents differ.

Genetic variability is prerequisite for crop improvement as it provides

raw material to plant breeders to recombine the genes of different characters

in same plant for development of desirable variety. Plant genetic resources

are the basis of global food security. They contain diversity of genetic

5

material contained in traditional varieties, modern cultivars, crop wild

relatives and other wild species. To fulfill the demand of increasing

population and to produce more food, it would be essential to make better

use of a broader range of the world’s plant genetic diversity (Farshadfar and

Farshadfar, 2008).

1.9 Nodulation and nitrogen fixation

Plant requires amino acids to form proteins and for the synthesis of

amino acids nitrogen is one of the most important elements. Plants cannot

take the atmospheric nitrogen as such they primarily take nitrogen in the

ionic form of either ammonium (NH+,) or nitrate (NO3) (Cleyet-marel et al.

1990). Leguminous plants form nitrogen-fixing root nodules post

embryonically with symbiotic bacteria called rhizobia.

The cross-kingdom symbiosis is commenced by reciprocal signal

exchange between the two organisms (Oldroyd and Downie, 2004). Root of

legumes secrets flavonoid which is sense by rhizobia. Flavonoids trigger the

secretion of nod factor by rhizobia, that in turn, recognized by the host plant

which can lead to root hair deformation and many other cellular reactions

such as ion fluxes. The rhizobia enter through a deformed root hair just like

endocytosis, making an intracellular tube known as infection thread. The

infection starts cell division in the cortex of the root where a new organ, the

nodule appears as a result of successive processes. Infection threads infect

the central tissue of nodule and release the rhizobia in these cells where

they differentiate morphologically into bacteroids and start fixation of

atmospheric nitrogen into plant useable form ammonia (NH4+), by using

nitrogenase enzyme. In return the plant provides carbohydrates, proteins

6

and oxygen to the bacteria. Plant protein leghaemoglobins (similar to human

hemoglobins) helps in providing oxygen for respiration by keeping the free

oxygen concentration very low so that nitrogenase activity may not be

inhibited.

The fact that about 50 million tons of nitrogen is manufactured

industrially each year against an estimated 90 million tons fixed by plant

processes, has make the phenomenon of nitrogen fixation tremendously

important and the value of this “free” fertilizer N, can be placed in global

perspective. (Cleyet-marel et al., 1990)

1.10 Inoculation of chickpea with rhizobium

On farmer’s fields, yield of Chickpea is very low as compared to its

potential. The basic reported reasons responsible for its low yield are the

unavailability of high-quality seed, absence of effective rhizobial inoculation

and severe damage by blight and pod borer attack. In those soils lacking

native effective rhizobia artificial seed inoculation is a very useful practice

for the improvement of root nodulation and yield of the chickpea (Rupela

and Dart, 1979; Sharma et al., 1983; Shamim and Ali, 1987; Shah et al.,

1994). Legume inoculation is a means of guarantee that the strain of

Rhizobium appropriate for the cultivar being planted is present at the proper

time in numbers sufficient to assure effective nodulation and nitrogen

fixation (Cleyet-marel et al., 1990).

It has been reported that the soils of Pakistan are generally deficient

in nitrogen which is an essential element in the plants metabolism and

protein synthesis. The deficiency of nitrogen is one of the basic reasons of

7

the low crop yield. In order to stimulate early growth of leguminous crops

and to induce the activity of nitrogen fixing bacteria a starter dose of

fertilizer nitrogen is frequently used in most legumes (Ali et al., 1998; Jefing

et al., 1992).

It is important that agricultural scientists learn to manipulate this

symbiotic relationship in agronomic practice, employing selected

combinations of bacteria and legumes in specific situations to obtain

maximum crop production on land which is of low fertility and frequently

unsuitable for growth of non-legume crops. Nitrogen fixation rates from 75

to 300 kg of N per hectare per year are common in various combinations.

1.11 Nodulation mutants in chickpea

Non-nodulating, ineffectively nodulating and/or super nodulating

mutants have been identified in several species of soybean, chickpea,

peanut, red clover, and alfalfa. Variants in nodulation have been produced

either spontaneously or from induced mutagenesis (Methews at al., 1989).

These altered nodulation mutants play an important role in understanding the

physiological processes and genetic control of nodule development and

function.

1.12 Linkage study in chickpea

By studying inheritance patterns in pedigrees, it is possible to identify,

which genes are near each other on a chromosome. This information is

called linkage, which provides a way to map genes on chromosomes.

Linkage is important when used with other techniques to determine the

8

location of certain genes on chromosomes, especially for genes, which have

not been well studied, and for understanding the mode of transmission for

many genetic disorders. Linkage study between non-marker characters like

nodulation in chickpea and morphological markers like flower color, stem

color etc will help the breeders and biotechnologists to do selection of better

genotypes and to map the locus and gene controlling important characters

(Thomas, 1999; Pundir and Reddy, 1998)

1.13 Molecular study in chickpea

DNA marker analyses are of increasing importance in modern plant

breeding, and, as the methods become more widely adopted, the capacity

for high-throughput analyses at low cost is crucial for its practical use. DNA

marker techniques are non-destructive, and heterozygous individuals may

be identified and maintained more easily than by conventional means

(Helentjaris, 1991). Molecular marker technology is a valuable tool for plant

breeding. A number of techniques [e.g. restriction fragment length

polymorphism, amplified fragment length polymorphism, simple sequence

repeats (SSR), single nucleotide polymorphisms] can be used as DNA

markers linked to characters/traits of interest, directing selection towards

these markers instead of a phenotypic reaction (Edwards and Mogg, 2001).

Important breeding steps can be considerably improved by molecular

marker tools, now available in plant breeding.

Chickpea is an important system for genomics research because of

its small genome size (740 Mb) and high economic importance as a food

crop legume. Molecular markers are the prerequisites for undertaking

molecular breeding activities. However, till now the reasonable number of

9

molecular markers could not be developed in cultivated species of

chickpea. One of the major reasons for this may be the detection tools that

are currently available which can sense a low level of genetic variability in

the cultivated gene pools of these species (Huttle et al., 1999; Winter et al.,

1999). Although through various markers and genomic tools several genetic

linkage maps have become available, but still sequencing efforts and their

use are limited in chickpea genomic research. Among various molecular

markers currently available, microsatellite or SSR markers are often chosen

as the favored markers for a variety of applications in breeding because of

their multi-allelic nature, co-dominance inheritance, relative abundance and

extensive genome coverage (Gupta and Varshney, 2000). As a result

several hundred of SSR markers have been developed in chickpea (Huttle

et al., 1999; Winter et al., 1999).

Due to the extreme importance of nodulation in nitrogen fixation,

restoring soil fertility and increased crop production, Furthermore, owing to

the increasing importance and utilization of molecular study via molecular

markers, this project was planed with the following major objectives:

1. Characterization of chickpea germplasm for morphological markers and

quantitative traits

2. Categorization of chickpea genotypes for nodulation and evaluation of

the effect of rhizobial inoculation on nodules number and seed yield

plant-1

3. To investigate the inheritance of nodulation and genetic linkage

between loci responsible for nodulation and leaf color

4. Molecular Characterization of chickpea genotypes using microsatellite

markers

10

CHAPTER 2

REVIEW OF LITERATURE

Research findings regarding genetic diversity of nodulation, effect of

rhizobial inoculation and other important agronomic and morphological

parameters in chickpea is reported from most parts of the world but such

information are lacking for the germplasm being grown in Pakistan. The

information about genetics and other related studies of nodulation conducted

at the agro-climatic conditions of the country are rarely available in literature.

Since chickpea is marginal areas’ crop in Pakistan and is grown on sandy

soils thus its production is entirely dependent on timely rain fall during its

growing season. Due to high risk of chickpea production because of

uncertain rains, the growers are reluctant to use inputs and thus government

gives very low attention toward research for the improvement of this crop.

Research findings related to nodulation and other important relevant

parameters reported in literature are briefly reviewed below.

2.1 Characterization of chickpea germplasm for morphological markers and other quantitative traits

In chickpea sufficient genetic variability for number of days taken to

flowering, days to maturity, plant height, total dry weight of the plant and

grain yield plant-1 and high heritability for plant height (96.24%) and total dry

weight of the plant (80.26%) has also been reported (Iqbal et al., 1994).

Considerable amount of variations were also reported in characters like plant

color, flower color, dots on seed testa, seed testa texture, days to maturity,

pods per plant, 100-seed weight and plot yield among all the three groups

11

(kabuli, desi and intermediate). However, the Kabuli and Intermediate types

did not differed significantly for seed color and growth habit (Upadhyaya et

al., 2002). Sufficient genetic variability was reported for various quantitative

traits including plant height, number of pods plant-1, 100- seed Weight,

biological yield and seed yield plant-1. Heritability for these characteristics

indicated that selection could be more effective for genetic improvement

(Nawab, 2002). Highly significant differences among genotypes were also

reported for days to flowering, number of secondary branches plant-1,

number of pods plant-1, 100-seed weight seed yield plant-1 (Shahzad et al.,

2002; Shaukat et al., 2003; Arshad et al., 2004), pods plant-1, days to

maturity (Burli et al., 2004), seed color, flower color and plant growth (Afsari

et al., 2004).

Significant differences for yield and yield attributes among genotypes

for high productivity, wide adaptability and multiple resistances can be

achieved simultaneously by using potentially complementary approaches

(Yadav et al., 2004). Extensive amount of genetic diversity was also

observed in grain yield and yield components of chickpea (Imtiaz et.al.,

2004; Bakhsh et al., 1999). Significant amount of genetic variability was

recorded among chickpea genotypes for pods plant-1, 100-seed weight;

biological yield plant-1 and seed yield plant-1 (Jeena et al., 2005).

In chickpea high genetic variability with considerable amount of

heritability was registered for seed yield plant-1 followed by biological yield

plant-1, pods plant-1, 100-seed weight, secondary branches plant-1 plant

height (Sharma et al., 2004) flowering, days to maturity, 100 seed weight,

secondary branches and biological yield (Ghafoor et al., 2004; Burli et al.,

2004). High coefficients of variation (CVs) were recorded in pods branch-1,

12

seeds pod-1, pods plant-1, yield plant-1, seeds plant-1and branches plant-1

(Naghavi and Jahansouz, 2005) 100-seed weight and seed yield kg ha-1

(Hakim et al. 2006) and some other morphological traits (Farshadfar, 2008)

showing low environmental impact for these characters. Highest genotypic

variance was recorded for 1000 seed weight, followed by seed number plant-

1. Minimum (5.47%) broad-sense heritability was reported for days to

flowering while maximum (51.66%) for seed number plant-1 (Derya and

Emin, 2006).

Significant amount of variability were observed for means and

components of variability for yield and various yield components in chickpea.

Lower genotypic and phenotypic coefficients of variation were found for days

to maturity and days to flowering. While moderate PCV and GCV were

observed for seeds pod-1, plant height, branches plant-1, 100 seed weight,

total weight of the plant, pods plant-1 and seed yield plant-1. High heritability

estimates were observed for branches plant-1, pods plant-1, seed yield

(Durga et al., 2007), days to flowering (Saleem et al., 2008), days to maturity,

grain yield plant-1 and secondary branches (Atta et al., 2008). On the other

hand in another report estimates of heritability were moderate for days to

maturity and pods plant-1 (Saleem et al. 2008). This variation has

necessitated the establishment of a core collection of chickpeas in the world.

2.2 Characterization of genotypes for nodulation and effect of rhizobial inoculation on nodulation

According to Rupela (1992) the frequency of Nod- plants ranged from

120 to 490 per million in four genotypes (ICC 435, –4918, –5003 and –4993)

of chickpea. The Nod- selections and their respective parent genotypes

13

were identical in plant growth they only differed in their nodulation trait, and

most Nod- plants yielded likewise to their Nod+ genotypes when supplied

with 50 to 100 kg N ha–1. While in the field with low nitrogen fertilizer or

without N fertilizer, the Nod- plants grew poorly, light green, had small

leaves and leaflets, had short internodal distance and had reddish brown

pigment on margine of leaflets, rachis and sometime branches. In another

report sinorhizobium fredii strains was found to produce nodules and can

fix nitrogen significantly, for symbiosis S.fredii-soybean, compatibility is

must (Vidieria et al., 2001)

Bhatia et al. (2004) examined the Prospects of using the

nodulation mutants in developing grain legume cultivars that combine high

yield with high residual N, within the bioenergetic constraints, for developing

sustainable cropping systems. Nodulation mutants have contributed to the

understanding of plant-microbe symbiotic interactions, nitrogen fixation, and

nodule development. Breeding of genotypes with higher yield and nitrogen

fixation rate becomes possible through nodulation mutants. They found that

after inoculation with specific bacterial strains or a mixture of strains, the

nodulation mutants show either: no nodulation (nod-), few nodules (nod+/-),

ineffective nodulation (fix-), or hyper nodulation (nod++). In most of the

nodulation mutant’s monogenic recessive inheritance has been observed,

however semi-dominant and dominant inheritance was also reported. The

process of autoregulation controlled the nodule number; relaxed

autoregulation have been reported in hypernodulating mutants.

Significant variation has been reported with respect to nodulation

among chickpea genotypes (Yadav et al., 2004; Gallani et al., 2005).

Nodulation also showed high heritability along with significant variation

14

among different genotypes of chickpea (Mensah and Olukoya, 2007). C.

arietinum and its root nodule bacteria should be considered in a separate

cross-inoculation group (Gaur and Sen, 1979). There is a significant

correlation between nodulation and seed yield (Corbin et al., 1977).

Nitrogen fixed by nodules is the most important source supplying nitrogen

to the grain as compare to the other sources like nitrogen fertilization

(Hungria and Neves, 1987).

Symbiotic relationship in chickpea is highly specific, with a unique

group of rhizobia necessary for the formation of nodules and nitrogen

fixation. Absence of appropriate strains, low population numbers,

infectiveness, and poor survival of rhizobia are basic reasons for

nodulation problems. Legume inoculation assures presence of appropriate

rhizobial strain at proper time in sufficient number which confirms nodules

formation and nitrogen fixation (Cleyet-Marel et al., 1990). Numbers of

indigenous rhizobia inversely affect the response of the plant to

inoculation and the competitive success of inoculant rhizobia. The

considerable response to N application, significant increases in nodule

parameters, and more than 50% nodule occupancy by inoculant rhizobia

did not essentially correspond to significant inoculation responses (Thies

et.al., 1991).

Liquid inoculant gave a very uniform coverage of the seeds and yield

of lentil and peas increased upto 29% and 9%, respectively (Hynes et al.,

1995). Significant variation was reported for plant growth in response to

inoculation with Rhizobium strains (Rodríguez-Navarro et al., 1999).

Equally effective establishment of successful symbiosis was reported with

peat as well as granular inoculants in chickpea (Stephen et al., 2002). On

15

the other hand in another study the performance of peat based powder

inoculant was better than the liquid inoculant in terms of seed yield. The

effect of inoculant formulation on nodules number, nitrogen accumulations

etc were in order: granular > peat powder > liquid = un-inoculated

(Clayton et al., 2003).

An increase in 1000 seed weight, seed yield and biological yield in

chickpea was observed with seed inoculation as compared to un-

inoculated seed (Hakoomat et al., 2004). Inoculation also enhanced

nodules formation on the root system of chickpea (Khattak et al., 2006). In

the same way significantly enhanced yield as well as nitrogen uptake was

recorded with rhizobial inoculation in sainfoin over the control (Tufenkci et

al, 2006).

Significant increase in nodules number, nodule fresh weight and

nitrogen uptake along with grain yield was recorded in chickpea with

rhizobium inoculation (Romdhane et al., 2007). The average increase due

to inoculation was 110% over the un-inoculated control. Different

genotypes responded differently to inoculation, the effect of various

rhizobium strains was also diverse (Tellawi et al., 2007). Stover yield also

showed positive response to inoculation along with seed yield, nodule

number and nodule weight in chickpea (Bhuiyan et al., 2008).

Requirement of competitive rhizobium was reported for enhanced

nodulation and yield of chickpea and is economically promising to

increase chickpea production (Romdhane et al., 2008).

16

2.3 Inheritance and linkage study of gene responsible for nodulation in chickpea

Non-nodulation trait is controlled by two tetrasomically inherited

recessive genes (nn1, and nn2) in Alfalfa. At both loci the nulliplex condition

resulted in non-nodulation. The non-nod trait segregates independently of

one another (Peterson and Barnes, 1981). In chickpea nodulation study

through combining ability showed that nodulation was predominantly under

the control of non-additive gene action although considerable additive effect

was also present (Bhapkar and Deshmukh, 1982). In another study

resistance race 1 of Fusarium oxysporum f.sp was found to be controlled by

at least two genes in chickpea. Presence of both genes in homozygous

recessive form is must for complete resistance (Updhyaya, 1983). In

soybean the gene for ineffective nodulation in Kent was dominant over the

gene responsible for normal nodulation in Pecking with the fast-growing

rhizobial strain USDA 205 (Devin, 1984).

Host genetic control of nodulation in the chickpea rhizobium

symbiosis was tested by nodulation mutants PM665, PM679 and PM 233 at

different root temperatures (24°C, 29°C and 34°C). Mutant PM 233 did not

form nodules at any temperature tested while other mutants altered their

nodulation response at different root temperature (Devis et al., 1986).

These mutants of chickpea were characterized by rhizobium strain CC1192.

Genetic studies showed that the Nod– phenotypes in PM233, PM665, and

PM679 were controlled by recessive alleles at three different loci. They

proposed that the symbols rn1, rn2, and rn3 be assigned to the loci

producing the non-nodulated phenotype in mutants PM233, PM665, and

PM679, respectively (Thomas et al., 1986). Similarly in mutants PM405 and

17

PM796 of chickpea ineffective nodulation is controlled by two recessive,

non allelic root nodulation genes. Recessive epitasis was observed in

cross PM405 x PM796 (Devis, 1988).

A new genetic model involving three genes in the inheritance of non-

nodulation was proposed in pea. Nodule formation is controlled by two

genes while the third genes inhibit nodulation only when it is dominant and

the former two genes (that control nodule formation) are in recessive

homozygous condition. They suggested that nodulation intensity appears to

be controlled quantitatively (Dutta and Reddy, 1988).

Non-nodulation in mutant ICC 435 of chickpea is controlled by single

recessive gene. Recessive gene that control non-nodulation in mutant ICC

435 is different from the recessive gene that control non-nodulation in

mutant PM 233 (Singh et al., 1992). In another study monogenic dominant

gene action was reported for nodulation in chickpea genotypes, Annegeri,

Rabat, PM 233 and P 319-9. The recessive gene controlling non-nodulation

of roots in previously identified mutants (PM 233, P319-9 NN, and Anigeri

NN) is different from the one that identified in Rabat NN (Singh and Rupela,

1998). However, in pea, lines UF 487A, PI 262090, and Florunner,

nodulation is controlled by three genes at three independent loci (Gallo-

Meagher et al., 2001).

Nodulation in promiscous (produce functional nodules and green

leaves) type of soybean is controlled by two dominant alleles at two

segregating loci and thus in non-promiscous (no or nonfunctional nodules

and yellow leaves) it is controlled by recessive alleles at each locus (Gwata

et al., 2005).

18

Genetic linkage was reported among gene controlling restricted root

nodulation (Rj1) and gene responsible for fascinated stem (F) in soybean.

The two loci were separated by a distance of 40 ± 2.2 genetic map units

(Dvine et al., 1983). Allelic and linkage associations among five

morphological markers of chickpea showed linkage between allele for white

flower color (w2) and filiform leaf trait (fil). Linkage was also exhibited

between simple leaf trait (slv) and root nodulation gene (rn3). They also

observed a loose linkage between the w2-fil and the rn3-slv (Devis, 1991).

Linkage relationship between principle subtypes of histone (H1) and

the cotyledon protein FG which is soluble in perchloric acids was recorded.

The two groups of genes were found to be linked with a recombination value

ranging from 6.5 to 9.5. The corresponding genes of Pisum sativum (garden

pea) were also found to be linked (Gorel and Berdnikov, 1993). Flower type

(open or closed) and leaf type (small or large) of chickpea were found to be

controlled by single recessive gene. Linkage was reported between these

two valuable traits (Pundir and Reddy, 1998).

Linkage was reported in B. oleracea among loci for genetic male

sterility and seedling markers. Linkage was also observed between male

sterility gene ms-1 of green sprouting broccoli (var. italica) and gene c

(antheocyanin free plant with bright green hypocotyls) of curly kale (var.

acephala) and variegated ornamental kale (Sampson, 1966a). Furthermore,

linkage was recorded between genes po (polycotyledony) and le (leaf

excrescened), among loci gl-3 (glossy) and pg (pale green) and between the

glossy foliage gene gl-y and gl-e3 of the linkages group 1(Sampson, 1967b).

In B. oleracea Linkage was also detected between the genes Fn (fern-leaf)

and c-2 (antheocyanin suppressor), and genes cp-1 (crinkly petal) and A

19

(pigmented overy) (Wills and Smith, 1972). Genes responsible pg-2 (pale

green foliage) and Hr-1 (hairy leaf) (Wills and Smith, 1974), cp-1 (crinkily

petal) and as (anther spot) and gl-el (glossy foliage) also showed linkage in

B. oleracea. Genes fc1 and fc2 (fused cotyledon) were found to be unlinked

(Wills and Smith, 1973). A dominant gene for heavy pubescence, designated

Hr –2, from curly kale (var. acephala) acted in an additive manner with gene

Hr-1 (hairy leaf margins) to produce relatively long hairs on leaves, petioles

and stems. Linkage (group 4) between the two genes was tentatively

proposed (Sampson, 1978).

2.4 Molecular characterization of chickpea germplasm using Microsatellite (SSR) markers

Microsatellite markers were found to be useful in distinguishing

diverse genotypes. The large amount of polymorphisms permits a quick

and efficient detection of barley genotypes (Struss and Plieske, 1998).

High level of interspecific polymorphism was reported with microsattelite

markers as compare to isozymes, RAPDs and conventional RFLPs which

revealed slight or no polymorphism. The amount of variation revealed by

microsattelite had significant positive correlation with the average number

of repeats, showing that replication slippage may be the molecular

mechanism involved in generation of variability at the loci (Udupa et al.,

1999).

Simple sequance repeat (microsatellite) markers were reported to

be efficient for assessing the genetic variability patterns within chickpea

(Winter et al,. 1999). Significant proportion (14.6%) of genetic variation

20

among cultivar was recorded by analysis of molecular variance (AMOVA)

with SSR markers in raygrass (Christine et al., 2001). SSR markers

detected an average of five alleles per locus and had mean polymorphism

information content (PIC) value of 0.67 in rice (Coburn et al., 2002).

Microsatellite markers have great value in the evaluation of

polymorphic information content, diversity index, and probability of identity

in legumes (Jan et al., 2002). With RAPD markers analysis of molecular

variance (AMOVA) showed 18% of the total variation by species, 15% by

populations within species, and 67% by individuals within populations. G.

max and G. soja groups are separated completely by principal component

analyses and cluster analysis (Zenglu and Randall, 2002). Different levels

of variability were recorded with SSR markers while studying bean types.

Lower level of variation was recorded for fine French bean, indicating the

effect of breeder’s intensive selection (Métais et al., 2004)

Simple sequence repeats (SSR) were reported to cause mostly the

modification of the gene with which they are connected. The number of

repeats is responsible for the influence of SSRs on gene regulation,

transcription and protein function. However there are frequent and reversible

mutations that add or subtract repeat units, thus SSRs provide a productive

source of qualitative and quantitative variation. Over the last decade,

researchers have reported that this spontaneous variation has been tapped

by artificial and natural selection to adjust almost every aspect of gene

function. These studies support the hypothesis that SSRs, by asset of their

special mutational and functional qualities, have a most important role in

producing the genetic diversity underlying adaptive evolution (Kashi and

King 2006).

21

Evaluation of chickpea genotypes differing in reaction to ascochyta

blight by simple sequence repeat (SSR) markers linked to quantitative trait

loci (QTL) for resistance, revealed that it is possible to enhance the level of

resistance by crossing moderately resistant parents with distinct genetic

backgrounds at the QTL for resistance to ascochyta blight (Tar’an et al.,

2007). SSR markers are well suited for studies of genetic diversity and

cultivar identification in chickpea because SSR markers are polymerase

chain reaction (PCR)-based markers, extremely polymorphic, and amenable

to high-throughput technologies (Imtiaz et al., 2008).

22

CHAPTER 3

MATERIALS AND METHODS

The research project was conducted in the chickpea growing seasons

of 2006 to 2009. The project consisted of classical breeding (carried out at

Peshawar, Pakistan) and molecular study (accomplished at Faculty of

Agriculture, Ehime University Matsuyama, Japan).

3.1 Procurement of seed

Forty-nine local and exotic genotypes were procured from Nuclear

Institute for Food and Agriculture (NIFA), Peshawar, Grain Research Station

(GRS), Ahmadwala Karak and International Crops Research Institute for the

Semi-Arid Tropics (ICRISAT), India.

Out of 40 genotypes procured from NIFA, two genotypes (NKC-262-

26 and NKC-452-2) did not germinate. Out of remaining 38 genotypes, 22

(NDC-122 to NDC 4-20-7, NIFA 88 to NIFA 2005 and Hassan 2K) were

developed through induced mutation through gamma irradiation of four

genotypes i.e. C-44, Pb-91, 6153 and ILC-195. The remaining 16 genotypes

obtained from NIFA were the selection from hybrids originated at ICRISAT,

India and ICARDA, Syria. Genotypes, NDC 5-S10 and NDC 5-S11 were

developed through hybridization of Desi and Kabuli genotypes at ICRISAT.

Genotypes NKC-10-99 to NKC-5-S24, were developed through hybridization

of Kabuli x Kabuli lines at ICARDA. Five genotypes (Karak 1 to Lawaghar)

procured from ARS were the selection from land races of Pakistan. Four

genotypes (ICC4993 to ICC19181) were obtained directly from ICRISAT, out

of these two genotypes (ICC19183 and ICC19181) were developed at

23

ICRISAT while the other two are land races, one from India (ICC4993) and

one (ICC4918) from Morocco (Detailed description along with pedigree of

these genotypes is presented in Table 3.1.).

3.2 Experimental sites

Field experiment was conducted in department of Plant Breeding and

Genetics (PBG), Khyber Pukhtunkhwa Agricultural University Peshawar

(KPK-AUP) in the season of 2006-07. In this experiment germplasm was

characterized for genetic markers and other quantitative traits.

In the same season (2006-07), the second experiment was conducted

in pots in the net house facility of Institute of Biotechnology and Genetic

Engineering (IBGE) KP-AUP, to evaluate genotypes for presence or absence

of nodules without rhizobium inoculation as well as with rhizobium

(Rhizobium leguminosarium) inoculation and grouped into nodulated and

non-nodulated genotypes. The germplasm was also characterized for

number of nodules plant-1 (without inoculation) to recognize the highly

nodulated genotypes. In this experiment the effect of inoculation on number

of nodules and seed yield plant-1of diverse chickpea genotypes was also

studied.

In experiment 4 all the 47 genotypes were also characterized at

molecular level for confirmation of groupings (nodulated/non-

nodulated).Also, to study diversity in the germplasm and among nodulated

and non-nodulated genotypes at molecular basis. This experiment was

performed in the Forest Resource Biology (FRB) laboratory and green house

facilities of Ehime University Matsuyama, Japan during March-October 2008

24

Third experiment was consisted of study of inheritance pattern of

nodulation and linkage of gene responsible for nodulation with morphological

marker (leaf color) using selected highly nodulated and non-nodulated

genotypes (experiment 2).

These genotypes were crossed in the experimental fields of the

department of PBG, during growing season of 2007-08. In the subsequent

season (2008-09), the F1 generation was grown in the fields of Nuclear

Institute for Food and Agriculture (NIFA), Peshawar to get the F1 population.

Back crosses were also made. During September-October 2009, F1,

backcross and F2 populations along with concerned parents were evaluated

for presence (+) or absence (-) of nodules and leaf color, in the net house of

the department of PBG, KP-AUP.

25

Table 3.1 Pedigree and origin of genotypes/accessions used in the study Genotype name

Parentage Origin Genotype name

Parentage Origin

NDC-122 C-44 x ILC-195

NIFA, Pakistan

NKC-10-99 Flip98-138c x Sel99th15039

ICARDA/,Syria

NDC-727 C-44/M NIFA, Pakistan

NKC-5-S12 BAHODIR x SEL99TER85530

ICARDA, Syria

NDC-728-5 C-44/M NIFA, Pakistan

NKC-5-S13 SEL99TH15039 x S98008

ICARDA, Syria

NDC-730-2 C-44/M NIFA, Pakistan

NKC-5-S14 SEL99TH15039 x S98008

ICARDA, Syria

NDC-15-1 Pb-91/M NIFA, Pakistan

NKC-5-S15 FLIP98-15C x S98033

ICARDA, Syria


NKC-5-S16 S99456 x SEL99TER85314

ICARDA, Syria


NKC-5-S17 S99456 x SEL99TER85314

ICARDA, Syria


NKC-5-S18 (ILC4291xFLIP98-129C) x S98008

ICARDA, Syria

NDC-4-15-1 C-44/M NIFA, Pakistan

NKC-5-S19 (ILC4291xFLIP98-129C) x S98008

ICARDA, Syria


NKC-5-S20 (FLIP98-138C x SEL99TH15039)

ICARDA, Syria


NKC-5-S21 GLK95069 x SEL99TER85530

ICARDA, Syria


NKC-5-S22 CA9783007 x SEL99TER85534

ICARDA, Syria


NKC-5-S23 CA9783007 x SEL99TER85534

ICARDA, Syria


NKC-5-S24 CA9783007 x SEL99TER85534

ICARDA, Syria


HASSAN-2K ILC-195/M NIFA, Pakistan


Karak 1 Local selection Karak, Pakistan





NDC-5-S10 JG 74 x ICC 12071.

ICRISAT, India

Sheenghar Local selection Karak, Pakistan

NDC-5-S11 JG 74 x ICC 12071.

ICRISAT, India

Lawaaghar Local selection Karak, Pakistan

NIFA-88 6153/M NIFA, Pakistan

ICC 4993 Rabat Karnataka, India

NIFA-95 6153/M NIFA, Pakistan

ICC 19183 ICC 4993 ICRISAT

NIFA-2005 PB-91/M NIFA Pakistan

ICC 4918 Annigeri Morocco

NKC-262-26 ILC-195/M NIFA/,Pakistan

ICC 19181 ICC 435 ICRISAT

NKC-452-2 (ILC4291 x Flip98-129c) x S98008

ICARDA, Syria

26

3.2. Experiment 1 Characterization of germplasm for various

morphological markers and quantitative traits

This experiment was carried out using a Randomized Complete

Block Design (RCBD) with three replications. Each replication consisted of

forty nine blocks of 4 m2, having a block to block distance of 40 cm. Each

block was divided in three rows, each of which was of 4 m long. Plant-to-

plant spacing between and within rows was kept 30 cm and 10 cm,

respectively.

Morphological or genetic markers

All genotypes were characterized for three morphological markers

i.e. flower color (pink / white), stem color (green / green with purplish

tinge) and seed coat color (brown /dark brown / yellow / white).

Quantitative characteristics

Genotypes were also evaluated for quantitative traits including days

to 50% flowering, days to maturity, plant height, number of leaflets leaf-1,

leaf area, seed yield plant-1,100 seed weight and biological yield.

Procedures for recording observations

Morphological markers

Flower color and stem color were recorded at flowering stage while

seed coat color was noted at pod maturity.

27

Quantitative traits

Ten equally competitive plants were ear marked from each genotype

replication-1 for recording data on the quantitative traits. Days to 50%

flowering were recorded as number of days from the date of sowing to the

date on which at least 50% of the plants had at least one flower. Days to

maturity were counted from the date of sowing to the date on which 90% of

the plants had mature pods. Data on plant height was documented in cm at a

stage when plants turned brown and ceased further growth. Plant height of

the already selected plants was measured from the ground level to the tip of

the plant with the help of a meter rod. Average number of leaflets leaf-1 were

noted for five randomly selected leaves selected from each plant, while Leaf

area of the same leaves was measured in cm using leaf area meter (Licor

model 2000).

After about 175 days, most of the genotypes were mature. Pods from

the already selected plants were harvested and threshed separately. Seeds

of these selected plants genotype-1 were weighed using electric balance and

recorded as Seed yield plant-1. A random sample of 100 seeds from each

genotype was also weighed. Selected plants of all genotypes were carefully

uprooted and sun dried. Total biomass of each selected plant was weighed

and average weight plant-1 (g) was calculated as biological yield plant-1.

Data analysis

Data were analyzed using statistical software SAS (statistical analysis

system) version 9 following the model for a Randomized Complete Block

Design (RCBD). Least significant difference (LSD) test at 5% probability

level was applied for mean differences (James et al., 1997).

28

The genetic parameters (genotypic and phenotypic variances, and

their coefficient of variation) and heritability (broad-sense) were estimated

as suggested by Burton (1952) and Hanson et al. (1956), respectively.

Details of the equations used are as follows

Genotypic Coefficient of Variation

Phenotypic coefficient of variation

Heritability

Where

Vg = Genotypic variance; Vp = Phenotypic variance; Ve = Environmental

variance; MSG = mean squares of genotypes; MSE = mean squares of

error; r= number of replications; Ū= mean value for a particular trait

3.4.1 Experimsnt.2 Characterization of genotypes for nodulation and effect of rhizobial inoculation on nodules number and seed yield plant-1

Treatments and design

To study presence or absence of nodules, the effect of rhizobium

inculcation on nodules number and seed yield plant-1 and to select highly

nodulated and non-nodulated genotypes for further study, a pot

100x(GCV)u

gV 2

100)( xPCVu

Vp

r

MSeMSgVg

Vp

Vg

VeVgVp

29

experiment was carried out in the net house of Institute of Biotechnology

and Genetic Engineering (IBGE), KP Agricultural University, Peshawar

during 2006-07. The inoculum (Rhizobium leguminosarium) for seed

treatment was provided by Soil Microbiology section of Agriculture

Research Institute, Tarnab, Peshawar.

The experiment comprised of two treatments (un-inoculated and

inoculated). Pots were arranged in three replications using Completely

Randomized Design (CRD). Seeds of each genotype were sown in two

pots replication-1 @ 5 seeds pot-1 in each treatment. Pots having a

diameter of 22 cm were filled with 4.5 KG soil. Potting soil was comprised

of 50% clay and 50% sand. All genotypes (Table 3.1) were evaluated for

number of nodules plant-1 genotype-1 in un-inoculated pots as well as in

the pots treated with inoculum. Moreover effect of inoculation on seed

yield plant-1was also recorded.

Inoculant preparation and use

Chickpea inoculation slurry was prepared by adding 40 g of

inoculant in 300 ml of 5% sugar solution. The contents were stirred well.

Sugar solution improves the adhesion of inoculant to the seed. Slurry was

then poured on seed, and mixed in a clean vessel or on a plastic sheet

until all the seeds were uniformly coated. The whole inoculation procedure

was completed in shade as sunlight damages the bacteria. Seed were

then dried in shade for about an hour before sowing in pots.

30

Procedure for data collection

Plants were removed from pot at flowering stage. Soil was washed

out by dipping the plants along with the soil into a tub of clean tap water.

This procedure was repeated three times for each entry in order to remove

soil thoroughly. All genotypes were checked for presence or absence of

root nodules. Number of nodules plant-1 was also recorded for inoculated

and un-inoculated treatments. Seed yield plant -1 was recorded in g, after

harvest by weighing seeds of each plant pot -1 for all genotypes in both

treatments.

Figure 3.1 Washing Procedure of roots to check nodules

31

Data analysis

Data of number of nodules plant-1 was analyzes by SAS (statistical

analysis system version 9). Genotypic and phenotypic variances, their

coefficient of variation and heritability (broad-sense) were estimated as

suggested by Burton (1952) and Hanson et al. (1956), respectively

(formulas are given in experiment 1)

The effect of rhizobium inoculation on the number of nodules plant-1

and seed yield plant-1 of chickpea and difference among treatments (un-

inoculated and inoculated) were analyzed using analysis of variance

procedures (SAS, statistical analysis system version 9) for two factorial

design by using PROC GLM. The differences among the treatments

mean, genotypes mean and interaction of treatment and genotype were

compared, using least significant difference (LSD) test at 5% probability

level.

Percent change (Percent increase or decrease) in number of

nodules plant-1 and seed yield plant-1 after rhizobium inoculation of each

genotype was obtained by the following formula

% change = Mean of inoculated – Mean of un-inoculated X 100 Mean of un-inoculated

On the basis of presence or absence of nodules (with rhizobium

inoculation and without inoculation) in each genotype the germplasm was

divided into nodulated and non-nodulated groups. Among nodulated

genotypes highly nodulated genotypes were selected, for studying

inheritance and linkage study of nodulation.

32

3.5 Experiment.3 Inheritance and linkage study of nodulation

Three chickpea genotypes viz., NDC 5-S-10 (nod+), NDC 4-20-4

(nod+) and ICC 19181, (nod-) selected in experiment 2. Selection was

based on high nodulated and non-nodulated genotypes.

Hybridization of Nod+ with Nod- chickpea genotypes

These genotypes were crossed in the following two combinations

during 2007-08 in the field of PBG, AUP.

Production of F1 and backcross seeds

The hybrid seeds obtained from both cross combinations were

grown during 2008-09 in the field of Nuclear Institute for Food and

Agriculture (NIFA), Peshawar. The parents and F1 generation (seeds of

hybrid A and hybrid B) were raised in separate rows as follows:

Row 1: Male parent of hybrid A (NDC 5 S 10)

Row 2: Hybrid A seeds (ICC19181 x NDC 5-S-10)

Row 3: Female parent of hybrids A and B (ICC 19181)

Row 4: Hybrid B seeds (ICC19181 x NDC 4-20-4)

Row 5: Male parent of hybrid B (NDC 4-20-4)

F1 plants of both cross combinations (hybrid A and hybrid B) were

backcrossed to their respective female as well as male parents in order to

S.No Cross combination Hybrid Name

1 ICC19181♀ x NDC 5-S-10♂ Hybrid A

2 ICC19181 ♀x NDC 4-20-4♂ Hybrid B

33

produce backcrossed population (BC11F1, BC12F1) for assessing the

inheritance pattern of nodules.

Seeds from the F1 plants (including backcross seeds as well as F1

seeds) were collected separately.

Segregating population

In the season of 2009, the following populations were tested for

presence/absence of nodulation in the screen house of the Department of

Plant Breeding and Genetics, KP Agricultural University Peshawar.

Parents (ICC 19181, NDC 5-S-10 and NDC 4-20-4)

F1 (of hybrids A and B),

Backcross, BC11F1a (hybrid A X ♀ parent),

Backcross, BC12F1a (hybrid A X ♂ parent)

Backcross, BC13F1b (hybrid B X ♀ parent),

Backcross, BC14F1b (hybrid B X ♂ parent)}

F2 (of hybrids A and B)

Data was recorded for the presence/absence of nodules on 120

plants of parents (40 plant parent-1), 57 plants of F1 (27 plants of hybrid A

and 30 plants of hybrids B), 270 backcross plants (84 plants of BC11F1a, 48

of BC12F1a and 79 of BC11F1b, 59 BC12F1b) and 226 F2 plants (109 from

hybrid A and 117 from hybrid B). Three seeds were sown pot-1 [22 cm-

diameter pots filled with 4.5 KG soil (50% sand + 50% clay)]. Seeds were

inoculated at the time of sowing with rhizobium leguminosarium strain. Pots

of each entry were placed in eleven separate rows as per following details:

34

Row 1: 14 pots (40 plants) of ICC 19181 (♀ parent of both hybrid A and B)

Row 2: 14 pots (40 plants) of NDC 5 S 10 (♂ parent of hybrid A)

Row 3: 14 pots (40 plants) of NDC 4-20-4 (♂ parent of hybrid B)

Row 4: 9 pots (27 plants) of hybrid A (ICC19181 x NDC 5-S-10)

Row 5: 10 pots (30 plants) of hybrid B (ICC19181 x NDC 4-20-4)

Row 6: 28 pots (84 plants) of Backcross BC11F1a (Hybrid A x ICC 19181)

Row 7: 16 pots (48 plants) of Backcross BC12F1a (Hybrid A x NDC 5-S-10)

Row 8: 27 pots (79 plants) of Backcross BC11F1b (Hybrid B x ICC 1918)

Row 9: 20 pots (59 plants) of Backcross BC12F1b (Hybrid B x NDC 4-20-4)

Row 10: 37 pots (109 plants) of F2 population of hybrid A

Row 11: 40 pots (117 plants) of F2 population of hybrid B

Plants were evaluated after six weeks of sowing for the presence or

absence of nodules (Singh and Rupela, 1998)

Data collection

Data leaf color was also recorded six weeks after sowing (used for

linkage study in experiment 3 part 2) for all populations. Plants along with

soil were removed from pots by holding pots upside down (to study the

presence or absence of nodules). The soil was washed out thoroughly

(washing procedure is same as presented in section 3.4). All plants were

carefully scored for leaf color and presence or absence of root nodules.

Data analysis

The Chi-square (χ2) test using Yates correction factor to adjust small

population size was used to test the goodness of fit for appropriate genetic

hypotheses (Steel et al., 1980). Data on presence or absence of nodulation

35

in F2 and backcross populations were tested based on the hypothesis that

it will fit to the expected ratios of 3:1, 1:1, respectively. The test statistics

was calculated as described by Yates (1934).

Where:

χ2 Yates= test statistic that asymptotically approaches a χ2 distribution

with yates correction factor.

Oi = observed frequency

Ei = expected (theoretical) frequency, asserted by the null hypothesis

N = number of distinct event

For linkage study between gene responsible for nodulation and leaf

color, the data recorded in F1 and F2 generations was analyzed through

Linkage 1 computer program (Sulter et al., 1983).

3.6 Experiment 4 Molecular characterization of chickpea germplasm using microsatellite markers

Molecular analysis of chickpea germplasm was performed in Forest

Resource Biology Laboratory at Faculty of Agriculture, Ehime University

Matsuyama, Japan.

DNA Extraction

Seeds of chickpea were sown in 27 cm diameter pots containing 7.5

Kg of sandy soil with 5 seeds per pot in a green house. DNA was extracted

36

separately from fresh leaves of 2 to 5 week old plants for each genotype

using modified CTAB method described by Doyle and Doyle (1990). Details

of the method are given below:

1. 0.5 g of leaves were weighed and placed into a pestle. Liquid

nitrogen was poured on leaves and grinded with a mortar to convert

them into powder form. Then 10 ml Cold isolation Buffer I (CIBI) was

added and further grinded.

2. The material was filtrated with cheese cloth.

3. The solution was transferred to 15 ml spits tube, and centrifuged at

3,500 rpm for 10 min. Then the upper solution was discarded.

4. 2.5 ml of Cold isolation Buffer (CIB) II was added and kept in

refrigerator for 10 min.

5. 2.5 ml of 2 x CTAB (cetytriammoniumbromide) was added. Mixed

well and kept in water bath at 60°C for 30 min.

6. Equal volume (5 ml) of CIA (Chloroform: L isoamylalchohol, 24:1)

was added. Shacked for 15 min on shaker.

7. Centrifuged at 3,500 rpm for 10 min.

8. The supernatant was transferred to a new 15 ml spits tube. The

debris was discarded to a determined container.

9. 2/3 vol. of ice-cold isopropanol was added and mixed gently. DNA

appeared (Isopro-precipitation).

10. Isopro-precipitation was centrifuged at 3,500 rpm for 10 min and the

upper solution was discarded carefully, not to lose the precipitation

(DNA).

37

11. Alcohol was removed completely. 250µl of TE (10 mM TrisCl PH8.0,

1mM EDTA PH8.0) was added to dissolve DNA. DNA solution was

transferred into a new 1.5 mL Eppendorf tube.

12. 0.5 µl of RNase (10mg/mL) was added and incubated at 37°C for 30

min in a water bath.

13. Equal volume (250 µl) of Tris-EDTA saturated phenol was added.

Mixed well for 5 min.

14. Centrifuged at 10,000 rpm for 10 min. The supernatant was

transferred to a new 1.5 mL Eppendorf tube.

15. 1/10 volume (18 µl) of 3M sodium acetate was added; mixed well

and then 2.5 volumes (500 µl) of 100% ethanol was added. Mixture

was kept in a freezer at -20°C for at least 30 min. Mixed gently and

DNA precipitation were appeared (this step is called “ethanol

precipitation”).

16. Mixture was centrifuged at 12,000 rpm for 10 min. The upper

solution was discarded with care so that not to lose DNA.

17. 70% ethanol was added gently on top of DNA (rinsing). Kept at

room temperature for 5 min without mixing.

18. DNA along with 70% ethanol was centrifuged at 12,000 rpm for 10.

The upper solution was discarded. Kept at room temperature with

the tube lid open (air dry).

19. 100µl of TE (10 mM TrisCl PH8.0, 1mM EDTA PH 8.0) was added to

dissolve DNA. Then kept in refrigerator.

38

Amplification of DNA

Ten SSR (microsatellite) markers including 5 dinucleotide and 5

trinucleotide repeats were used for genotyping. Eight markers were screened

from C. arietinum BAC libraries (Lichtenzveig et al., 2005) and 2 were Cicer

arietinum sequence-tagged microsatellite site (CaSTMS) markers screened

from a genomic library of the same species (Hüttle et al., 1999). Primers were

labeled with FAM, HEX, and NED fluorescent dyes (Applied Biosystems, Foster

City, USA). Sequences of the primer pairs for each marker are listed in Table

3.2. The reaction mixture for amplification of markers contained 8.4 µl of distilled

water, 1.25 µl of 10× PCR buffer, 1.25 µl of 2 mM deoxynucleotide mix, 0.5 µM

forward and reverse primers, 0.1 U of Taq DNA polymerase (Blend Taq,

Toyobo, Osaka, Japan) and 5 ng of genomic DNA in a total volume of 12.5 µl.

Microsatellite amplification was carried out using the following cycling

parameters: preheating for 3 min at 95°C followed by 25 cycles of denaturing at

94°C for 30 sec, annealing at primer specific temperatures of 48–60°C for 30 sec

(Table 3. 2), and extension for 1 min at 72°C. Reactions were completed by

incubation at 72°C for 1 min and holding at 4°C. The PCR products were

denatured for 2 min at 95°C and separated by capillary electrophoresis on an

ABI Prism 310 Genetic Analyzer (Applied Biosystems). GeneMapper software

(Applied Biosystems) was used for sizing and genotyping microsatellite

alleles.

39

Table 3.2 Details of primers sequencing

Locus Sequence Annealing

temperature Motif Repeats

H1116f GACATGAAATTCGGTGCATT 52°C GA 20

H1116r AACGCCCTAAACCTCTTGGT

H1F17f GGGGAGGAAGAAGATGGAA 48°C TA 27

H1F17r GCGTTATGGGTGGAAATGGTA

H3C06f AATTTCGTGAATCATTAAAAATAGAGG 55°C TAA 23

H3C06r CACATGACTATCTAGACATTTTATTTATC

H3A10f TTTAAGGCTTCAGGTATTGATTTCT 55°C TTA 24

H3A10r TCACACATGCCAACTTAAAATAAAA

H3A07f GCGACACCTATTCCTCTTTTCTA 58°C TTA 20

H3A07r TCATTTTTGGAATATTTTAGTGACAA

H2J09f AACGAAAAACAAGGGAGAAAAA 52°C GA 18

H2J09r TATTTCTTTGACTCCCCCTAACTT

H1B11f GCAGCTGTTGACATCTAATTTTG 60°C TAA 20

H1B11r ACCGAAAACACTTGTGATTGTTA

H6G07f TCTATCAGAGATATTAAGTTGAACG 60°C TAA 23

H6G07r CGTGACAGAATTAGCCTCTTGT

CaSTMS19f TGAAGCTGGGGGTTCCTTG 50°C AT 15

CaSTMS19r TCAATTGAGTCGCGACGAGAG

CaSTMS25f TACACTACTGCTATTGATATGTGGT 50°C CT 19

CaSTMS25r GACAATGCCTTTTTCCTT

40

Microsatellite data analysis

Genetic diversity parameters were computed using GenAlEx 6

software (Peakall and Smouse, 2006). The following statistics were

estimated: average number of alleles (Na), and effective number of alleles

(Ne). Polymorphic information content (PIC) of each microsatellite locus was

determined as described by Weir (1996):

PIC 1 pi2

where pi is the frequency of the ith allele in the examined test lines. The

estimation of genetic differentiation was performed using AMOVA and

Principal coordinate analysis (PCoA) implimented in GenAlex 6. Random

permutation test with 999 shuffles was performed under the assumption of

stepwise mutation model (Slatkin, 1995). Phylogenetic analysis was

completed using UPGMA and ME method using MEGA4 software (Tamura

et al., 2007) based on the genetic distance matrix constructed by the method

of Smouse and Peakall (1999).

41

Chapter 4 Results and Discussions

Experiment.1. Characterization of chickpea germplasm for various morphological markers and quantitative traits

4.1.1 Introduction

The assemblage of germplasm with a wide range of genetic

variability is preliminary and prerequisite step to initiate a successful

breeding program. The efficacy of a germplasm collection would be

enhanced if the distinctive features of each accession were to be

described and recorded, so that researcher could choose those

accessions from the collection, which have the desired genetic

characteristics for the specific objectives of a program.

The characterization of diversity in germplasm collections is

important to plant breeders for crop improvement and to gene bank

curators for efficient and effective management of collection (Updhaya,

2003). The presence of genetic variability is of utmost importance for any

breeding program and for that reason the plant breeders have recognized

the evaluation and characterization of germplasm for the improvement of

crop yield (Virmani et al., 1983; Bakhsh et al., 1992) as well as for

selection of core collection for utilization in breeding programs. Thus the

evaluation of germplasm is not only useful in selection of core collection

but also for its utilization in breeding programmes.

Chickpea has high variation for various qualitative and quantative

traits i.e. grain color and shape, color of flower, podding, seed coat color,

earliness, insect pests resistance, like any other crop of different

ecological zones, that can help breeders to release better and superior

42

lines and varieties (Dasgupta et al., 1987; Singh, 1997). For maintenance

and efficient utilization of germplasm, it is important to investigate the

extent of genetic variability and its magnitude for the determination of the

success of a breeding program (Smith et al., 1991).

An initial step in a breeding program is the assembly of germplasm

with a wide range of genetic variability. The utility of a germplasm

collection would be enhanced if the unique features of each genotype

were to be described and recorded, so that the researcher could choose

those genotypes in the collection, which have the genetic characteristics,

desired for his specific objectives (Shah, 1999).

Evaluation and characterization of chickpea germplasm has received

attention of plant breeders due to increased recognition and its

importance (Virmani et al., 1983; Bakhsh et al., 1992). Thus due to utmost

importance of genetic diversity, the present study was planned with

following objectives

1. Characterization of genotypes for various genetic markers

2. Characterization of genotypes for other quantitative characters

3. To estimate heritability of different important traits

4.1.2 Materials and Methods

Forty nine local/exotic genotypes obtained from NIFA, Peshawar;

GRS, Ahmedwala Karak and ICRISAT, India were evaluated in the field of

Plant Breeding and Genetics department, Agricultural University

Peshawar, during the season of 2006-07. The material was planted in

randomized complete block design (RCBD) with three replications. Out of

49 genotypes two genotypes failed to germinate therefore data were

43

collected on 47 genotypes (Table 4.1.7). Detailed materials and methods

including data collection, data analyses etc of this experiment are

presented in chapter 3 section 3.3.

4.1.3 Results

4.1.3.1 Characterization for morphological markers

Characterization for flower, stem and seed coat color of all

genotypes is presented in Table 4.1.1. Significant variation in color of

three traits among genotypes was observed. Out of 47 genotypes, 29

genotypes including 23 from NIFA, four from Karak and two from

ICRISAT, India (Table 4.1.7) produced pink flowers, green with a purplish

tinge stem and light brown to dark brown or yellowish seed coat color

(Table 4.1.1). These markers are typical for Desi type chickpea. Rest 18

genotypes including 15 from NIFA, one from Karak and two from ICRISAT

(Table 4.1.7) produced white flowers with green stem and white seed coat

(Table 4.1.1). These characters are distinctive for Kabuli type chickpea.

These markers play a vital role in identification of land races and

estimation of out-crossing percentage in chickpea.

4.3.2 Characterization for quantitative characters

All genotypes were also evaluated for days to flowering, days to

maturity, plant height, leaflets leaf-1, leaf area, seed yield plant-1, 100-seed

weight, and biological yield. Analysis of variance (Table 4.1.1 and 4.1.2)

and mean values (Table 4.1.3 and 4.1.4) for each trait showed highly

significant differences and broad range of variation for various traits for

44

genotypes, which is amenable for genetic improvement of chickpea

through selection. Flowering in genotype ICC19183 was earlier (90 days),

while in NDC-4-20-6 was late (122). Genotype ICC19181 matured earlier

(163 days) while genotype Sheenghar matured later (178 days). Plants of

the accession NDC 5-S11 were the shortest (47 cm) whilst those of NKC-

5-S15 were the tallest (94 cm). Leaf area of accession NKC-5-S18 was

the largest (10.92 cm) whereas of genotype ICC4918 was the smallest

(1.73 cm). Genotypes NDC-730-2 and NDC-4-20-2 produced the

maximum number of leaflets leaf-1 (16.33) while genotype NDC-5-S11

produced the minimum (11.66) leaflets leaf-1. On average seed yield of

Karak 3 was the maximum (28.9 g) while of NDC-15-2 was the lowest (4.1

g). Weight of 100 seed of NKC-5-S18 was the maximum (39 g) whereas of

NDC-4-20-1 was the minimum (13 g). NIFA-2005 produced the highest

biological yield (68 g) whilst NKC-5-S19 yielded the lowest (14.3 g).

Heritability estimates (Table 4.1.6) were high for days to 50%

flowering (93%), biological yield plant-1 (89%), plant height (88%), 100

seed weight (82%), seed yield plant-1 (77%) and number of leaflets leaf-1

(75%). Moderate heritability was recorded for days to maturity (68%) and

leaf area (51%). All traits revealed higher phenotypic coefficient of

variation (PCV) as compare to genotypic coefficient of variation (GCV)

(Table 4.1.6). However, differences among PCV and GCV were low for

days to 50% flowering (7.1 and 6.9), days to maturity (2.3 and 1.9), plant

height (14.5 and 13.4), leaflets leaf-1(13.4 and 11.l6), 100 seed weight

(23.3 and 21.1) and biological yield plant-1 (36.4 and 34.5) while only leaf

area (29.91 and 21.2) and seed yield plant-1 (49.9 and 43.6) revealed

comparatively more difference among PCV and GCV. Majority of traits

45

revealed high heritability and low level of differences among PCV and

GCV which indicate less environmental influence on these traits and

showed that genotypes had more influential role in the expression of these

traits. This suggests a great chance of genetic improvements of these

traits in chickpea.

46

Table 4.1.1 Flower color, stem color and seed coat color of evaluated chickpea germplasm Genotype Name Flower color Stem color Seed coat color NDC-122 Pink Green with purplish tinge light brown NDC-727 " " dark brown NDC-728-5 " " dark brown NDC-730-2 " " dark brown NDC-15-1 " " " NDC-15-2 " " " NDC-15-3 " " " NDC-15-4 " " " NDC-4-15-1 " " " NDC-4-15-2 " " " NDC-4-15-3 " " " NDC-4-20-1 " " " NDC-4-20-2 " " " NDC-4-20-3 " " " NDC-4-20-4 " " " NDC-4-20-5 " " " NDC-4-20-6 " " " NDC-4-20-7 " " " NDC-5-S10 " " dark brown NDC-5-S11 " " yellowish NIFA-88 " " brown NIFA-95 " " brown NIFA-2005 " " yellowish NKC-10-99 white Green white NKC-5-S12 " " " NKC-5-S13 " " " NKC-5-S14 " " " NKC-5-S15 " " " NKC-5-S16 " " " NKC-5-S17 " " " NKC-5-S18 " " " NKC-5-S19 " " " NKC-5-S20 " " " NKC-5-S21 " " " NKC-5-S22 " " " NKC-5-S23 " " " NKC-5-S24 " " " HASSAN-2K " " " Karak 1 Pink Green with purplish tinge brown Karak 2 " " " Karak 3 " " " Sheenghar " " " Lawaghar white Green white ICC4993 " " " ICC 19183 " " " ICC4918 Pink Green with purplish tinge brown ICC19181 Pink “ brown

47

Table 4.1.2. Mean square for days to 50% flowering, days to maturity, plant height and number of leaflets leaf-1 of chickpea germplasm

Mean square values Source of variation

Degrees of freedom Days to 50%

flowering Days to maturity

Plant height

Number of leaflets leaf-1

Replication 2 25.45 12.27 0.900 0.57

Genotype 46 186.64** 38.4** 260.05** 8.61**

Error 92 4.36 5.75 60.42 0.84

Table 4.1.3. Mean square for leaf area, seed yield plant-1, 100 seed weight and biological yield plant-1 of chickpea germplasm

Mean Square values Source of variation

Degrees of

freedom Leaf area Seed yield plant-1

100 seed weight

Biological yield plant-1

Replication 2 0.45 3.64 1.75 10.82

Genotype 46 9.09** 70.75** 88.02** 359.36**

Error 92 2.23 6.58 5.93 13.5

48

Table 4.1.4: Mean values of days to 50% flowering, days to maturity, plant height and leaflets leaf-1 in chickpea germplasm

Genotype Days to 50%

Flowering Days to maturity Plant height No of Leaflets

leaf-1

NDC-122 116 173 70 16 NDC-727 118 167 66 12.6 NDC-728-5 118 168 77 15.6 NDC-730-2 114 166 73 16.3 NDC-15-1 118 171 66 15.0 NDC-15-2 118 175 76 15.6 NDC-15-3 118 169 62 16.0 NDC-15-4 118 168 67 15.6 NDC-4-15-1 115 173 64 16.0 NDC-4-15-2 116 171 50 16.0 NDC-4-15-3 118 172 58 15.3 NDC-4-20-1 116 169 70 15.3 NDC-4-20-2 119 170 67 16.3 NDC-4-20-3 118 172 65 14.6 NDC-4-20-4 117 173 67 16.0 NDC-4-20-5 115 170 67 16.0 NDC-4-20-6 122 173 63 14.0 NDC-4-20-7 116 172 67 15.6 NDC-5-S10 105 177 51 12.0 NDC-5-S11 104 176 47 11.6 NIFA-88 116 175 73 12.0 NIFA-95 115 174 67 12.0 NIFA-2005 113 171 64 13.0 NKC-10-99 116 175 88 12.0 NKC-5-S12 112 176 69 12.3 NKC-5-S13 113 176 72 12.3 NKC-5-S14 111 175 66 12.0 NKC-5-S15 113 175 94 12.0 NKC-5-S16 97 174 60 16.0 NKC-5-S17 110 176 57 14.0 NKC-5-S18 99 175 62 12.0 NKC-5-S19 120 172 65 12.3 NKC-5-S20 101 175 72 13.0 NKC-5-S21 112 176 60 14.0 NKC-5-S22 107 175 64 13.3 NKC-5-S23 107 174 73 13.3 NKC-5-S24 110 173 73 11.6 HASSAN-2K 116 174 68 15.6 Karak 1 115 175 69 12.0 Karak 2 117 174 67 12.6 Karak 3 114 172 67 13.0 Sheenghar 120 178 70 14.3 Lawaghar 118 176 60 14.0 Rabat 97 165 88 12.3 ICC 90 164 89 11.0 Annigeri 96 168 59 12.3 ICC19181 92 163 60 12.3

LSD (0.05) 3.38 3.89 2.42 1.49

49

Table 4.1.5: Mean values of chickpea germplasm leaf area, seed yield plant-1, 100 sed weight and biological yield-1 in chickpea germplasm.

Genotype Leaf area Seed yield plant -1 100-seed weight

Biological yield plant-1

NDC-122

8.3

10.0

27.0

39.7

NDC-727 6.2 7.0 20.7 32.0 NDC-728-5 9.5 7.8 21.7 30.4 NDC-730-2 8.2 7.4 20.9 36.0 NDC-15-1 7.1 15.0 23.0 39.6 NDC-15-2 7.6 4.1 21.1 20.4 NDC-15-3 8.0 5.0 20.3 23.1 NDC-15-4 7.8 9.0 25.0 22.2 NDC-4-15-1 7.7 5.3 20.1 24.0 NDC-4-15-2 9.9 7.0 23.0 18.2 NDC-4-15-3 6.5 5.4 23.0 24.1 NDC-4-20-1 6.5 18.0 13.0 56.7 NDC-4-20-2 8.3 6.2 21.3 29.0 NDC-4-20-3 8.8 5.3 22.3 22.0 NDC-4-20-4 8.0 5.0 20.6 24.3 NDC-4-20-5 6.8 6.0 21.7 28.0 NDC-4-20-6 7.5 15.6 25.0 65.7 NDC-4-20-7 7.7 5.2 21.5 47.4 NDC-5-S10 5.7 9.1 27.0 24.4 NDC-5-S11 5.4 9.0 27.0 23.0 NIFA-88 7.1 7.5 14.6 20.1 NIFA-95 4.4 7.0 14.1 19.7 NIFA-2005 7.2 14.7 22.0 68.0 NKC-10-99 8.3 7.2 33.0 43.9 NKC-5-S12 5.2 13.6 23.0 34.6 NKC-5-S13 8.3 15.5 35.0 39.6 NKC-5-S14 7.0 14.5 29.0 32.3 NKC-5-S15 9.0 13.1 32.0 36.7 NKC-5-S16 8.5 12.0 25.0 30.0 NKC-5-S17 9.8 13.3 34.0 34.4 NKC-5-S18 10.2 13.2 39.0 32.1 NKC-5-S19 7.1 10.0 26.0 14.3 NKC-5-S20 6.4 13.1 29.0 27.4 NKC-5-S21 5.6 12.0 25.0 27.1 NKC-5-S22 6.4 9.2 25.0 26.4 NKC-5-S23 7.1 12.5 28.0 31..0 NKC-5-S24 6.7 15.0 33.0 36.2 HASSAN-2K 5.0 13.2 23.0 26.3 Karak 1 7.2 18.2 22.0 31.3 Karak 2 8.1 16.9 24.0 28.5 Karak 3 6.3 28.9 25.0 27.6 Sheenghar 7.5 14 22.0 33.4 Lawaghar 7.5 13.7 36.0 25.3 Rabat 3.6 7 21.0 28.7 ICC 3.8 6 22.0 24.6 Annigeri 1.7 8.4 28.0 25.1 ICC19181 4.6 5.9 26.0 27.7

LSD (0.05) 6.18 4.16 4.44 5.95

50

Table 4.1.6: Estimates of genotypic variance (Vg), phenotypic variance (vp), genotypic coefficient of variability (PCV), phenotypic coefficient of variability (GCV) and heritability ( h2

(bs) ) for various agronomic traits

Traits

Vg

Vp

GCV

PCV

%h2

(bs)

Days to 50% flowering

60.76

65.06

6.9

7.18

93

Days to maturity

10.88 15.93 1.9 2.3 68

Plant height 81.84 96.37 13.41 14.56 88

Leaflets leaf-1

2.58

3.43

11.66

13.42

75

Leaf area

2.28 4.519 21.26 29.91 51

Grain yield plant-1

21.3 27.9 43.6 49.9 77

100 seed weight

27.34

33.34

21.16

23.37

82

Biological yield plant-1 115.28 128.78 34.51 36.47 89

51

Table 4.1.7 Description of chickpea genotypes used in present study

No. Entry Phenotype Genotype ParentageA Origin HB

1 NDC-122 Desi Nod+ C-44 × ILC-195 NIFA 02 NDC-727 Desi Nod+ C-44/M NIFA 0.253 NDC-728-5 Desi Nod+ C-44/M NIFA 0.3754 NDC-730-2 Desi Nod+ C-44/M NIFA 0.1255 NDC-15-1 Desi Nod+ Pb-91/M NIFA 06 NDC-15-2 Desi Nod+ Pb-91/M NIFA 07 NDC-15-3 Desi Nod+ Pb-91/M NIFA 08 NDC-15-4 Desi Nod+ Pb-91/M NIFA 09 NDC-4-15-1 Desi Nod+ C-44/M NIFA 010 NDC-4-15-2 Desi Nod+ C-44/M NIFA 011 NDC-4-15-3 Desi Nod+ C-44/M NIFA 012 NDC-4-20-1 Desi Nod+ C-44/M NIFA 013 NDC-4-20-2 Desi Nod+ C-44/M NIFA 0.37514 NDC-4-20-3 Desi Nod+ C-44/M NIFA 015 NDC-4-20-4 Desi Nod+ C-44/M NIFA 0.516 NDC-4-20-5 Desi Nod+ C-44/M NIFA 0.12517 NDC-4-20-6 Desi Nod+ C-44/M NIFA 018 NDC-4-20-7 Desi Nod+ C-44/M NIFA 0.12519 NDC-5-S10 Desi Nod+ JG74 × ICC12071 ICARDA 020 NDC-5-S11 Desi Nod+ JG74 × ICC12071 ICARDA 0.37521 NIFA-88 Desi Nod+ 6153 NIFA 022 NIFA-95 Desi Nod+ 6153/M NIFA 0.12523 NIFA-2005 Desi Nod+ Pb-91/M NIFA 0

24 NKC-10-99 Kabuli Nod+ FlIP98-138C × SEL99TH15039

ICARDA 0

25 NKC-5-S12 Kabuli Nod+ BAHODIR × SEL99TER85530

ICARDA 0

26 NKC-5-S13 Kabuli Nod+ SEL99TH15039 × S98008 ICARDA 0.2527 NKC-5-S14 Kabuli Nod+ SEL99TH15039 × S98008 ICARDA 0.12528 NKC-5-S15 Kabuli Nod+ FLIP98-15C × S98033 ICARDA 0.375

29 NKC-5-S16 Kabuli Nod+ S99456 × SEL99TER85314

ICARDA 0.625


ICARDA 0

31 NKC-5-S18 Kabuli Nod+ (ILC4291 × FLIP98-129C) × S98008

ICARDA 0.375


ICARDA 0

33 NKC-5-S20 Kabuli Nod+ FLIP98-138C × SEL99TH15039

ICARDA 0

34 NKC-5-S21 Kabuli Nod+ GLK95069 × SEL99TER85530

ICARDA 0

35 NKC-5-S22 Kabuli Nod+ CA9783007 × SEL99TER85534

ICARDA 0


ICARDA 0


ICARDA 0

38 HASSAN-2K Kabuli Nod+ ILC-195/M NIFA 039 Karak 1 Desi Nod+ Local selection Karak 040 Karak 2 Desi Nod+ Local selection Karak 041 Karak 3 Desi Nod+ Local selection Karak 042 Sheenghar Desi Nod+ Local selection Karak 043 Lawaaghar Kabuli Nod+ Local selection Karak 0 44 ICC 4993 Kabuli Nod– Rabat Morocco 045 ICC 19183 Kabuli Nod– ICC 4993NN ICRISAT 046 ICC4918 NN Desi Nod– Annigeri India 047 ICC19181NN Desi Nod– ICC 435NN ICRISAT 0

AThe lines indicated by /M is mutation induction lines. BHeterozygosity.

52

4.1.4 Discussion

Characterization based on flower, stem and seed coat color clearly

differentiated chickpea germplasm in two types i.e. desi and kabuli.

Genotypes with pink flowers, green with purplish tinge stem and dark brown

to light brown seed coat were Desi type. Kabuli type had white flowers, green

stem and white seed coat. Upadhyaya et al. (2002) found significant

variation in core collection of chickpea for flower and plant color (stem color).

In their collection dots on seed testa were also present. Variation in flower

and seed color is also reported by Farshadfar and Farshadfar (2008) while

evaluating 360 chickpea land races. Variability in seed coat and flower color

was also observed by Afsari et al (2004) in chickpea germplasm.

Plant growth duration (flowering and maturity) plays important role in

increasing seed yield of chickpea. Variation in climatic factors like

temperature and photoperiod in different environments affect maturity of

genotypes and so the overall yield. The variability in days to 50% flowering

and days to maturity were significant among all the genotypes. Other studies

have confirmed significant variation in days to 50% flowering and days to

maturity in Chickpea (Atta et al., 2008; Saleem et al., 2008; Hakim et al.,

2006; Shaukat et al., 2003) and soybean (Muhammad et al., 2007).

Plant height is desirable trait to reduced lodging in crops; similarly,

higher seed weight, leaf area and more leaflets leaf-1 contribute to higher

seed yield. In the present study an adequate variability for plant height and

other yield contributing traits among various genotypes was found. Earlier

reports in chickpea by Atta et al. (2008), Hakim et al. (2006), Arshad et al.

(2004), Nawab et al. (2002) and Raza and Mahdi (1992) and in soybean by

Muhamad et al. (2007) also confirm these results.

53

Biomass (total dry weight) plant-1 gives significant information about

chickpea and other crops. Significant variation in biomass/biological yield

plant-1 was found among genotypes in the collected germplasm for the

current project. Similarly Jeena et al. (2005) and Arshad et al. (2004) also

reported significant differences in biological yield plant-1 among their

chickpea collection.

Components of variance for entire studied parameters are depicted in

table 4.1.6. Phenotypic coefficient of variation as well as the genotypic

coefficient of variation was found lower for days to flowering and days to

maturity. Moderate values of PCV and GCV were observed for 100 seed

weight, leaf area, leaflets leaf-1 and plant height and their range were high for

grain yield plant-1 and biological yield. Saleem et al. (2008), Atta et al.

(2008), Hakim et al. (2006) and Burli et al. (2004) also reported low PCV and

GCV for days to flowering and days to maturity in chickpea. Similar results

were registered by saleem at al. (2008) and Atta et al. (2008) for seed yield

plant-1, plant height, 100 seed weight and biological yield of chickpea, while

Hakim et al. (2006) observed high values of PCV and GCV in chickpea for

100 seed weight and seed yield plant-1 but lower for plant height. Burli et al.

(2004) also observed high PCV and GCV values for seed yield plant-1 but he

registered low values for 100 seed weight in chickpea. The difference in

result for 100 seed weight could be attributed to difference in environment.

Phenotypic coefficient of variability was greater than genotypic

coefficient of variability for all the parameters but the difference is less in all

the cases except leaf area and seed yield plant-1. All traits showed high

heritability except days to maturity and leaf area which exhibited moderate

heritability. High heritability of all the traits was also reflected by the minor

54

difference in magnitudes of PCV and GCV. High estimates of heritability of

the traits under study could be due to the greater genetic variability of the

germplasm. Saleem et al. (2008), Durga et al. (2007), Hakim et al. (2006),

Ghafoor et al. (2004), Noor et al. (2003), Sable et al. (2003), Arrora et al.

(2001) and Raza and Mehdi (1992) found similar results and observed high

heritability in chickpea for days to flowering, plant height, 100-seed weight,

seed yield plant-1 and biological yield plant-1.

The present study concluded that there is a great amount of genetic

diversity in the germplasm under study. High values of heritability indicate

that these parameters are less influenced by the environment and have

greater chance of transmission to the next generation. Therefore this

variability could be utilized successfully in different breeding programs for the

betterment of existing genotypes and for the development of new more

desirable genotypes through hybridization and other modern breeding

techniques.

55

Experiment 2. Characterization for nodulation and effect of rhizobium inoculation on nodules number and seed yield plant-1

4.2.1 Introduction

Grain legumes have an important characteristic of fixing atmospheric

nitrogen to ammonium nitrogen that can be used by the plant. In agro-

ecosystem biological nitrogen fixation (BNF) is a vital component of nitrogen

cycle. Traditionally legumes were grown as manure crop, to restore soil

fertility.

Root nodules are formed in most of the legumes when they are infected

by suitable rhizobium strain and they become able to fix atmospheric

nitrogen. However, either spontaneously or through mutagenesis non-

nodulated mutants have been reported from different species that generally

produce nodules including soybean (Glycine max L.), wild pea (Pisum spp.),

common bean (Phaseolus vulgaris L.), sweetclover (Melilotus alba Desr.),

chickpea (Cicer spp), red clover (Trifolium pratense L.) alfalfa (Medicago

sativa L.) and peanut (Arachis hypogaea L.) (Gallo-Meagher., 2001). Non-

nodulating (Nod–) lines are an important reference for estimating the amount

of biologically fixed N2 in a legume (Rupela, 1991).

Chickpea plants have a strong taproot system, grows 1.5-2.0 m deep with

3 or 4 rows of lateral roots (Cubero, 1987). Chickpea roots bear rhizobium

nodules. They are branched with laterally flattened ramifications,

occasionally forming a fanlike lobe (Corby, 1981). Nodulated mutants are

important for basic studies of plant-microbe symbiotic interactions, nitrogen

fixation and breeding of cultivars with higher yield and nitrogen fixation rate

(Bhatia et al., 2004).

56

Chickpea has the capacity to fix sufficient atmospheric nitrogen to

replace the nitrogen removed in harvested grains (Schwenke et al., 1998).

It has highly specific symbiotic association, with a unique group of rhizobia

necessary for formation of nodules and nitrogen fixation. (Cleyet-marel et

al., 1990). Absence of suitable strains, small population size and poor

survival of rhizobia cause problems in nodules formation. Legumes

Inoculation assures the presence of some appropriate strain of rhizobium

compatible with cultivar, at proper time and in sufficient numbers that

guarantee effective nodulation and nitrogen fixation (Cleyet-Marel et al.,

1990).

Appropriate nodule forming bacteria presence in the soil is essential for

management and utilization of atmospheric nitrogen. If the nodulated crop

has not been sown in recent past and grown for the first time then seed

inoculation is essential before sowing. Further to avoid uncertainty about

natural inoculation, the seed should be inoculated every time (Richard and

Henery, 1967)

Nitrogen fixing potential of chickpea genotypes can be increased

significantly by inoculation (Hussain, 1983) and grain yield of chickpea

increased significantly with rhizobial application (Khattak et al., 2006).

Nodulation chickpea yield can also be enhanced by inoculation with

competitive rhizobia and is especially economical promising to increase

chickpea production (Romdhane et al., 2008). In the light of above and

existing situation, the said research work was planned to:

1. categories the genotypes for the presence or absence of nodules

57

2. identify highly nodulated genotypes on the basis of number of

nodules plant-1

3. study the influence of seed inoculation with rhizobium on the number

of nodules plant-1 in all the genotypes.

4. evaluate the change in seed yield plant-1 genotype-1 due to rhizobium

inoculation

5. select suitable parents for inheritance and linkage studies of

nodulation in chickpea.


The experiment was carried out in pots in the net house facility of

Institute of Biotechnology and Genetic Engineering (IBGE) with

completely randomized design (CRD) during 2006-07. The experiment

was comprised of two treatments (inoculated and un-inoculated) each

consisted of three replications. Procedure for data collection, data

analyses etc of the experiment are presented in chapter 3 section 3.4.

4.2.3 Results

4.2.3.1 Characterization of genotypes for the presence or absence of nodules

Genotypes were examined without rhizobium inoculation as well as

with rhizobium inoculation for the presence or absence of nodules (Table

4.2.1). Results (both un-inoculated and inoculated) showed that genotypes

procured from NIFA Peshawar and GRS Ahmedwala Karak were nodulated

(produce nodules) while genotypes received from ICRISAT India failed to

produce any nodule (non-nodulated). Two groups were formed on the basis

of nodulation i.e. nodulated and non-nodulated. The nodulated group

58

consisted forty three genotypes i.e., from NDC 122 to Lawaghar (Table 5.1)

while non-nodulated group contained four genotypes (ICC4993, ICC19183,

ICC4918 and ICC19181).

4.2.3.2 Number of nodules plant-1 genotype-1

Analysis of variance revealed highly significant differences for

nodules number plant-1 among genotypes (un-inoculated) shown in table

4.2.1. Mean values of the genotypes for number plant-1 of nodules are

given in table 4.2.2., while components of variance and heritability

percentage for number of nodules plant-1 are presented in table 4.2.3.

The highest and lowest values for nodules plant-1 revealed wide range of

variation and showed highly significant differences among the genotypes

(Table 4.2.1). Maximum numbers of nodules plant-1 (14.53) were produced

by NDC-5-S10 followed by NDC 4-20-4 (12.53) whereas Lawaghar showed

minimum number of nodules plant-1 (2.43).

Components of variance for number of nodules plant-1 (Table 5.3)

revealed moderate phenotypic coefficient of variation (PCV) and genotypic

coefficient of variation (GCV) i.e. 32.88 and 24.08 respectively, while

heritability percentage was high i.e. 53.64%.

Figure 5.1: Nodulated Root Figure 5.2 Non-nodulated: Root

59

4.2.3.3 Effect of rhizobial inoculation on number of nodules plant-1

Among 47 genotypes 43 were nodulated while four genotypes were

recorded as non-nodulated. The non-nodulated genotypes were excluded

from the analysis. According to analysis of variance (Table 4.2.4) the

interaction means of genotypes with two treatments (un-inoculated and

inoculated) were highly significant. The genotype means as well as

treatment means also showed significant differences (Table 4.2.4). In

genotypes versus treatment interaction (Table 4.2.5), the maximum

nodules plant-1 (16.66) was exhibited by genotype NDC 4-20-1 followed by

genotype NDC 4-20-7 and NDC 15-4 with 16.5 and 16.4 nodules plant-1

respectively. The minimum number of nodules plant-1 was produced by

genotype Lawaghar (2.433) followed by genotype NKC 5-S-18 (3.100). In

genotype means, the genotype NDC 5-S10 showed best performance

followed by NDC 4-20-4 with 14.8 and 13.6 nodules plant-1 respectively.

Genotype Lawaghar showed poor performance with 4.8 nodules plant-1 in

genotype means. The treatment means were greater for inoculated

treatment (10.8 nodules palnt-1) than that of un-inoculated treatment which

showed 7.8 nodules palnt-1.

Among genotypes different levels of percent increase (Table 4.2.5)

were observed for number of nodules plant-1.With rhizobium application,

above 100% increase in nodules plant-1 was noticed in 3 genotypes (NKC 5-

S20, NKC 5-S23 and Lawaghar), 50 – 100% in 10 genotypes (NDC 727,

NDC 15-1,NDC 15-2, NDC 15-4, NDC4-15-2, NDC 4-20-7, NIFA 88, NKC

10-99, Karak 3 and Sheenghar) while less then 50% in 28 genotypes (NDC

60

122, NDC 728-5, NDC-730-2, NDC-15-3, NDC-4-15-, NDC-4-15-3, NDC-4-

20-1to NDC 4-20-6, NDC-5-S10, NDC 5-S11, NIFA 95, NIFA 2005, NKC-5-

S112 to NKC-5-S18, NKC-5-S21, Hassan 2K to Karak 2). Two genotypes

(NKC-5-S22, NKC-5-S24) did not show any positive response to rhizobium

inoculation, even a small decrease was observed in their number of nodules

plant-1. The greatest response to seed inoculation was recorded for genotype

Lawaghir (196%) whilst lowest by genotype NIFA 2005 (2.1%). The

treatment means showed that the inoculated genotypes produced 38.1%

more nodules plant-1 over the un-inoculated.

4.2.3.4 Effect of rhizobium inoculation on seed yield plant-1

Rhizobium inoculation also had extremely significant effect on seed yield

plant1 as observed in analysis of variance (Table 4.2.6). Highly significant

differences were recorded in interaction means of genotypes with two

treatments (control and inoculated); genotype means and in treatment

means (Table 4.2.6). In the interaction means of genotypes with treatments

as well as in the genotype means Karak 3 was reported as genotype with

highest seed yield plant-1 (33 and 30.4 g seed yield plant-1 ), respectively.

Genotype sheenghar stood second to Karak 3 in seed yield plant-1 (30.2 and

21.8 g). The genotype NDC 15-2 was recorded as a low yielding genotype in

the interaction means of genotypes with treatments followed genotype NDC

4-20-4 with 3.6 and 4.3 g seed yield plant-1 respectively. NDC 15-2 also

remained a poor yielder in genotype means with 3.8 g seed yield plant-

1.Treatment mean for seed yield plant-1 was 10.0 g for un-inoculated and

15.1 g for inoculated treatments.

61

Like nodulation, varied increase for seed yield plant-1 amongst genotypes

was also observed after seed inoculation with rhizobium. Seven genotypes

(NDC-727, NDC-15-4, NDC 4-15-1, NDC-5-S11, NKC-5-S13, NKC-5-S21,

NKC-5-S24, and Sheenghar) were in a position to have more than 100%

increase in seed yield plant-1, Six genotypes (NDC 4-20-3, NDC-4-20-5,

NIFA 95, NKC-5-S15, NKC-5-S20 and NKC-5-S23) provided 50 to 100%

greater seed yield plant-1, twenty five genotypes (NDC 122, NDC 728-5 to

NDC 15-3, NDC 4-15-2 to NDC 4-20-1, NDC 4-20-4, NDC 4-20-6 to NDC 5-

S10, NIFA 88 to NKC 5-S12, NKC 5-S16, NKC 5-S17, NKC 5-S19, NKC 5-

S22, Hassan 2K, Karak 2, karak 3 AND Lawaghar) produced less than

50% seed yield plant-1 after inoculation. Four genotypes (NDC-4-20-2,

NKC-5-S14, NKC-5-S18, and Karak 1) were observed with no response to

seed inoculation and there was even somewhat decrease in their seed

yield plant-1. Maximum response of 195.3% increase in seed yield plant-1 to

inoculation was recorded for genotype NKC 5-S13 while genotype NIFA

2005 revealed minimum percent increase (2.1) followed by Karak 1 (2.4) for

seed yield plant-1. The overall increase in seed yield plant-1 of inoculated

treatment over the un-inoculated treatment was 50.5%.

Table 4.2.1 Mean square for number of nodules plant-1 of chickpea germplasm

Source of variation Degrees of freedom Mean square values

Genotype 42 13.64**

Error 86 3.1

62

Table 4.2.2: Presence or absence of nodules and number of nodules plant-1 (without inoculation) in chickpea genotypes

Genotype name Nodules Present/absent

No of Nodules plant-1

(uninoculated) NDC-122 Present 8.0 NDC-727 Present 7.8 NDC-728-5 Present 7.2 NDC-730-2 Present 7.9 NDC-15-1 Present 8.1 NDC-15-2 Present 7.3 NDC-15-3 Present 9.3 NDC-15-4 Present 9.9 NDC-4-15-1 Present 8.1 NDC-4-15-2 Present 8.8 NDC-4-15-3 Present 8.7 NDC-4-20-1 Present 11.0 NDC-4-20-2 Present 12.0 NDC-4-20-3 Present 10.4 NDC-4-20-4 Present 12.5 NDC-4-20-5 Present 9.2 NDC-4-20-6 Present 8.8 NDC-4-20-7 Present 9.0 NDC-5-S10 Present 14.5 NDC-5-S11 Present 9.6 NIFA-88 Present 6.4 NIFA-95 Present 6.7 NIFA-2005 Present 9.1 NKC-10-99 Present 7.2 NKC-5-S12 Present 6.6 NKC-5-S13 Present 6.9 NKC-5-S14 Present 6.9 NKC-5-S15 Present 7.7 NKC-5-S16 Present 4.7 NKC-5-S17 Present 9.6 NKC-5-S18 Present 3.1 NKC-5-S19 Present 6.8 NKC-5-S20 Present 4.0 NKC-5-S21 Present 5.1 NKC-5-S22 Present 7.4 NKC-5-S23 Present 4.9 NKC-5-S24 Present 4.9 HASSAN-2K Present 7.8 Karak 1 Present 8.1 Karak 2 Present 7.9 Karak 3 Present 6.7 Sheenghar Present 5.8 Lawaghar Present 2.4 ICC 4993 (Rabat) Absent 0 ICC 19183 Absent 0 ICC 4918 (Annigeri) Absent 0 ICC19181 Absent 0 LSD (0.05%) 2.8

63

Table 4.2.4. Mean square for inoculation effect on nodules plant-1 and seed yield plant-1

Mean squares

Source of variance Degrees of freedom Nodules plant-1 Seed yield plant-1

Treatment 1 648.3** 1104.01**

Genotype 42 28.6** 201.59**

Treatment X Genotype 42 8.0** 28.72**

Error 172 2.19 11.86

Table 4.2.3. Estimates of variability parameters of number of nodules plant-1 in various chickpea genotypes Variability parameters

Number of nodules plant-1

Genotypic variance

3.51

Phenotypic variance

6.62

Genotypic coefficient of variation

17.48

Phenotypic coefficient of variation

32.85

Heritability (broad sense)%

53.2

64

Table 4.2.5. Effect of control and rhizobial inoculation on nodules plant-1 in chickpea gengotypes

Genotype name Un-inoculated Inoculated Genotype mean % change NDC-122 8.0 9.0 8.5 12.5 NDC-727 7.8 14.9 11.4 90.0 NDC-728-5 7.2 8.0 7.6 11.1 NDC-730-2 7.9 11.0 9.4 39.2 NDC-15-1 8.1 13.1 10.6 61.7 NDC-15-2 7.3 12.6 10.0 72.6 NDC-15-3 9.3 12.3 10.8 32.2 NDC-15-4 9.9 16.4 13.2 65.6 NDC-4-15-1 8.1 11.2 9.6 38.2 NDC-4-15-2 8.8 14.9 11.9 69.3 NDC-4-15-3 8.7 10.6 9.6 21.8 NDC-4-20-1 11 16 13.5 45.4 NDC-4-20-2 12 15 13.5 25.0 NDC-4-20-3 10.4 12.4 11.4 19.2 NDC-4-20-4 12.5 14.8 13.65 18.4 NDC-4-20-5 9.2 11.2 10.2 21.7 NDC-4-20-6 8.8 12.4 10.6 40.9 NDC-4-20-7 9.0 16.5 12.7 83.3 NDC-5-S10 14.5 15.1 14.8 4.1 NDC-5-S11 9.4 12.9 11.1 37.7 NIFA-88 6.4 9.9 8.1 54.6 NIFA-95 6.7 9.9 8.3 47.7 NIFA-2005 9.2 9.4 9.3 2.1 NKC-10-99 7.2 10.8 9.0 50.0 NKC-5-S12 6.6 9.0 7.8 36.3 NKC-5-S13 6.9 9.6 8.3 39.1 NKC-5-S14 6.9 7.2 7.1 4.3 NKC-5-S15 7.7 10.9 9.3 41.5 NKC-5-S16 4.7 6.9 5.8 46.8 NKC-5-S17 9.6 11.7 10.6 21.8 NKC-5-S18 3.1 7.5 5.3 141.9 NKC-5-S19 6.8 8.4 7.6 23.5 NKC-5-S20 4.0 10.0 7.0 150 NKC-5-S21 5.1 5.8 5.5 13.7 NKC-5-S22 7.4 5.4 6.4 -27.0 NKC-5-S23 4.9 12.5 8.7 155.1 NKC-5-S24 8.2 7.5 7.8 -8.5 HASSAN-2K 7.8 8.4 8.1 7.7 Karak 1 8.1 8.3 8.2 2.4 Karak 2 7.9 9.0 8.5 13.9 Karak 3 6.7 10.5 8.6 56.7 Sheenghar 5.8 11.0 8.4 89.6 Lawaghar 2.4 7.1 4.8 196.0 ICC 4993 0 0 0 0 ICC 19183 0 0 0 0 ICC 4918 0 0 0 0 ICC19181 0 0 0 0 Treatment mean

7.86 10.86 38.1

LSD(0.05) values Interaction LSD = 1.81; Genotype means = 1.34; Treatment means = 0.36

65

Table 4.2.6. Effect of control and rhizobial inoculation on seed yield plant-1 in chickpea genotypes

Genotype Name Un-inoculated Inoculated Genotype means % change

NDC-122 9.4 14 11.7 48.0 NDC-727 6.4 15 10.7 134.0 NDC-728-5 6.8 8 7.4 17.6 NDC-730-2 6.8 10 8.4 47.0 NDC-15-1 14.8 14.2 14.5 4.0 NDC-15-2 3.6 4.1 3.85 13.8 NDC-15-3 4.7 7 5.85 48.9 NDC-15-4 8.8 18.2 13.5 106.8 NDC-4-15-1 4.9 14.1 12.0 187.7 NDC-4-15-2 6.8 12 9.4 76.4 NDC-4-15-3 4.5 8 6.25 77.7 NDC-4-20-1 17.9 22.2 20.0 24.0 NDC-4-20-2 5.6 5 5.3 -10.7 NDC-4-20-3 4.5 7.2 5.8 60.0 NDC-4-20-4 4.3 6.2 5.2 44.1 NDC-4-20-5 5.3 10.1 7.7 90.5 NDC-4-20-6 14.8 24 19.4 62.1 NDC-4-20-7 4.4 6.16 5.2 40.0 NDC-5-S10 8.3 11.2 9.7 34.9 NDC-5-S11 8.6 18.1 13.3 110.4 NIFA-88 6.7 6.2 6.4 7.4 NIFA-95 6.0 9.06 7.5 51.0 NIFA-2005 13.8 25 19.4 81.1 NKC-10-99 6.8 11.1 8.9 63.2 NKC-5-S12 12.7 20.2 16.4 59.0 NKC-5-S13 6.5 19.2 12.8 195.3 NKC-5-S14 13.7 13.1 13.4 -4.3 NKC-5-S15 12.3 23 17.6 86.9 NKC-5-S16 11.4 12 11.7 5.2 NKC-5-S17 12.5 18.2 15.3 45.6 NKC-5-S18 12.6 12.1 12.3 -3.9 NKC-5-S19 9.3 13 11.1 39.7 NKC-5-S20 12.4 24 18.2 93.5 NKC-5-S21 11.1 22.2 16.6 100.0 NKC-5-S22 9.5 13.1 11.3 37.8 NKC-5-S23 11.3 18 14.6 59.2 NKC-5-S24 14.1 29.1 20.1 106.0 HASSAN-2K 12.1 14 13.0 15.7 Karak 1 17.1 17 17.0 0.0 Karak 2 15.5 17.1 16.3 10.3 Karak 3 27.8 33 30.4 18.7 Sheenghar 13.4 30.2 21.8 125.3 Lawaghar 12.6 16.3 14.4 29.3

Treatment means 10.0 15.1 50.5

LSD (0.05) values Interaction = 1.11; Treatment mean = 0.84; Genotypes mean = 3.93

66

4.2.4 Discussion

There was no report about non-nodulated chickpea earlier than 1985

(Thomas et al., 1985). Rupela and Sudarahana (1986) observed the

genetics of non-nodulation in a spontaneous mutant ICC 435M in chickpea.

The frequency of natural occurrence of non-nodulation in chickpea was

studied by Rupela (1992) he observed a low number of non-nodulated plants

in genotype ICC 435. The non-nodulated chickpea genotypes may originate

through spontaneous or induced mutations (Thomas et al., 1985). In the

present study the nodulation of genotypes in the un-inoculated pots showed

that there are native (local) rhizobia in the soil used in the pots which

produced nodules in chickpea genotypes.

Significant differences were recorded in the number of nodules plant-1

among the genotypes. This confirmed that these genotypes showed different

response to rhizobia. The reason of this difference is the bacteria-plant

genotype interaction or compatibility of bacteria (rhizobium leguminosarum)

and genotype of plant. The variation in nodulation among chickpea

genotypes have also been reported by Tellawi et al. (2007), Mensah and

olukoya, (2007), Gallani et al. (2005), and Rodriguez-Navarro (1999).

Values of both phenotypic coefficient of variability (PCV) and

genotypic coefficient of variability (GCV) were moderate but PCV was rather

greater than GCV. Moderate heritability indicated that genetic improvement

of number of nodules plant-1 in chickpea can be achieved successfully.

Rhizobium leguminosarium was used to test the 47 genotypes for

their nodulation expression. All genotypes developed normal nitrogen fixing

nodules except four procured from ICRSAT, India which didn’t produce any

67

nodule in control as well as inoculated treatments. Results revealed that 42

genotypes were nodulated and only four were found non-nodulated.

In un-inoculated pots majority of genotypes showed nodulation which

confirming the presence of existing indigenous rhizobia in the soil. The

nitrogen fertilizer was not applied to the pots, however the plants didn’t show

any nitrogen deficiency symptoms which confirmed the role of indigenous

rhizobia appeared to fix atmospheric nitrogen at reasonable levels. Still the

level of indigenous rhizobia might be less than 10 rhizobia g-1 of soil because

rhizobium inoculation significantly increased the nodules number and seed

yield plant-1. Thies et al. (1991) observed that once the soil holds more than

10 rhizobia g-1 of soil then the response to rhizobium inoculation become

negligible in many soils. Tufenkci et al. (2006) observed that inoculation had

not significantly influenced any of the parameters measured as the soil

already contained sufficient and an efficient native rhizobial population.

The seed inoculation with rhizobium significantly increased the

nodules and seed yield plant-1. After inoculation, the nodules plant-1 and seed

yield plant-1 increased at the rate of 38.1% and 50.5 % respectively, over the

un-inoculated chickpea genotypes. The possible reason for significant

increase in the nodules plant-1 was the increased rhizobial population in g-1

of soil. Due to increased rhizobia in the soil, the nodules plant-1 increased

which result in greater nitrogen fixation and eventually the yield was

influenced positively and resulted in significant increase in the seed yield

plant-1. A similar promotive effect of inoculation on nodules and seed yield

plant-1 in chickpea was also observed by Bhuiyan et al. (2008), Romdhane et

al. (2008), Tellawi et al. (2007), Khattak et al. (2006), Stephan et al. (2002)

and Cleyet-marel et al. (1990). However, the findings of McKenzie et al.

68

(1992) were contradictory and observed no-positive effect of rhizobial seed

inoculation on the seed yield of chickpea. The contradiction might be due to

different genotypes and climatic condition, soil fertility levels and existing

rhizobial population in soil.

The effect of inoculation on nodules number and seed yield plant-1

was different in different genotypes; in some genotypes it was very high

(more than 100%). Many genotypes were recorded with medium to high (50

to 100%) response. A large number of genotypes showed low to medium

(less than 49%) increase and at the same time a small number of genotypes

were observed with no response or somewhat decrease in nodules and seed

yield plant-1 when seed was inoculated with rhizobium. The possible

explanation of differences in response of miscellaneous genotypes to

rhizobium inoculation could be the compatibility and interaction between

rhizobium and genotypes. Romdhan et al. (2007) also found variable

response to inoculation in different chickpea cultivars. Rodríguez-Navarro et

al. (1999) findings on bean also showed significant effect of strain x cultivar

interaction for number of nodules and seed yield. They concluded that the

performance of rhizobia strain is significantly modified by the plant genotype,

the strain which was highly effective in one cultivar and could be rated

moderately or less efficient in other cultivar. Hungría and Neves (1987) also

detected significant rhizobia × plant cultivar interaction for symbiotic

parameters and plant growth in Phaseolus vulgaris. The reports of Videria et

al. (2001) in soybean also authenticated the results of this study by reporting

that nodulation was not related to rhizobium species but influenced by

interaction among rhizobium and cultivar. Otieno et al. (2009) also indicated

the variable influence of inoculation on grain legumes that depends on

69

specie, parameter being measured and other environmental factors. The

above mentioned observations identified the importance of considering both

symbiotic partners while attempting to improve different plant parameters.

The results of genotype means showed partial correlation of

nodulation with yield (Tables 4.2.5 and 4.2.6). One possible explanation may

be the complexity of yield character which is the product of several other

traits. The results are also in line with those of Bhuiyan et al. (2008) who

didn’t observe any significant correlation between seed yield plant-1 and

nodules plant-1in chickpea. Videira et al. (2001) also reported that the

increase in number of nodules was not directly reflected in soybean yield.

They suggested that the reason for such response of yield to nodules

number might be that induction of more nodules may provoke a diversion of

carbohydrate to maintain the metabolic activities of a larger mass of nodules.

Three genotypes (two highly nodulated i.e. NDC 5-S-10 and NDC 4-

20-4, and one non-nodulated genotypes, ICC1918) were selected on the

basis of current evaluation. ICC19181 was selected because most of the

non-nodulated mutants like PM 665 and PM 679 were reported to have

produced normal root nodules at a temperature of 2°C. Mutants ICC19181

(ICC 435M) when grown on at ICRISAT, India, under ambient temperature

varying between 3 and 32°C during the first 60 days after sowing, did not

produce root nodules (Singh et. al., 1992). This confirmed the stability in

resistance of this mutant to a mixture of rhizobium strains that probably

occurred in the fields.

The results suggested that nodulation and seed yield of chickpea

can be improved by inoculation with compatible rhizobia which could be

70

economically feasible to increase chickpea production. It is concluded

from the current findings that breeding efforts are needed to develop

chickpea genotypes that nodulate consistently across a wide range of

chickpea growing regions in Pakistan to overcome nitrogen deficiency

facing during growing this crop.

71

Expt. 3 Inheritance and linkage studies of nodulation in

chickpea

4.3.1 Introduction

Low availability of nitrogen is responsible for limited growth of

agricultural crops. Synthetic fertilizers like ammonia, nitrate, or urea met at

least 50% of the global requirement. However, there is great need to exploit

biological nitrogen fixation which is one of the most important sources of

nitrogen for agriculture. The primary source of biological nitrogen fixation are

rhizobium or rhizobia specie and the actinobacterium Frankia spp, Biological

nitrogen fixation through the endosymbiotic association reduces the need for

expensive nitrogen fertilizers in legume crops and is an important feature of

sustainable agriculture. Crops of family leguminocea, like chickpea do not

respond to high doses of nitrogen fertilizer as they mainly depend upon

atmospheric nitrogen. During selection of genotypes having high nitrogen

fixation efficiency was given due consideration when an attempt was being

made for breeding high yielding genotypes in legumes crops (Bhapkar and

Deshmukh, 1982)

In several legumes, genes controlling the formation and function of

nodules have been identified (Nutman, 1981). An independent single

recessive gene, determining the non-nodulation characteristic of chickpea

mutants PM233, PM665, and PM 679, has also been reported (Devis et al.,

1985; Rupela and Sudarshana, 1986). A genetic locus characterized by

Mendelian segregation with a major phenotypic effect on nodulation was

found to govern the nodulation response of soybeans (Devine, 1984).

72

Selection of plants with desirable characters is the basic step of plant

breeding. However, problems arise in selection of traits, evaluation of which

is difficult and have to be selected indirectly. That difficulty might be

overcome by the identification of markers closely linked to the desired traits;

indirect selection then becomes possible, if location of the desired gene in

relation to the other is known. Moreover, linkage map of various organisms

can be constructed with the help of linkage study.

Two genes said to be linked if they are located on the same

chromosome. Different chromosomes segregate independently during

meiosis. Therefore, for two genes located at different chromosomes, we may

assume that their alleles also segregate independently. The chance that an

allele at one locus co-inherits with an allele at another locus of the same

parental origin is 50% and such genes are unlinked.

Major component of genetic characterization of agronomic crops is

the evaluation of genetic linkage relationship among agronomically important

genes. More than sixty mendellian genes controlling morphological

characteristics such as the shape, color and the size of leaves, flower, fruits,

and other organs (reviewed by Muehlbauer and Singh, 1987), and the

formation and function of root nodules (Davis et al.,1986; Devis, 1988) have

been reported. The unclear allelic relationship among genes described, and

the inconsistent genetic terminology used by different workers, makes it

difficult to ascertain how many different linkage groups actually have been

identified in chickpea (Muehlbauer and Singh, 1987).

Polymorphic monogenic traits were some of the earliest genetic

markers used in scientific studies and they may still be best for genetic,

breeding and plant germplasm management. Although morphological

73

markers are limited in nature but their evaluation neither require complicated

equipment nor difficult procedures (Singh and Singh, 1992). Morphological

markers (Monogenic or oligenic) are usually simple, fast and cheap to score

(Ghafoor, 1999). Some of these traits may serve as genetic markers

appropriate for plant germplasm management (Stanton et al., 1994). The

information provided by markers based approach depend on the type and

number of markers and their linkage relationship (Singh and Singh, 1992)

Nodulation is an imperative character of legumes due to which

leguminous plants are capable of growing under nitrogen-limiting conditions.

Due to the utmost importance of biological nitrogen fixation and high cost of

synthetic fertilizers, breeding for an efficient nodulation in legume crops is an

important trait. Information on the inheritance of nodulation would facilitate to

incorporate this important character in a high yielding and well adapted

genotypes. Moreover, nodulation can’t be evaluated until plants are

uprooted, though, this problem can be solved by the identification of a

marker traits linked to nodulation. In the light of above mentioned facts the

present experiment was designed with the following objectives

1. To understand the mode of inheritance of nodulation in chickpea.

2. To know the inheritance pattern of leaf color in chick pea.

3. To identify linkage among loci controlling nodulation and leaf color.


The selected chickpea genotypes i.e. ICC19181 (non-nodulated with

dark green leaf color), NDC 5-S10 (nodulated with light green leaf color) and

NDC 4-20-4 (nodulated with light green leaf color) were hybridized during the

74

chickpea growing season of 2007-08 at PBG department, AUP. In 2008-09

the F1 generation was raised at NIFA, Peshawar to produce F1,, BC1 (P1)

and BC1 (P2) seed as per following details:

Parents, F1, F2 and backcross generations were raised in the net

house of the department of Plant Breeding and Genetics during September

2009. Data for leaf color and nodulation was recorded after six weeks of

planting. The distribution of phenotypic classes was tested for goodness-of-

fit through Chi square test. Detailed materials and methods including

procedure for data collection, data analysis etc has been mentioned in

chapter 3.

Figure 4.3.1 Chickpea crossed flower; Figure 4.3.2 Roots of nodulated (left) and non- nodulated plants (right)

S.No Cross combination Cross designation

1 ICC19181 x NDC 5-S-10 Hybrid A (F11)

2 ICC19181 x NDC 4-20-4 Hybrid B (F12)

3 (ICC19181 x NDC 5-S-10) x ICC19181 BC11(P1)

4 (ICC19181 x NDC 5-S-10) x NDC 5-S-10 BC12(P2)

5 (ICC19181 x NDC 4-20-4) x ICC19181 BC13(P1)

6 (ICC19181 x NDC 4-20-4) x NDC 4-20-4 BC14(P2)

75

4.3.3 Results

4.3.3.1 Inheritance of nodulation

The results presented in table 4.3.1 for hybrid A (ICC 19181 x NDC 5-

S10) and Table 4.3.2 for hybrid B (ICC19181 x NDC 4-20-4) revealed that all

F1 plants of both hybrids produced normal nodules (Table 4.3.1) indicating

dominant gene effect for nodulation in chickpea..

The segregation in F2 population for nodulated and non-nodulated

phenotypes was in the ratio of 3:1 in hybrid A (Table 4.3.2) indicated

monogenic inheritance of nodulation. The distribution of nodulated and non-

nodulated phenotypes in the backcross progenies (Table 4.3.2) further

confirmed this result which showed a good fit as 1 Nod+: 1 Nod- the same as

expected for single gene inheritance. The chi-square values for F2 data of

cross ICC 19181 x NDC 5-S10 had probability between 68 - 70%, which

authenticate the presence of single dominant gene for nodulation or

monogenic inheritance of nodulation in genotype NDC 5-S10.

In hybrid B, F2 segregation for nodulation fit well to the ratio of 15

nodulated: 1 non-nodulated (Table 4.3.3). This segregation pattern was

supported by the backcross progenies in which the ratio for nodulated and

non-nodulated phenotypes showed a good fit to the expected ratio of 3 Nod+:

1 Nod- (Table 4.3.3). Thus in hybrid B, the nodulation in genotype NDC 4-20-

4 is controlled by two alleles at two independent loci with dominant gene

action. Normal nodules production in the F1 generations, and segregation of

backcross and F2 progenies for nodulated and non-nodulated phenotypes

confirmed that nodulation in genotype NDC 4-20-4 is controlled by duplicate

dominant genes. The probability of chi-square values for F2 data of cross

76

ICC19181 x NDC 4-20-4 was between 15 - 20% which strongly supports the

duplicate gene action of nodulation in genotype NDC 4-20-4.

4.3.3.2 Inheritance of leaf color

F1 population of both the crosses (ICC19181 x NDC 5-S10 and

ICC19181 x NDC 4-20-4) showed light green color (Table 4.3.1). The F2

populations segregated for leaf color as light green and dark green.

Segregating ratios (Table 4.3.4 and 4.3.5) of both F2 populations were in

good agreement with a Medelian dominant genic model and fit to 3:1 ratio.

The backcross progenies of both crosses also verified the result of F2

segregating population by showing a good fit to the ratio of 1 light green: 1

dark green. Thus it is concluded that the leaf color in chickpea is controlled

by a single dominant gene. F2 data showed high level of probability for chi

square values which confirmed the monogenic inheritance of leaf color in

chickpea.

4.3.3.3 Leaf color Vs nodulation

Results of both the crosses (ICC19181 x NDC 5-S10 and ICC19181 x

NDC 4-20-4) presented in table 4.3.6 revealed that there is a close linkage

between nodulation and leaf color traits. The recombination value between

genes for leaf color and root nodulation in ICC19181 x NDC 5-S10 cross was

observed to be 0.1575 + 0.0928 (Table 4.3.6). This showed that the distance

between two genes is about 15 centi Morgan (cM). On the other hand cross

ICC19181 x NDC 4-20-4 showed recombination value of 0.264 + 0.084

among genes responsible for nodulation and leaf color. This suggests that

the space between genes controlling nodulation and leaf color in genotype

77

NDC 4-20-4 is 26 cM. From the present study results it is concluded that

both genes controlling nodulation in genotype NDC 4-20-4 are present on

the same chromosome and the space between these two genes (responsible

for nodulation) is 11cM.

Table 4.3.1: Morphological markers of parents and their F1 used in linkage studies of Chickpea

Parents/Hybrids Leaf Color Nodulation

ICC 19181 l rn

NDC 5-S10 L RN

NDC 4-20-4 L RN

ICC 19181 x NDC 5-S10 (F1)

L RN

ICC 19181 x NDC 4-20-4 (F1)

L RN

L = Light green; l = Dark green; RN + Nodulated; rn = Non-nodulated

Table 4.3.2: Nodulation response of non-nodulated (Nod-) and nodulated (Nod+) parents, F1, F2, and backcross progenies of ICC 19181 X NDC 5-S10 cross to rhizobial inoculation.

Plants

Generation

Parent or Cross Nod+ Nod-

Expected ratio

χ2

<P <

Parent (P1) ICC 19181 0 40 -

Parent (P2) NDC 5-S10 40 0 -

F1 P1 X P2 27 0 -

F2 P1 X P2 84 25 3:1 0.14 0.70-0.68

BC1(P1) F1 X P1 45 39 1:1 0.29 0.70-0.50

BC1(P2) F1 X P2 48 0 -

Nod+:=Nodulated; Nod-= Non-nodulated; χ2= Chi Square; <P <= Probability

78

Table 4.3.3: Nodulation response of non-nodulated (Nod-) and nodulated (Nod+) parents, F1, F2, and backcross progenies of ICC19181 X NDC 4-20-4 cross to rhizobial inoculation

Plants

Generation

Parent or Cross

Nod+ Nod-

Expected ratio

χ2

<P <

Parent (P1) ICC 19181 0 40 -

Parent (P2) NDC 4-20-4 40 0 -

F1 P1 X P2 30 0 -

F2 P1 X P2 105 12 15:1 2.55 0.20-0.15

BC1(P1) F1 X P1 53 26 3:1 2.22 0.20-0.15

BC1(P2) F1 X P2 59 0 -

Nod+:=Nodulaed; Nod-= Non-nodulated; χ2= Chi Square; <P <= Probability

Table 4.3.4: Inheritance of leaf color in light green and dark green leaf colored parents, F1,

F2, and backcross progenies of cross ICC 19181 X NDC 5-S10

Plants

Generation

Parent or Cross Light

green Dark green

Expected ratio

χ2

<P <

Parent (P1) ICC 19181 0 40 -

Parent (P2) NDC 5-S10 40 0 -

F1 P1 X P2 27 0 -

F2 P1 X P2 78 31 3:1 0.49 0.50

BC1(P1) F1 X P1 47 37 1:1 0.96 0.50 – 0.30

BC1(P2) F1 X P2 48 0 -

χ2= Chi Square; <P <= Probability

79

Table 4.3.5: Inheritance of leaf color in light green and dark green leaf colored parents, F1 F2, F2,and backcross progenies of ICC19181 X NDC 4-20-4 cross

Plants

Generation

Parent or Cross Light green

Dark green

Expected ratio

χ2

<P <

Parent (P1) ICC 19181 0 40 -

Parent (P2) NDC 4-20-4 40 0 -

F1 P1 X P2 30 0 -

F2 P1 X P2 88 29 3:1 0.45 0.50

BC1(P1) F1 X P1 46 33 1:1 1.82 0.30

BC1(P2) F1 X P2 59 0 -

χ2= Chi Square; <P <= Probability

Table 4.3.6: Joint segregation for markers of chickpea F2 population for Leaf color and nodulation

Crosses Gene A- B- A- bb aa B- aa bb Sum χ2 P R+SE

ICC 19181 x NDC 5-S10

L: N 73 5 11 20 109 42.37 0.00 0.1575+0.092

ICC 19181 x NDC 4-20-4

L: N 85 3 20 9 117 18.084 0.00 0.264+0.084

χ2: Chi square tests for assuming ratio 9:3:3:1; P: probability of a greater chi square; SE: Stander error; R:

Recombination value

80

4.3.4 Discussion

The results of hybrid A in this study fit to the monohybrid Mendalian

ratio 3 nodulated: 1 non-nodulated which indicates that nodulation in

genotype NDC 5-S10 was controlled by a single dominant gene. In chickpea,

monogenic inheritance in a cross of Annigeri and Rabat genotypes is also

reported by Singh and Rupela (1998). In another study Devis (1988) got

similar results from crosses of PM405 and PM 769 (non nodulated mutants

with ineffective nodulation) with P502 (produce effective nodules), he also

recorded monogenic inheritance of nodulation in chickpea, fit to monohybrid

expected ratio of 3 (effective nodules): 1(ineffective nodules). In a cross of

ICC435M (non-nodulated mutant) with its normal nodulated parent Singh et

al. (1992) found the same single gene inheritance pattern of nodulation in

chickpea. In addition to chickpeas, it was found that the nodulation in

soybeans is also govern by Mendelian segregation with a major phenotypic

effect in a cross of Kent and Peking with using the fast-growing rhizobial

strain USDA 205 (Devin, 1984). The F1BC1 data and the highest probability

level (68-70%) of chi square values also provided strong evidence to support

the monogenic inheritance of nodulation in genotype NDC 5-S10.

Results of second cross (hybrid B) revealed normal nodulation of the

parents, F1 and backcross progenies, and the F2 segregation ( 15 Nod+: 1

Nod-) for nodulated and non-nodulated phenotypes showed that nodulation

in genotype NDC 4-20-4 of chickpea is controlled by two independent

dominant genes. In chickpea, this is the first report about duplicate gene

action controlling nodulation. Data of backcross progenies (distributed into 1

Nod+: 1Nod-) strengthened the result of F2 segregating population. Moreover

the probability level of chi square values of segregating data as well as of

81

backcross data was between 15–20% which strongly support the control of

nodulation by duplicate gene action in genotype NDC 4-20-4.

The digenic inheritance behaviour of nodulation in chickpea in the cross

ICC19181 x NDC 4-20-4 is also confirmed by the molecular characterization

of genotypes under study in experiment 4. Molecular investigations through

Simple Sequence Repeats (SSR) or microsatellite markers revealed that the

two genotypes NDC 5-S10 and NDC 4-20-4 fall into distinct groups in the

cluster analysis or phylogenetic tree constructed by UPGMA (unweighted

paired-group method) and by ME (minimum evolution) methods. Principal

coordinate analysis (PCoA) based on microsatellite allele frequencies also

showed these two genotypes in completely separate classes. Thus the

molecular investigations also confirmed the high level genetic diversity

among these two nodulated genotypes. Therefore it provides evidence of

different genetic control for nodulation in two chickpea genotypes i.e., NDC

5-S10 and NDC 4-20-4 in this study and probably other genotypes (may be

reported by some other investigators in future endeavors). However, in

chickpea the presence of two genes for certain other traits like resistance to

wilt and ascochyta blight and leaf type have been reported. Duplicate gene

action (Updhyaya et al., 1983) for the inheritance of resistance to Race 1 of

Fusarium oxysporum f.sp. ciceris and control of leaf type by two genes

(Danehloueipoueet et al., 2008) in chickpea further strengthens the results of

the present project.

In other crops like pigeon peas, Gallo-Meagher et al. (2001) proposed

a three gene model for nodulation and suggested that three genes control

nodulation at three independent loci, with nodulation being a product of two

genes and inhibited by a third gene when it is dominant and the others are

82

homozygous recessive. In Soybean two dominant alleles were observed to

control promiscuous nodulation (Gwata et al., 2005). Two duplicate

recessive genes were also reported by Nigam et al. (1982) that control non-

nodulation in groundnut. Dutta and Reddy (1988) also reported duplicate

genes action as well as single recessive gene controlled non-nodulation in

Peanut. They stated that duplicate genes action as well as single recessive

gene controlled non-nodulation in Peanuts.

In the present study leaf color of chickpea showed monogenic

inheritance in both cross combinations. Previous studies also reported single

gene inheritance for morphological markers including characteristics of leaf

(other than color) in chickpea. Davis (1991) reported monogenic inheritance

for simple leave, filform leave, ineffective nodulation and white color flower

traits in chickpea similarly Pundir and Reddy (1998) observed single gene

inheritance for chickpea’s small leaf size and open flower characteristics.

Results of this study revealed that both the qualitative characters

(nodulation and leaf color) deviated from the expected Mendelian

segregating ratio of 9:3:3:1. Monogenic markers are useful in estimating the

rate of out crossing in predominantly self pollinating crops. They also help in

identification of F1 hybrids in the breeding programs. In case of complete

dominance, detection of heterozygotes for morphological markers is not

possible (Arshad et al. 2005).

Rare linkage study has been reported among genes controlling

nodulation with other morphological markers in chickpea. The first report on

genetic linkage of a locus controlling nodulation in the higher plants was

reported by Thomas et al. (1983) who observed linkage between Rj1 locus

(controlling restricted nodulation) and the F locus (controlling fasciated stem)

83

in soybean (Glycine max L.). Gwata et al. (2005) studied the inheritance of

nodulation with the help of leaf color in soybean, they found that yellow leaf

color with non-promiscous nodulation (which forms no or nonfunctional

nodules) is dominant over green leaf color with promiscuous nodulation

(which form functional nodules). From plant breeding stand point, such

method of identifying nodulated genotypes is most rapid, less expensive and

effective. The present study is the first effort to report linkage between genes

for nodulation and leaf color in chickpea. In 1991, Davis reported linkage

between genes for simple leave (slv) and root nodulation (rn3) as well as

among genes controlling filform leave and open flower (fil and w2) traits of

chickpea. He also detected loose linkage between w2-fil and slv-rn3 linkage

groups. Pundir and Reddy (1998) found that two traits i.e. open flower and

small leaf size in chickpea have monogenic recessive inheritance and both

the traits have showed some linkage, while flower color and flower type

revealed no linkage. Linkage was also reported by other scientists between

characteristics of leaf and nodulation in legumes. Sarker (1999) reported

linkage of early flowering gene (Sn) with seed coat pattern gene (Scp) and

tendrilled leaf (Tnl) with colored stem (Gs) in linkage group 1 of lentil

genome.

It is concluded from the current study that nodulation in chickpea is

controlled by a single dominant gene or two dominant genes, depending on

the nature of germplasm. Leaf color in chickpea also has monogenic

inheritance. Moreover, the It is concluded from the current study that

nodulation in chickpea is controlled by a single dominant gene or two

duplicate dominant genes, depending on the nature of germplasm. It is

concluded from the current study that nodulation in chickpea is controlled by

84

a single dominant gene or two duplicate dominant genes, depending on the

nature of germplasm. Presence of linkage between genes for nodulation and

leaf color is of great importance, it could be used efficiently in plant breeding

programs for rapid- screening method of effective nodulation.

85

Experiment 4 Molecular Characterization of Chickpea Germplasm using microsatellite markers

4.4.1 Introduction

Over recent decades, molecular marker technology has developed into

a valuable tool for plant breeding. A number of techniques (e.g. RFLP,

RAPD, AFLP, SSR) can be used as DNA markers linked to traits of interest,

directing selection towards these markers instead of selecting for a

phenotype (Edwards and Mogg, 2001). The small genome size (740 Mb),

and high economic importance as a food crop legume make chickpea an

important species for genomics research. Molecular markers and linkage

maps are the prerequisites for undertaking molecular breeding activities.

However, the progress towards development of a reasonable number of

molecular markers has been very slow in cultivated varieties of chickpea.

One of the main reasons for this may be attributed to the low level of genetic

diversity present in the cultivated gene pools of this species, at least with the

detection tools that are currently available (Sharma et al., 1995, Rajesh et al,

2002). Although several genetic linkage maps using various markers and

genomic tools have become available, sequencing efforts and use of

available resources have been limited in chickpea genomic research. Among

various molecular markers currently available, SSR or microsatellite markers

are often chosen as the preferred markers for a variety of applications in

breeding because of their multi-allelic nature, co-dominant inheritance,

relative abundance and extensive genome coverage (Gupta and Varshney,

2000). As a result, several hundred SSR markers have been developed and

are available in chickpea (Hüttle et al., 1999; Winter et al., 1999). By

86

examining the vast collection of chickpeas covering a broad geographic

range, a sufficient amount of genetic variation has been reported (Serret et

al., 1997; Udupa et al., 1999; Imtiaz et al., 2008; Upadhyaya et al., 2008).

Together with availability of high density linkage map (Winter et al., 2000),

highly polymorphic marker system like SSR would be of great value in QTL

mapping and marker assisted selection for various important traits (Singh et

al., 2008).

Genetic variation not only provides crop varieties with the capacity to

adapt to various environments, and resistant pests and diseases, but is also

a necessary resource for improving yield and quality required for food. In

order to enhance the genetic diversity of cultivars, it is necessary to utilize

exotic and diverse germplasm in breeding programs. Genetically diverse

lines provide ample opportunity to create favorable gene combinations, and

the probability of producing a unique genotype increases in proportion to the

number of genes by which the parents differ.

The objective of the study was to explore the genetic diversity of all the

composite genotypes of chickpea and also to examine desi and kabuli

morphs in comparison with each other for their genetic variability. Due to the

utmost importance of nodulation in nitrogen fixation, restoring soil fertility and

increased crop production it is important to study the genetics of nodulation

at molecular level in chickpea also just like other crops (Soybean,

Medicago). The present study was planned with the following objectives:

1. To investigate the genetic diversity in the composite genotypes of

chickpea

87

2. To evaluate genetic variability among desi and kabuli morphs of

chickpea

3. To check the grouping pattern of nodulated and non-nodulated

genotypes of chickpea.


A total of 47 genotypes of chickpea were used for this study (Table

4.4.1). They included 29 Desi and 18 Kabuli genotypes. Seeds of chickpea

were sown in 27 cm diameter pots containing 7.5 Kg of sandy soil with five

seeds per pot in a green house. DNA was extracted separately from fresh

leaves of two-five week-old plants for each genotype using modified CTAB

method described by Doyle and Doyle (1990). Ten SSR (microsatellite)

markers including five dinucleotide and five trinucleotide repeats were used

for genotyping. Primers were labeled with FAM, HEX, and NED fluorescent

dyes (Applied Biosystems, Foster City, USA). Details’ regarding reaction

mixture for amplification of markers, cycling parameters for markers

amplification, capillary electrophoresis and data analysis has been given in

chapter 3.

4.4.3 Results

4.4.3.1 Genetic variation

All the primer pairs generated reproducible and easily readable

microsatellite patterns. Eight of the ten primer pairs generated more than two

alleles (polymorphic), whilst two SSR primer pairs (H1B11 and H6G07)

amplified only one allele (monomorphic). These two monomorphic loci were

88

excluded from the analysis. A total of 58 alleles were detected in the 47

genotypes of chickpea (Table 4.4.3). The number of alleles per locus ranged

from 2 (CaSTMS19) to 16 (H3A10), with the average of 7.4 per locus. The

effective number of alleles (Ne) ranged from 1.29 (CaSTMS25) to 8.06

(H3A10) with an average of 3.66. Between Desi and Kabuli morphs, 46

alleles were found in Desi, and 42 alleles were found in the Kabuli, whilst 30

alleles were shared by both. A total of 28 private alleles (16 in Desi and 12 in

Kabuli) were detected. The average numbers of allele per locus in Desi and

Kabuli morphs were 5.87 and 5.25, respectively, while their mean effective

numbers were 2.78 and 3.40, respectively (Table 4.4.3). Across all the

polymorphic loci, 6 alleles (0.3%) of the 58 alleles were classified as rare

(present at a frequency of <1%), whereas 36 alleles (70.1%) were common

alleles (1–20%) and 16 alleles (27.6%) were frequent alleles (> 20%).

The PIC values ranged from 0.227 (CaSTMS25) to 0.876 (H3A10), with

an average of 0.636 (Table 4.4.3). Both CaSTMS markers showed relatively

low PIC values. The average PIC was 0.582 in Desi and 0.577 in Kabuli, and

these values were very similar. AMOVA was used to partition the genetic

variation between morphs, between individual genotypes within each morph

and within individuals (Table 4.4.4). Significant differentiation (RST = 0.239, P

≤ 0.001) was obtained between the two morphs.

Segregating loci were detected in 14 of our composite chickpea

genotypes, ranging from 1 to 5 loci per genotype with an average

heterozygosity from 0.125 to 0.625 (Table 4.4.1). This resulted in an overall

mean heterozygosity of 0.088.

89

4.4.3.2 Genetic relationship among genotypes

The UPGMA tree based on the genetic distance (Fig. 1a) shows

that all the genotypes were divided into 6 monophyletic groups (A, B, C, D, E

and F) and 2 outliers (genotype ICC19183 and ICC19181). Group A consists

of 3 Desi (genotypes NDC-122, Karak 1 and Sheenghar) and 1 Kabuli

(Hassan 2K) genotype. Genotypes Karak 3 and Sheenghar were the local

collections of Karak, Pakistan. Groups B (7 genotypes) and C (10 genotypes)

both consist of only Desi lines. Among Group B genotypes, NDC-727, NDC

4-15-1, NDC 4-15-3, NDC 4-20-3 and NDC 4-20-4 were derived from the

same induced mutation line (C-44), while NDC 15-3 and NDC 15-4 were

from another induced mutation line (Pb-91). Nine genotypes (NDC 728-5,

NDC 730-2, NDC 15-1, NDC 15-2, NDC 4-15-2, NDC 4-20-1, NDC 4-20-2,

NDC 4-20-5, NDC 4-20-6, and 2NIFA 2005) in Group C had the same

parentage of C-44, with genotype NIFA 2005 located in the basal position.

Group D includes 12 genotypes containing six Desi (NDC 4-20-7, NDC 5-

S10 to NIFA 95) and eight Kabuli (NKC 10-99 to NKC 5-S13, NKC 5-S15 to

NKC 5-S19) genotypes. Lines NKC 5-S15 and NKC 5-S16 had the same

parental origin. Group E is composed of seven Kabuli lines (genotypes NKC

5-S14, NKC 5-S20 to NKC 5-S24 and ICC4993) with genotype ICC4993

(local collection from Morocco) in the basal position. All the genotypes

included in the Group F are the local collections from Karak, Pakistan, and

this group consists of two Desi (Karak 1 and Karak 2) and one Kabuli

(Lawaghar) genotype. The genotypes ICC19183 and ICC19181 are basal in

the tree. The UPGMA tree locates the root at the middle point of the branch

connecting genotype 47; however, the branching associations among the

groups are ambiguous because they are connected with very short

90

branches. Bootstrap values for all these branches are very low (less than

50%).

An ME tree was constructed and is shown in Fig. 1b. In this tree, six

monophyletic groups (A', B', C', D', E' and F') were recognized. Genotype

NDC-727 was a sister to the cluster of Groups A' and B'. These groupings

are mostly the same as the grouping of the UPGMA tree (Group A in

UPGMA corresponds to A' in ME, and so on) with some minor differences.

Genotype ICC4993 in Group E of the UPGMA tree was displaced to Group

A' in the ME tree. Genotype ICC19181 was included in Group B' in the ME

tree. The composition of Group C' in the ME tree is the same as for Group C

in the UPGMA tree. Genotype NKC 5-S16, which was in Group D of the

UPGMA tree, was included in Group F' in the ME tree. Genotype NKC 5-

S12, which was in Group E' of the ME tree was in Group D in the UPGMA.

Branching associations among the groups in the ME tree were very different

from that of the UPGMA tree, reflecting ambiguous associations among the

groups. Non-nodulating lines (genotypes ICC4993, ICC19183, ICC4918 and

ICC19181) were not closely related in either of the trees.

The genetic relationships among 47 chickpea genotypes were also

determined using PCoA of GD estimates and are presented in figure 3. The

first (PC 1) and the second (PC 2) principal coordinate axes accounted for

28.12% and 42.70% of the total variation, respectively. So the first two

components of the PCoA between chickpea genotypes represented 70.82 %(

71%) of the variance, therefore PCoA was useful for graphical

representation, as it adequately summarize the microsatellite data. The two

axes separated the chickpea genotypes into 6 groups, with three outliers,

line number 31, 46 and 49(fig 3). Grouping of PCoA was same to that of

91

cluster analysis. Group A'' include three Desi and one Kabuli genotypes. The

genotypes of group B in cluster analysis were distributed into two

groups in PCoA that is group B'' and B'''. Group C'' of PCoA is

comparable to the group C of cluster analysis (UPGMA tree) containing ten

desi genotypes. There were 13 genotypes in group D'', this group was also

identical to group D of cluster analysis. Group E'' consisted of eight

genotypes, identical to cluster analysis only genotypes NKC 5-S15 (30)

which was in group A in cluster analysis fell into group E'' in PCoA, may be

due to some statistical error. Group F'' of PCoA was indistinguishable from

group F of cluster analysis. The non-nodulated lines 46, 49 and the

nodulated genotype 31 remains as an out liar in PCoA, this is also

comparable with UPGMA which showed genotype 49 as an out liars.

4.4.3.3 Grouping of chickpea genotypes based on morphological traits Means of the 4 morphological traits in Desi and Kabuli genotypes are

shown in Table 4.4.5 with their standard errors. ANOVA showed that there

were significant difference in the number of leaflets and nodules per plant.

Both were larger in Desi genotypes. Correlations among the 4 morphological

traits (number of nodules per plant, seed weight, number of leaflets per leaf,

and leaf area) are summarized in Table 6. Nodulation was significantly

positively correlated with the leaf area and the leaflet number (P ≤ 0.01 for

both). The correlation between leaf area and leaflet number was also highly

significant (P ≤ 0.01). The seed weight did not show any significant

correlation with any of the other traits. PCA was performed based on the

covariance matrix. PCA based on the 4 morphological traits allocated 72.1%

92

and 16.4% of the variance to Principal component 1 (PC1) and PC2,

respectively, accounting for 88.5% of the total variance (Table 7). Nodule

number showed the largest eigenvector for PC1. Similarly, leaf area and

leaflet number were the first and second largest eigenvectors for PC2,

respectively. Using PC1 and PC2 as X and Y axes, respectively, chickpea

genotypes were clustered graphically as shown in Fig. 2. This clearly

differentiates the Nod– genotypes as an isolated group from the Nod+

genotypes. Among the Nod+ genotypes, Desi and Kabuli genotypes formed

overlapping groups, but their center was clearly distinguished.

93

Fig.4.4.1

Figure 4.4.1. (a) UPGMA tree showing phylogenetic relationship between 47 chickpea

genotypes. (b) ME tree showing phylogenetic relationship between 47 chickpea genotypes.

In both trees branch length is proportional to the genetic distance.

94

Fig. 4.4.2

Figure 4.4.2. Scatter plot of principal component analysis based on

morphological data. PC1: principal component 1; PC2: principal component

2. Dotted line and dashed line circles show the 90% density ellipse of Nod+-

Desi and Nod+-Kabuli genotypes, respectively. The genotypes with asterisk

show Nod- genotypes with 90% density ellipse of solid line.

95

Figure 4.4.3

Principal Coordinates

as1

as2

as3as4

as5

as6

as7

as8as9

as10

as11

as12

as13

as14

as15

as16

as17

as18

as19

as20

as21as22

as23

as26

as27

as28

as29

as30

as31

as32

as33as34

as35as36

as37

as38

as39

as40

as41as42

as43

as44

as45 as46

as47

as48

as49

PC 1

PC

2

A''

B''

B'��''

C'' D''

E''F''

Figure 4.4.3: Scatter plot of principal coordinate analysis based on

microsatellite allele frequencies. PC1: principal coordinate 1; PC2: principal

coordinate 2. Circles show the clusters shown by alphabets. A=3 Desi & 1

Kabuli, B= 4 Desi, B’= 3 Desi, C=10 Desi, D= 6 Desi & 7 Kabuli, E= 8 Desi,

and F=2 Desi & 1 kabuli

96

Table 4.4.1 Description of chickpea genotypes used in this study

No. Entry Phenotype Genotype ParentageA Origin HB

1 NDC-122 Desi Nod+ C-44 × ILC-195 NIFA 02 NDC-727 Desi Nod+ C-44/M NIFA 0.253 NDC-728-5 Desi Nod+ C-44/M NIFA 0.3754 NDC-730-2 Desi Nod+ C-44/M NIFA 0.1255 NDC-15-1 Desi Nod+ Pb-91/M NIFA 06 NDC-15-2 Desi Nod+ Pb-91/M NIFA 07 NDC-15-3 Desi Nod+ Pb-91/M NIFA 08 NDC-15-4 Desi Nod+ Pb-91/M NIFA 09 NDC-4-15-1 Desi Nod+ C-44/M NIFA 010 NDC-4-15-2 Desi Nod+ C-44/M NIFA 011 NDC-4-15-3 Desi Nod+ C-44/M NIFA 012 NDC-4-20-1 Desi Nod+ C-44/M NIFA 013 NDC-4-20-2 Desi Nod+ C-44/M NIFA 0.37514 NDC-4-20-3 Desi Nod+ C-44/M NIFA 015 NDC-4-20-4 Desi Nod+ C-44/M NIFA 0.516 NDC-4-20-5 Desi Nod+ C-44/M NIFA 0.12517 NDC-4-20-6 Desi Nod+ C-44/M NIFA 018 NDC-4-20-7 Desi Nod+ C-44/M NIFA 0.12519 NDC-5-S10 Desi Nod+ JG74 × ICC12071 ICARDA 020 NDC-5-S11 Desi Nod+ JG74 × ICC12071 ICARDA 0.37521 NIFA-88 Desi Nod+ 6153 NIFA 022 NIFA-95 Desi Nod+ 6153/M NIFA 0.12523 NIFA-2005 Desi Nod+ Pb-91/M NIFA 0

24 NKC-10-99 Kabuli Nod+ FlIP98-138C × SEL99TH15039

ICARDA 0

25 NKC-5-S12 Kabuli Nod+ BAHODIR × SEL99TER85530 ICARDA 0

26 NKC-5-S13 Kabuli Nod+ SEL99TH15039 × S98008 ICARDA 0.2527 NKC-5-S14 Kabuli Nod+ SEL99TH15039 × S98008 ICARDA 0.12528 NKC-5-S15 Kabuli Nod+ FLIP98-15C × S98033 ICARDA 0.375


ICARDA 0.625


ICARDA 0


ICARDA 0.375


ICARDA 0

33 NKC-5-S20 Kabuli Nod+ FLIP98-138C × SEL99TH15039 ICARDA 0

34 NKC-5-S21 Kabuli Nod+ GLK95069 × SEL99TER85530

ICARDA 0


ICARDA 0


ICARDA 0


ICARDA 0

38 HASSAN-2K Kabuli Nod+ ILC-195/M NIFA 039 Karak 1 Desi Nod+ Local selection Karak 040 Karak 2 Desi Nod+ Local selection Karak 041 Karak 3 Desi Nod+ Local selection Karak 042 Sheenghar Desi Nod+ Local selection Karak 043 Lawaaghar Kabuli Nod+ Local selection Karak 0 44 ICC 4993 Kabuli Nod– Rabat Morocco 045 ICC 19183 Kabuli Nod– ICC 4993NN ICRISAT 046 ICC4918 NN Desi Nod– Annigeri India 047 ICC19181NN Desi Nod– ICC 435NN ICRISAT 0

AThe lines indicated by /M is mutation induction lines. BHeterozygosity.

97

Table 4.4.2 Primers used for amplifying chickpea microsatellite regions

Locus Primer Sequence (5'-3') Annealing temp.

Motif Repeat

H1116 F GACATGAAATTCGGTGCATT 52°C GA 20

R AACGCCCTAAACCTCTTGGT

H1F17 F GGGGAGGAAGAAGATGGAA 48°C TA 27

R GCGTTATGGGTGGAAATGGTA

H3C06 F AATTTCGTGAATCATTAAAAATAGAGG 55°C TAA 23

R CACATGACTATCTAGACATTTTATTTATC

H3A10 F TTTAAGGCTTCAGGTATTGATTTCT 55°C TTA 24

R TCACACATGCCAACTTAAAATAAAA

H3A07 F GCGACACCTATTCCTCTTTTCTA 58°C TTA 20

R TCATTTTTGGAATATTTTAGTGACAA

H2J09 F AACGAAAAACAAGGGAGAAAAA 52°C GA 18

R TATTTCTTTGACTCCCCCTAACTT

H1B11 F GCAGCTGTTGACATCTAATTTTG 60°C TAA 20

R ACCGAAAACACTTGTGATTGTTA

H6G07 F TCTATCAGAGATATTAAGTTGAACG 60°C TAA 23

R CGTGACAGAATTAGCCTCTTGT

CaSTMS19 F TGAAGCTGGGGGTTCCTTG 50°C AT 15

R TCAATTGAGTCGCGACGAGAG

CaSTMS25 F TACACTACTGCTATTGATATGTGGT 50°C CT 19

R GACAATGCCTTTTTCCTT

98

Table 4.4.3 Genetic diversity statistics of chickpea genotypes

Locus

Paramet

ersA H1116 H1F17 H3C06 H3A10 H3A07 H2J09 CaSTMS19 CaSTMS25 Mean

All Genotypes

N 47 47 47 47 47 47 47 47 47

Na 5 7 12 16 6 7 2 3 7.37

Ne 3.261 3.648 5.381 8.062 3.344 2.543 1.641 1.294 3.66

PIC 0.693 0.726 0.814 0.876 0.701 0.607 0.39 0.227 0.636

Kabuli vs Desi

Desi N 29 29 29 29 29 29 29 29 29

Na 4 7 10 10 5 6 2 2 5.87

Ne 2.346 3.228 3.526 5.021 2.339 2.507 1.859 1.312 2.78

PIC 0.574 0.69 0.716 0.801 0.573 0.601 0.462 0.238 0.592

Kabu

li N

18 18 18 18 18 18 18 18 18

Na 3 5 8 12 5 4 2 3 5.25

Ne 2.418 2.582 2.838 7.714 3.682 2.445 1.246 1.256 3.40

PIC 0.586 0.613 0.829 0.87 0.728 0.591 0.198 0.204 0.577

AN, no. genotypes examined; Na, actual number of alleles; Ne, effective

number of alleles; PIC, polymorphic information content.

99

Table 4.4.4 AMOVA analysis of microsatellite data of chickpea genotypes

SourceA d.f. SSB MSC Est. Var.D %

Among morphs 1 7087.7 7087.7 140.7*** 24%

Among genotypes 45 37647.6 836.6 389.3*** 66%

Within genotypes 47 2725.5 58.0 58.0*** 10%

Total 93 47460.9 588.0 100%

*** Significant at P ≤ 0.001.

AMorphs are Desi and Kubuli. BSum of square. CExpected mean square. DEstimated variance.

Table 4.4.5 ANOVA for the 4 morphological traits of chickpea genotypesA

*** P ≤ 0.001

ANon-nodulated lines are excluded from the analysis of nodulation. BThe values are mean ± standard error. CF-ratio testing the difference of Desi and Kabuli genotypes. The numbers in parenthesis are the degree of freedom.

MeanB

Leaf area (cm2) Leaflet/plant Seed weight (g)

Nodule/plant

Desi 7.14 ± 0.159 14.3 ± 0.098 0.719 ± 0.0165

8.89 ± 0.414

Kabuli 7.06 ± 0.202 12.9 ± 0.125 0.747 ± 0.0209 6.04 ± 0.538

F-ratioC 0.0878 (1, 45) 74.14 (1, 45)*** 1.116 (1, 45) 17.60 (1, 41)***

100

Table 4.4.6 Correlation among four morphological traits of chickpea

genotypes

Leaflet Seed weight Nodulation

Leaf area 0.3971** 0.0532 0.4365**

Leaflet 0.0024 0.4307**

Seed weight –0.0699

** P ≤ 0.01

Table 4.4.7 Principal component analysis on four morphological traits of

chickpea genotypes

PC1 PC2 PC3 PC4

Eigenvalue 11.5216 2.6242 1.7774 0.0614

Percent 72.0793 16.4170 11.1196 0.3841

Cum. Percent 72.0793 88.4963 99.6159 100.0000

Eigenvector

Leaf 0.29749 0.68135 –0.66861 0.01471

Leaflet 0.28387 0.60556 0.74345 –0.00211

Seed weight –0.00377 0.01524 –0.00814 0.99984

Nodulation 0.91154 –0.41088 –0.01335 0.00959

101

4.4.4 Discussion

A collection of chickpea experimental lines for breeding programs

was evaluated for their genetic variation, especially between Desi and Kabuli

genotypes. All the SSR markers examined were selected randomly from

previously published data. In this study, 8 out of the 10 selected SSR

markers showed polymorphism in our 47 chickpea genotypes. The allele

number per locus ranged from 2 to 16, averaging 7.32, and with PIC values

of 0.227–0.876, averaging 0.636. These values are lower than those

observed by Udupa et al. (1999), who reported an average number of alleles

of 14.1 per locus and an average PIC of 0.86 for 12 SSR loci in 78 genotypes

of chickpea including 72 landraces, 4 cultivars and 2 wild species of the

primary gene pool (i.e. C. reticulatum and C. echinospermum). Similarly,

Imtiaz et al. (2008), who evaluated 48 genotypes of chickpea comprising

cultigens, landraces, and wild relatives using 21 SSR loci, detected an

average of 16.9 alleles per locus and an average PIC value of 0.82. In a

more comprehensive study, Upadhyaya et al. (2008) examined 2915

genotypes from a vast collection of chickpea germplasm maintained in two

gene banks in ICRISAT and ICARDA using 48 SSR markers. They reported

that the number alleles per locus ranged from 14 to 67, with an average of

35, and PIC values from 0.467 to 0.974, averaging 0.854. In comparable

soybean studies, Narvel et al. (2001) reported an average of 10.2 alleles per

locus among 79 genotypes including 39 elite genotypes (Elites) and 40 plant

introductions (PIs) using 74 SSR loci. Average marker diversity among the

PIs was 0.56 and that of the Elites was 0.50. Likewise Wang et al. (2006)

calculated an average of 12.2 alleles per locus for 129 Chinese soybean

genotypes using 60 SSR loci and the PIC among genotypes varied from 0.5

102

to 0.92 with a mean of 0.78. Although allele number is very much dependent

on sample size, the possible explanation for the lower observed PIC value in

our study could be that most of the genotypes were advanced-generation

laboratory stocks derived from a limited number of parental lines or hybrids,

whereas many of the studies cited above used a larger number of genotypes

from geographically diverse areas including landraces and wild relatives.

However, the materials used here still reveal a considerable amount of

genetic variation. PIC values for the induced mutation lines derived from C-

44 was 0.443 and from Pb-91 was 0.422. These contain about 50% of the

variation that resides in the world collections of chickpea (Upadhyaya et al.,

2008), and the SSR markers could effectively discriminate the lines derived

from a same parental line. Thus the microsatellite technique proved to be a

useful system for managing our experimental lines. Our result also showed

the average heterozygosity of 0.088, which is rather high compared with

reported range between 0 and 1% in chickpea natural cross-pollination

(Singh, 1987). In this study, extensive care was taken to avoid inadvertent

seed mixture and a single plant from each genotype was selected and used

for DNA extraction and analysis. Probably in some lines, especially in the

lines of hybrid origin, genetic materials are still segregating.

Desi and Kabuli genotypes showed clear morphological difference

especially in the leaflet number and nodule number (Table 5, and Fig. 2).

The average PIC values for Desi and Kabuli morphs were 0.582 0.061 and

0.577 0.090, respectively, which are not significantly different from each

other. Although significant differentiation in their allele frequency constitution

was shown by AMOVA (Table 4), Desi and Kabuli groups were not clearly

divided in UPGMA and ME phylogenetic trees (Figs. 1a and 1b). Some

103

genotypes formed monophyletic groups in phylogenetic tree such as in

Group B and Group C for Desi genotypes and Group E for Kabuli genotypes

in UPGMA tree, but others are paraphyletic (Fig. 1a). Apparently in our

study, some genotypes were derived from hybrids between Desi and Kabuli

lines (such as genotypes 1, 19, and 20), so that they made a group of

mixture with both types. In the study of Upadhyaya et al. (2008) with 2915

genotypes including 1668 Desi and 1167 Kabuli groups, Desi and Kubuli

genotypes are largely separated but include some paraphyletic members.

This probably shows that these two groups have not been completely

isolated and occasional hybridization between the two morphs might have

occurred.

AMOVA also showed significant differentiation between Nod+ and Nod–

genotypes (RST = 0.270, P ≤ 0.01); however, Nod– genotypes are not

monophyletic in either the UPGMA, ME trees or PCoA (Figs. 1a, 1b and 3).

Probably mutations causing non-nodulation had occurred independently in

different genetic backgrounds. Rupela (1992) reported that the frequency of

Nod– plants in 4 Nod+ genotypes ranged from 120 to 490 per million. He also

reported that Nod– selections were indistinguishable from their respective

parents for plant growth except for nodulation, and yielded similarly to their

Nod+ genotypes when supplied with 50 to 100 Kg N/ha, but on a low-N field,

the Nod– plants were light green and grew poorly. Singh et al. (1992)

reported that a new Nod– mutation that occurred in 1 genotype (ICC435) was

inherited in Mendelian recessive manner, but was non-allelic to the formerly

reported mutants, and suggested that there are at least 6 loci controlling

nodulation. This verifies the high mutation rate observed for this character.

104

The high level of variability observed in microsatellite markers makes

them suitable for application in identification of germplasm of local varieties,

cultigens and cultivars (Udupa et al., 1999). Here, we have shown that all,

but 3 pairs (genotypes 5 and 10, genotypes 11 and 14, and genotypes 21

and 26, Figs. 1a and 1b), could be readily distinguished with these

microsatellite markers. Since the number of markers we used was very

limited, using a larger number of markers in even more closely related lines

could certainly improve resolution. Thus, this method is extremely useful for

breeding programs that utilize induced mutation lines and lines of hybrid

origin.

105

SUMMARY

Total of 49 genotypes of Chickpea (Cicer arietinum L) were procured

from various organizations for Genetic and Molecular Analysis of Nodulation.

Two accessions failed to germinate and evaluation was made on 47

genotypes. Out of 47 genotypes, 36 were from Institute for Food and

Agriculture (NIFA), Peshawar, five from Gram Research Station (GRS),

Ahmedwala Karak, and four from International Crops Research Institute for

Semi-Arid Tropics (ICRISAT), India.

Characterization of genotypes for quantitative and qualitative traits,

and inheritance and linkage study of nodulation were carried out at the KPK

Agricultural University Peshawar and NIFA, Peshawar. These genotypes

were also characterized at molecular level, in the Forest Resource Biology

laboratory Ehime University, Matsuyama, Japan.

All genotypes were characterized under field conditions using

Randomized Complete Block Design (RCBD) with three replications for

morphological markers (i.e. anthocyanin pigmentation on stem, flower and

seed coat color) and for quantitative traits (i.e., days to 50% flowering, days

to maturity, leaf area, number of leaflets leaf,-1 plant height, biological yield,

seed yield plant-1and 100 seed weight). The experiment was also conducted

in pots using Complete Randomized Design (CRD) with three replications

with two treatments (with and without rhizobium inoculation), to evaluate

genotypes for nodulation, number of nodules plant-1 and to study the effect of

rhizobium leguminosarum inoculation on nodules plant-1 and seed yield

plant-1.

106

Based on morphological markers, the germplasm was grouped as

desi (pink flower, green with purplish tings stem and colored seed coat) and

kabuli type genotypes (white flower, green stem and white seed coat). The

germplasm showed significant variation and high broad sense heritability for

all quantitative traits. Genotypes from NIFA, and GRS produced nodules

while accessions from ICRISAT did not produce nodules. Two genotypes

NDC 5-S10 and NDC 4-20-4 produced the maximum number of nodules

plant-1 and were selected for inheritance and linage study along with

genotype ICC 19181 (non-nodulated).

Inheritance of nodulation and its linkage with leaf color in chickpea was

thoroughly studied using F1, F2, backcross-I (BC11) and backcross-2 (BC12)

using two cross combinations i.e., ICC 19181(Nod- and dark green leaves) x

NDC 5-S10 (Nod+ and light green leaves) - Hybrid A and ICC 19181 x NDC

4-20-4 (Nod+ and light green leaves) - Hybrid B. The hybrid A showed

dominant monogenic inheritance for nodulation, while hybrid B showed

duplicate gene action nodulation. This clearly shows that the nodulated

genotypes of both crosses have different genetic background for nodule

formation; molecular analysis also placed these genotypes in separate

clusters. Both hybrid A and hybrid B revealed single gene dominant

inheritance for light green leaf color. Linkage was detected between the loci

for nodulation and leaf color in the F2 population of both the crosses.

Nodulation and light green leaf color were dominant over Nod- and dark

green leaf color in F1. In F2 population this segregation was diverted from the

normal ratio of 9:3:3:1, which is an indication of linkage between these two

loci. The recombination values (0.15 and 0.26) shows that the loci of

nodulation and leaf color are residing on the same chromosome in genotype

107

NDC 5-S10 and NDC 4-20-4. In genotype NDC 5-S10 genes of nodulation

and leaf color are at the distance of 15 cM, while In NDC 4-20-4 both genes

of nodulation and gene of leaf color looks like to be on one chromosome one

nodulation gene is at the distance of 15 cM and other at 26 cM from the gene

of leaf color.

Molecular characterization (with 10 SSR markers) revealed high level

of polymorphism in 47 accessions of chickpea. Rreproducible and easily

readable microsatellite patterns were recorded for all the primer pairs. Eight

primers were polymorphic (more than two alleles), whilst two primer pairs

(H1B11 and H6G07) were monomorphic (amplified only one allele). These

two monomorphic loci were not included in the analysis. The number of

alleles locus-1 ranged from 2 (CaSTMS19) to 16 (H3A10), with the average

of 7.4 locus-1. The effective number of alleles (Ne) ranged from 1.29

(CaSTMS25) to 8.06 (H3A10) with an average of 3.66 locus-1. A total of 46

alleles were found for desi, and 42 alleles for kabuli, whilst 30 alleles were

shared by both. The average number of alleles locus-1 were 5.87 in desi

types in which 2.78 were effective on average basis. In kabuli morphs, the

average was 5.25 alleles locus-1in which mean effective number of alleles

was 3.40. The Polymorphic Information Content (PIC) values ranged from

0.227 (CaSTMS25) to 0.876 (H3A10), with an average of 0.636. Among

chickpea accessions, 14 showed segregating loci ranging from 1 to 5 loci

genotype-1 with an average heterozygosity from 0.125 to 0.625. Thus the

overall mean heterozygosity was 0.088.

Genetic relationship among accessions was checked by Unweighted

Pair-Group Method using Arithmetic averages (UPGMA), the Minimum-

Evolution method (ME) and Principal Coordinate Analysis (PCoA). The

108

cluster analysis through these three methods revealed same grouping as

were determined through molecular characterization with negligible

deviations. Branching associations between groups in the UPGMA tree were

very different from that of the ME tree, reflecting ambiguous associations

among the groups. Non-nodulating lines ICC4993, ICC19183, ICC 4918 and

ICC19181 were not closely related in either of the trees. PCoA also revealed

almost same grouping of genotypes as UPGMA and ME analysis. Significant

variations between nodulated (Nod+) and non-nodulated (Nod–) accessions

were also reported in AMOVA (RST = 0.270, P ≤ 0.01); however, Nod–

accessions are not monophyletic in either the UPGMA or ME trees. Thus

non-nodulated genotype ICC 19181 and two nodulated genotypes NDC 5-

S10 and NDC 4-20-4 were selected for hybridization to study the inheritance

of nodulation in chickpea

109

Conclusions

The following conclusions have been drawn from the present investigations

1. Chickpea genotypes showed considerable variability for various

morphological markers, number of nodules plant-1 and quantitative

traits. Most of the traits revealed high heritability estimates.

2. Number of nodules and seed yield plant-1 increased extensively with

rhizobium inoculation.

3. Cluster analysis showed monophyletic as well as paraphylatic groups,

revealing that desi and kabuli genotypes are not separated

completely. Although AMOVA revealed significant differentiation in

their allele frequency constitution.

4. Nodulated and non-nodulated genotypes showed significant

differences; yet non-nodulated genotypes are not monophyletic in

either cluster analysis. Perhaps mutations causing non-nodulation

had occurred independently in different genetic backgrounds.

5. In chickpea nodulation is controlled by a single dominant gene

(monogenic) or two dominant genes (duplicate gene action),

depending on the nature of genotype.

6. Genes responsible for nodulation are located on the same

chromosome and are linked to the gene that controls leaf color in

chickpea.

110

RECOMMENDATIONS

1. Genetic diversity recorded among genotypes could be utilized

effectively for further investigations regarding genetic background of

various traits including nodulation parameters and in crop

improvement programs

2. Hybridization among the two groups (desi and kabuli) of chickpea

could be used successfully for development of the crop in terms of

high production, nitrogen fixation etc

3. Leaf color of chickpea can be used as an indicator marker for

presence/absence of nodules. It can further enhance use of other

morphological markers in construction of more linkage groups in

chickpea.

4. The genotypes used in inheritance study and the results obtained

could be useful in conducting further studies on nodulation in

chickpea.

111

BIBLIOGRAPHY

Achmad, S. 2005. Characterization of hyacinth bean (Lablab purpureus (L.) sweet) seeds from Indonesia and their protein isolate. Food chem. 95 (1): 65-70.

Afsari, S. Q., A. Shoukat, A. Bakhsh, M. Ardhad, and A. Ghafoor. 2004. An assessment of variability traits in chickpea (Cicer arietinum L.). Pak. J. Bot. 36 (4): 779-785.

Akkaya, M. S., A. A. Bhagwat, and P. B. Cregan. 1992. Length Polymorphisms of Simple Sequence Repeat DNA in Soybean. Genetics 132: 1131-1139

Al Khanjari, S., K. Hammer, A. Buerkert, and M. S. Röder. 2007. Molecular diversity of Omani wheat revealed by microsatellites: II. Hexaploid landraces. Gene. Res. Crop Evol. 54 (7): 1407-1417.

Ali, A., M. S. Zia, Rahmatullah, A. Shah, and M.Yasin. 1998. Nodulation in Sesbania bispinosa as affected by nitrogen application. Pak. J. Soil Sci. 15: 183-185.

Arora, P. P., and A. S. Jeena. 2001. Role of variability for improvement in chickpea. Leg. Res. 24 (2): 50-51.

Arshad, M., A. Bakhsh, M. Zubair, and A. Ghafoor. 2003. Genetic variability and correlation studies in chickpea (Cicer arietinum L.). Pak. J. Bot. 35 (4): 605-611

Arshad, M., A. Ghafoor, and S. Q. Afsari. 2005. Inheritance of qualitative trats and their linkage in blackgram (Vigna mungo (L.) Hepper). Pak. J. Bot. 37 (1): 41-45.

Arshad, M., A. S. Qureshi, A. Shaukat, A. Bakhsh, and A. Ghafoor. 2004. An assessment of variability for economically important traits in chickpea (Cicer arietinum l.). Pak. J. Bot. 36 (4): 779-785.

Ashrar, M. Z. 2008. Pulse production in Pakistan current situation. Available at htt://www.ndma.gov.pk.

Atta, B. M., M. A. Haq, and T. M. Shah. 2008. Variation and inter-relationships of quantitative traits in chickpea ((Cicer arietinum L.). Pak. J. Bot. 40 (2): 637-647.

Bakhsh, A., A. Ghafoor, and B. A. Malik.1992. Evaluation of lentil germplasm. Pak. J. Agic. Res. 12 (4): 245-251.

Bakhsh, A., T. Gull, A. Sharif, M. Arshad, and B. A. Malik. 1999. Genetic variability and character correlation in pure lines, F1 and F2 progenies of chickpea (Cicer arietinum L.). Pak. J. Bot. 31 (1): 41-53.

112

Bhapkar, D. G., and R. B. Deshmukh. 1982. Genetics of nodulation in chickpea (Cicer arietinum L.). Genetica 59: 89-92.

Bhatia, C. R., K. Nichterlein, and M. Maluszynski. 2004. Mutations affecting nodulation in grain legumes and their potential in sustainable cropping systems. Euphytica 120 (3): 415-432.

Bhuiyan, M. A. H., D. Khanam, M. F. Hossain, and M. S. Ahmed. 2008. Effect of rhizobium inoculation on nodulation and yield of chickpea in Calcareous soil. Bangladesh J. Agril. Res. 33 (4): 549-554

Bowcock, A. M., A. Ruiz-Linares, J. Tomfohrde, E. Minch, J. R. Kidd, and L. L. Cavalli Sforza. 1994. High resolution of human evolutionary trees with polymorphic microsatellites. Nature 368: 455-457.

Burli, A. V., S. M. More, B. N. Gare, and S. S. Dodake. 2004. Genetic variability and heritability in chickpea under residual soil moisture condition. J. Maharashtra Agric. Uni. 29 (3): 353-354.

Burton, G. W. 1952. Quantitative inheritance in grasses. Proc. 6th Int. Grassland Cong. 1: 277-283.

Cavalli Sforza, L. L. 1994. High resolution of human evolutionary trees with polymorphic microsatellites. Nature. 368: 455-457.

Chowdhry, K. A., K. S. Saraswat, S. N. Hassan, and R. C. Gaur. 1971. 4000-3500 year old barley, rice and pulses from Atranjikhera. Sci. and Cult. 37: 531-532.

Christine, K., M. Sawkins, W. A. Meyer, and B. S. Gaut. 2001. Genetic Diversity in Seven Perennial Ryegrass (Lolium perenne L.) Cultivars Based on SSR Markers. Crop Sci. 41:1565-1572

Christodoulou, V., V. A. Bampidis, B. Hucko, K. Ploumi, C. Iliadis, P. H. Robinson, and Mudrik Z. 2005. Nutritional value of chickpeas in relations of lactating ewes and growing lambs. Anim. Feed Sci. Technol. 118: 229-241.

Clayton, G., W. A. Rice, N. Z. Lupwayi, A. M. Johnston, G. P. Lafond, C. A. Grant, and F. Walley. 2003. Inoculant formulation and fertilizer nitrogen effects on field pea: Crop yield and seed quality. Can. J. Plant. Sci. 84: 89-96.

Cleyet-Marel, J. C., R. D. Bontto, and D. P. Beck. 1990. Chickpea and its root-nodule bacteria: implications of their relationships for legume inoculation and biological nitrogen fixation. Option Mediterranean’s series seminar: 101-106

Coburn, J. R., S. V. Temnykh, E. M. Paul, and S. R. McCouch .2002. Design and Application of Microsatellite Marker Panels for Semiautomated Genotyping of Rice (Oryza sativa L.) Crop Sci. 42: 2092-2099.

113

Corbin, E. J., J. Brockwell, and R.R. Gault. 1977. Nodulation studies on chickpea (Cicer arietinum L). Australian J. Exp. Agri. and Animal Husbandry 17(84): 126 - 134

Corby, R. 1981. Seeds of Leguminosae. Advances In legumes systemics. Part 2 (Polhill, R.M., and Raven, P.H., eds.). Kew, UK: Royal Botanic Gardens: 913-915

Cubero, J. I. 1987. Morphology of chickpea. p. 35-66. In: M.C. Saxena and K.B. Singh (eds.). The chickpea. CAB. International, Wallingford, Oxon, UK.

Cubero, J. I. 1975. The research on chickpea (Cicer arietinum) in Spain. Pages 117-122 in Proceedings of the International Workshop on Grain Legumes, 13-16 Jan 1975, ICRISAT, Hyderabad, India: International Crops Research Institute for the Semi-Arid Tropics.

Danehloueipour, N., H. J. Clarke, G. Yan, T. N. Khan, and K. H. M. Siddique. 2008. Leaf type is not associated with ascochyta blight disease in chickpea ( Cicer arietinum L.). Euphytica 162 (2): 281-289.

Dasgupta, T., M. O. Islam, P. Gayen, and K. K. Sarkak, 1987. Genetic divergence in chickpea(Cicer arietinum L.). Exp. Genet. 3: 15-21.

Davis, T. M. 1991. Linkage relationships of genes for leaf morphology, flower color, and root nodulation in chickpea. Euphytica 54 (1): 117-123.

Davis, T. M., K. W. Foster, and D. A. Phillips. 1985. Nodulation mutant in chickpea. Crop Sci. 25: 345-348.

Davis, T. M., K. W. Foster, and D. A. Phillips. 1986. Inheritance and expression of three genes controlling root nodule formation in chickpea. Crop Sci. 26: 719-723.

Derya, Z. Y., and A. Emin. 2006. Genetic variability, correlation and path analysis of yield, and yield components in chickpea (Cicer arietinum L.) Turk J. Agric. 30 (1): 183-188.

Devine, T. E.1984. Inheritance of soybean nodulation response with a fast-growing strain of Rhizobium. Heredity 75 (5): 359-361

Devis, T. M. 1988. Two genes that confer ineffective nodulation in chickpea (Cicer arietinum L.). J. heredity 79 (6): 476-478.

Doyle, J. J., and J. L. Doyle. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemistry Bulletin 19:11-15.

Doyle, J. J., and J. L. Doyle. 1990. Isolation of plant DNA from fresh tissue. Focus 12: 13-15.

114

Duke, J. A. 1981. Handbook of legumes of world economic importance. 2nd

Ed. Plenum Press New York: 52-57.

Durga, K. K., S. S. N. Muthy, Y. K. Rao, and M. V. Reddy. 2007. Genetic studies on yield and yield components of chickpea. Indian J. Agric. 27 (3):61-79.

Duschak, M. 1871. Zur Botanik des Talmud. I. Neuer, Pest: 105-106

Dutta, M., and L. J. Reddy. 1988. Further Studies on Genetics of Nonnodulation in Peanut. Crop Sci. 28:60-62

Edwards, K. J., and R. Mogg. 2001. Plant genotyping by analysis of single nucleotide polymorphisms. In: Henry RJ (ed) Plant Genotyping: The DNA Fingerprinting of Plants. CABI Publishing, Wallingford, UK.

Farshadfar, M., and E. Farshadfar. 2008. Genetic Variability and Path Analysis of Chickpea (Cicer arientinum L.) Landraces and Lines. J. Appl. Sci. 8 (21):3951-3956

FAO. 2004. Food and Agriculture Organization of the United Nations, (FAOSTAT). FAO Statistical Databases. Available at http://faostat.fao.org/ default.aspx.

Gallani, R., J. M. Dighe, R. A Sharma, and P. K Sharma. 2005. Relative performance of different chickpea (Cicer arietinum L.) genotypes grown on vertisols. J. Res. Crops. 6(2):211-213.

Gallo-Meagher, M., K. E. Dashiell, and D. W. Gorbet .2001. Parental Effects in the Inheritance of Nonnodulation in Peanut. J. Heredity 92 (1): 86-89

Gaur, P. M., and V. K. Gour. 2003. Broad-few-leaflets and outwardly curved wings: & nbsp; two new mutants of chickpea. Plant Breed. 122: 192-194.

Gaur, Y. D., and A. N. Sen. 1979. Cross inoculation group specificity in cicer-rhizobium symbiosis. New Phytologist 83:745-754

Geervani, P., 1991. Utilization of chickpea in India and scope for novel and alternative uses. p. 47-54. In: Uses of Tropical Grain Legumes: Proceedings of Consultants' Meeting, 27-30 March 1989. ICRISAT Center, Patancheru, Andhra Pradesh, India.

Ghafoor, A., A. S. Qureshi, and M. Zahoor. 1999. Crossing techniques in black gram (Vigna mungo ). Pak. J. Arid Agric. 2 (1):25-31.

Ghafoor, A., M. Arshad, and A. Bakhsh. 2004. Path coefficient analysis in chickpea (Cicer arietinum L.) under rainfed conditions. Pak. J. Bot. 36 (1):75-81.

Gorel, F.L., and V. A. Berdnikov. 1993. Linkage of loci encoding H1 histone and a cotyledon protein in lentil. Heredity 93 (1):342-346

115

Gupta, P. K., and R. K. Varshney. 2000. The development and use of microsatellite markers for genetic analysis and plant breeding with emphasis on bred wheat. Euphytica 113:163-185.

Gwata, E. T., D. S. Wofford, K. J. Boote, and P. L. Pfahle. 2005. Inheritance of promiscous nodulation in soybean. Crop Sci. 45:635-638

Hakim, K., S. Q. Ahmad, F. Ahmad, M. S. Khan, and N. Iqbal. 2006. Genetic variability and correlation among quantitative traits in gram. Sarhad J. Agric. 22 (1):55-59.

Hakoomat, A., M. A. Khan and S. A. Randhawa. 2004. Interactive effect of seed inoculation and phosphorus application on growth and yield of chickpea ( Cicer arietinum L.). Int. J. Agri. Biol., 6 (1):110-112.

Hamanda, H., and T. Kakunaga. 1982: Potential Z-DNA forming sequences are highly dispersed in the human genome. Nature 298:396-398.

Hanson, C. H., H. F. Robinson, and R. E. Comstock. 1956. Biometrical studies of yield in segregating populations of Korean lespedeza. Agron. J. 48 (6):268-272.

Hartl, D.L., and A.G. Clark. 1997. Principles of population genetics. 3rd edition. Sunderland (MA): Sinauer Associates.

Hawtin, G. C., and k. B. Singh (1981) Prospects and potential of winter sowing of chickpeas in Mediterranean region. In: Saxena MC, Singh KB (eds) Proceedings of Workshop on Ascochyta Blight and Winter Sowing of Chickpeas (International Center for Agricultural Research in the Dry Areas), Aleppo, Syria, May 1981. Martinus Nijhoff/Dr. W. Junk Publishers, The Hague, Netherland.

Helbaek, H. 1959. Domestication of food plants in the old world: Joint efforts by botanists and archeologists illuminate the obscure history of plant domestication. Science 130:365–372.

Hulse, J. H. 1991. Composition and utilization of grain legumes. In: Uses of tropical Legumes: Proceedings of a Consultants' Meeting, 27-30 March 1989, ICRISAT Center, India. Nature 11-27.

Hungría, M., and M. C. P. Neves, 1987. Partitioning of nitrogen from biological fixation and fertilizer in Phaseolus vulgaris. Physiol. Plant 69:55–63.

Hussain, A. 1983. Isolation and identification of effective root nodule bacteria for important grain legumes of Pakistan. Project report. Department of Soil Science, University of Agriculture, Faisalabad. P-73.

Hüttel, B., P. Winter, K. Weising, W. Choumane, F. Weigand, and G. Kahl. 1999. Sequence-tagged microsatellite site markers for chickpea (Cicer arietinum L.). Genome 42:210–217.

116

Hynes, R. K., K. A. Craig, D. Covert, R. S. Smith, and R. J. Rennie. 1995. Liquid rhizobial inoculants for lentil and field pea. J. Prod. Agric. 8:547-552.

ICRISAT. 1987. Agronomic practices to optimize yield of chickpea (Bengal gram) in peninsular India. Legumes program, ICRISAT Center, India. Patancheru, A.P. 502 324, India.

Imtiaz, M., M. Materne, K. Hobson, M. van Ginkel, and R. S. Malhotra . 2008. Molecular genetic diversity and linked resistance to ascochyta blight in Australian chickpea breeding materials and their wild relatives. Aust. J. Agri. Res. 59 (6):554–560.

Imtiaz, M., M. Materne, K. Hobson, M. van Ginkel, and R. S. Malhotra. 2008. Molecular genetic diversity and linked resistance to ascochyta blight in Australian chickpea breeding materials and their wild relatives. Aust. J Agric. Res. 59:554–560.

Imtiaz, A. K., S. Imtiaz, and B. A. Malik. 1990. Selection of diverse parents of chickpea (Cicer arietinum L.) by multivariate analysis and the degree of heterosis of their F1 hybrids. Euphytica 51(3):227-233

Iqbal, A., I. A. Khalil, N. Ateeq and M. S. Khan. 2006. Nutritional quality of important food legumes. Food Chem. 97:331-335.

Iqbal, J., M. Saleem, A. Khan and M. Anwar. 1994. Genetic variability and correlation studies in chickpea. J. of Animal & Pl. Sci. 4 (2):35-36.

James,T. M., H. D. Frank, and S. Terry. 1997. Statistics. 7th ed. pp. 429-43. Prentice-Hall International Inc. Co. New Jersey

Jeena, A. S., P. P. Arora, and O. P. Ojha. 2005. Variability and correlation studies for yield and its components in chickpea. Legume Res. 28 (2):146-148.

Jefing, Y., D. F. Herridge, M. B. Peoples and B. Rerkasem. 1992. Effects of N fertilization on N2 fixation and N balance of soybean grain after lowland rice. Plant and Soil 147:235-24

Johansen, C., B. Baldev, J. B. Brouwer, W. Erskine, W. A. Jermyn, L. Li-Juan, B. A. Malik, A. A. Miah, and S. N. Silim. 1994. Biotic and abiotic stresses constraining productivity of cool season food legumes in Asia, Africa and Oceania. p. 175-194. In: F.J. Muehlbauer and W.J. Kaiser (eds.). Expanding the production and use of cool season food legumes. Kluwer Academic Publishers. Dordrecht, The Netherlands.

Kaiser, W. J. 1992. Epidemiology of Ascochyta rabiei. In: K.B. Singh and M.C. Saxena (eds.). Disease resistance breeding in chickpea. ICARDA, Aleppo, Syria: 117-134

117

Kaish., and D. G. King. 2006. Simple sequence repeats as advantageous mutators in evolution. Trends Genet. 22 (5):253-9.

Kazan, K., F. J. Muehlbauer, N. F. Weeden, and Ladizinsky. 1993. Inheritance and linkage relationships of morphological and isozyme loci in chickpea (Cicer arietinum L). Theor. Appl. Genet. 86:417-126

Khattak, S., D. F. Khan, S. H. Shah, M. S. Madani and T. Khan. 2006. Role of Rhizobial inoculation in the production of chickpea crop. Soil & Environ. 25(2): 143-145.

Ladizinsky, G. 1975. A new Cicer from Turkey. Notes from the Royal Botanic Gardens, Edinburgh 34:201-202

Ladyzinsky, G., and A. Alder. 1976. The origin of chickpea (Cicer arietinum L). Euphytica 25:211-217.

Levinsen, G., and G. A. Gutman. 1987: Slipped-strand mispairing: a major mechanism for DNA evolution. Mol. Biol. Evol. 4:203-221.

Lichtenzveig, J., C. Scheuring, J. Dodge, S. Abbo, and H. B. Zhan. 2005. Construction of BAC and BIBAC libraries and their applications for generation of SSR markers for genome analysis of chickpea (Cicer arietinum L). Theor Appl Genet. 110:492–510.

Litt, M., and J. A. Luty. 1989: A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle action gene. Am. J. Hum. Genet. 44:397- 401

Malhotra, R. S., R. P. S. Pundir, and A. E. Slinkard. 1987. Genetic resources of chickpea. In: M.C. Saxena and K.B. Singh (ed.), The Chickpea. C.A.B. International Cambrian News Ltd, Aberystwyth, UK: 67-81.

Mauro, P., J. Kipe-Nolt, L. Frustiante, and L. Monti. 1993. Common bean (Phaseolus vulgaris L.) mutants defective in root nodule formation II genetic analysis. J. of exp. Bot. 44 (6):1007-1014.

Mayer, M., A. Gland, S. Ceccarelli, and H. H. Geiger. 1995. Comparison of doubled haploid lines and F2 bulks for the improvement of barley in the dry areas of North Syria. Plant Breed. 114:45-49.

McKenzie, B. A., M. Andrews, A. Z. Ayalsc, and J. R. Stokes. 1992. Leaf growth and canopy development in chickpea. Proc. Annual Conference. Agronomy Society of New Zealand 22:121-125.

Mensah, J. K., and R. T. Olukoya. 2007. Performance of mungbean (vigna mungo l. hepper) grown in mid-west Nigeria. American-eurasian J. Agric. & Env. Sci. 2 (6):696-701.

Métais, B. Hamon, R. Jalouzot & D. Peltier. 2004. Structure and level of genetic diversity in various bean types evidenced with microsatellite

118

markers isolated from a genomic enriched library. Theo. & Appl. Gen. 104 (8):1346-1352

Methews, A., B. J. Caroll, and P. M.Gresshoff. 1989. A new nonnodulation gene in Soybean. Heredity 80 (5):357-360.

MINFAL, 2007-2008. Agricultural statistics of Pakistan. Ministry of food, agriculture and livestock. Economic wing, Islamabad.

Moreno, M, T., and J. I. Cubero. 1978. Variation in Cicer arietinum L. Euphytica 27:465–485.

Moreno, M. T. 1985. Variabilidad intraespesifica in Cicer arietinum L. Unpublished thesis doctoral, University of Cordoba, Spain.

Muehlbauer, F. J., and K. B. Singh. 1987. Genetics of chickpea. In: M.C. Saxena and K.B. Singh (eds.), The Chickpea. CAB. International, Wallingford, Oxon, OX10 8DE, UK:99-125.

Muehlbauer, F. J., R.W. Short, and W. J. Kaiser. 1982. Description and culture of garbanzo beans. Coop. Ext. Publ. EB 1112, Washington State Univ., Pullman.

Muhamad, F., A. Malik, M. Ashraf, A. S. Qureshi, and A. Ghafoor. 2007. Assessment of genetic variability, correlation and path analysis for yield and its components in Soybean. Pak. J. Bot. 39 (2):405-413

Naghavi, M, R., and M. R. Jahansouz. 2005. Variation in the agronomic and morphological traits of Iranian chickpea genotypes. J. Integrative Plt. Bio. 47: 375-379.

Narvel, J. M., W. R. Fehr, W. C. Chu, D. Grant, and R. C. Shoemaker. 2000. Simple sequence repeats diversity among soybean plant introductions and elite genotypes. Crop Sci 40:1452–1458.

Nawab, N. B. 2002. Estimation of variability parameters and correlations for quantitative traits in Cicer arietinum L. Sarhad J. Agric. 27 (1):30-99.

Nguyen, T., T. Taylor, P.W.J, Redden, and R. J, Ford. 2008. Genetic diversity estimates in Cicer using AFLP analysis. Plt. Breed. 123:173-179.

Nigam, S. N., P. T. C. Nambiar, S. L. Dwivedi, R. W. Gibbons, and P. J. Dart. 1982. Genetics of nonnodulation in groundnut (Arachis hypogaea L.). Studies with single and mixed Rhizobium strains. Euphytica 31 (3):691-693.

Noor, F., M. Arshad, and A. Ghafoor. 2003. Path analysis and relationship among quantitative traits in chickpea (Cicer arietinum L). Pak. J. of Bio. Science. 6 (6):551-555.

119

Nutman, P. S. 1981. Hereditary factors affecting nodulation and nitrogen fixation. In A.H. Gibson and W. E. Newton (ed) Current perspective in nitrogen fixation. Aust. Acad. Sci., Canberra 194-204.

Oldroyd, G., and J. A. Downie. 2004. Calcium, kinases and nodulation signaling in legumes. Nat. Rev. Mol. Cell Biol. 5:566–576.

Otieno, P. E., J. W. Muthomi, G. N. Chemning’wa, and J. H. Nderitu. 2009. Rhizobia inoculation, Farm yard manure and nitrogen fertilizer on nodulation and yield of food grain legumes. J. Bio. Sci.: 9 (4):326-332.

Parmar, N., and K.R. Dadarwal. 1999. Stimulation of nitrogen fixation and induction of flavonoid-like compounds by rhizobacteria. J. Appl. Microb. 86:36-44.

Peakall, R., and R. E. Smouse. 2006. GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol Ecol Notes 6:288–295.

Peterson, M. A., and D. K. Barnes. 1981. Inheritance of Ineffective Nodulation and Non-nodulation Traits in Alfalfa. Crop Sci. 21:611-616

Powell, W., M. Morgante, C. Andre, M. Hanafey, J. Vogel, S. Tingey, and A. Rafalski. 1996. The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Mol. Breed. 2:225-238.

Pundir, R. P. S., and G. V. Reddy. 1998. Two new traits - open flower and small leaf in chickpea (Cicer arietinum L.). Euphytica 102 (3):357-361.

Rajesh, P. N., V. J. Sant, V. S. Gupta, F. J. Muehlbauer, and P. K. Ranjekar. 2002. Genetic relationships among annual and perennial wild species of Cicer using inter simple sequence repeat (ISSR) polymorphism. Euphytica 129:15–23.

Rathnaparkh, M. B., D. K. Santra, A. Tullu, and F. J. Muehlbauer. 1996. Inheritance of inter-simple sequence-repeat polymorphism’s and linkage with the fusarium wilt resistance in chickpea. Theor. Appl. Genet. 96:348-353.

Raza, S. M., and S. S. Mehdi. 1992. Genetic variations of yield and yield components of chickpea (Cicer arietinum L.) grown in two diverse environments. Pak. Agric. Sci. 2 (1):47-48.

Rent, R. S., and O. O. Kohire. 1991. Phosphorus response in chickpea (Cicer arietinum L) with rhizobium inoculation. Legume Res.14:78-82.

120

Richard, J. D., and L. Henery. 1967. In: Crop production (3rd Ed). Prentice Hall Inc. Eagle Wood Cliffs., N.J.:392.

Rodríguez-Navarro, D. N., C. Santamaríaa, F. Tempranoa, and E. O. Leidib. 1999. Interaction effects between Rhizobium strain and bean cultivar on nodulation, plant growth, biomass partitioning and xylem sap composition. Eur. J. Agron. 11 (2):131-143.

Romdhane, S. B., M. E. Aouani , M. Trabelsi , P. de Lajudie, and R. Mhamdi. 2008. Selection of High Nitrogen-Fixing Rhizobia Nodulating Chickpea (Cicer arietinum) for Semi-Arid Tunisia. J. Agron. & Crop Sci. 194 (6):413 – 420

Romdhane, S. B., F. Tajini, M. Trabelsi, M. E. Aouani, and R. Mhamdi. 2007. Competition for nodule formation between introduced strains of Mesorhizobium cicer and the native populations of rhizobia nodulating chickpea (Cicer arietinum L.) in Tunsia. World J. Microb. Biotech. 23:1195-1201.

Rupela, O. P. 1992. Natural Occurance and Salient Characters of Nonnodulating Chickpea Plants. Crop Sci. 32:349-352

Rupela, O. P., and P. J. Dart. 1979. Research on symbiotic nitrogen fixation by chickpea at ICRISAT. In: Proc. International Workshop on chickpea Improvement, Feb. 8 to March 2, Hyderabad, India 162-167.

Rupela, O.P., and M. R. Sudarshana. 1986. Identification of a non-nodulating spontaneous mutant chickpea. Int. chickpea Newsl. 15:13-14.

Sabaghpour, S. H., J. Kumar, and T. N. Rao. 2003. Inheritance of growth vigor and its association with other characters in chickpea. Plt. Breed. 122: 542-544.

Sabhyata. 2008. Development and utilization of DNA-based molecular markers in chickpea. Available at http://www.nipgr.res. In/ research1/Dr_S_Bhatia.htm

Sable, N. H., M. N. Narkhede, M. M. Wakode, and G. K. Lande.2003. Genetic parameters and selection indices in chickpea. Indian J. of Pulses Res. 16 (1):10-11.

Saiki, R. K., S. Scharf, F. A. Faloona, K. B. Mullis, G. T. Horn, H. A. Erlich, and N. Arnheim. 1985. Enzymatic amplification of f1-globin sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350-1354.

Salam, M. A., and R. K. Downey. 1978. Inheritance of the morphological and quality characteristics in the progeny of interspecific cross between Brassica napus and B. campestris. Bangladesh J. Sci. Ind. Res. 13:23-28.

121

Saleem, M., M. Arshad, and M. Ahsan. 2008. Genetic variability and interrelationship for grain yield and its various components in chickpea (Cicer arietinum l.). J. Agric. Res. 46 (2):109-116.

Sampson, D.R. 1978. A second gene for gairs in B. olerracea and its tentative locantion in linkage group 4. Can. J. Genet. Cytol. 20:101-109.

Sampson, R. D. 1966. Linkage of genetic male sterility with a seedling markers and its used in producing F1 hybrid seed of B. oleracea (Cabbage, Broccoli, Kali, etc.). Can . J. Plant. Sci. 46:703.

Saraladevi, M., S. Kanagarajan, and S. Ponnusamy. 2008. Efficiency of RAPD and ISSR markerssystem in accessing genetic variation of rice bean (Vigna umbellata) landraces. Elec.J. Biotech. 11(3):1-10.

Sarker, A., W. Erskine, B. Sharma, and M. C. Tyagi. 1999. Days to flowering an d morphological loci in Lentil (Lens culnaris medikus subsp. Culnaris). The Am. Genet. Soc. 90:270-275.

Savithri, R. 1976. Studies in Archaeobotany together with in bearing upon socio-economy and environment of India protohistoric cultures. Ph.D. Thesis, University of Lucknao, India.

Saxena, N. P., M. C. Saxena, C. Johansen, S. M Virmani, and H. Harris. 1996. Future research priorities for chickpea in WANA and SAT. In: Saxena, N.P., Saxena, M.C., Johansen, C., Virmani, S.M., Harris, H. (Eds.), Adaptation of Chickpea in the West Asia and North Africa Region. ICARDA, Aleppo, Syria: 257–263.

Schwenke, G.. D., M. B. Peoples, G.. L. Turner, and D. F. Herridge. 1998. Does nitrogen fixation of commercial, dryland chickea and faba beancrops in North-west New South Wales maintain or enhance soil nitrogen. Australian J. Exp. Agric. 38:61-70.

Serret, M. D., S. M. Udupa, F. Weigand. 1997. Assessment of genetic diversity of cultivated chickpea using microsatellite-derived RFLP markers: implications for origin. Plt. Breed. 116:573–578

Shah, G. S. 1999. Assessment of Genetic Variation, and heritability of yield components in Mungbean (Vigna radiata (L.) Wilczek),and Mode of Inheritance of Resistance to mymv in this crop. Thesis, submitted to the Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan.

Shah, S. H., D. F. Khan, and M. S. Madani. 1994. Effect of different Rhizobial strains on the performance of two chickpea cultivars under field conditions. Sarhad J. Agri. 10 (1):103-107.

Shahzad, K., M. Saleem, M. Javid, and A. R. Shehzad. (2002). Heritability estimates. for grain yield and quality characters in chickpea (Cicer arietinum). Int. J. Agric. and Bio. 4 (2):275-276.

122

Shamim, M., and N. Ali. 1987. Effect of seed inoculation with Rhizobium and NP fertilizer levels on the yield of gram. Pak. J. Agr. Res. 8 (4):383-386.

Sharma, L. C., S. Saxena, R. K. Jain, J. Prasad, and B. N. Reddy. 1983. Survey of nodulation in gram in Rajastan. Int. chickpea. Newsl. 9:24-25.

Sharma, M. K., P. P. Arora, and A. S. Jecna. 2004. Variability, heritability and genetic advance in chickpea. Agric. & bio. Res. 20 (2):160-181.

Sharma, P. C., P. Winter, T. Bünger, B. Hüttel, F. Weigand, K. Weising, and G. Kahl. 1995. Abundance and polymorphism of di-, tri- and tetra-nucleotide tandem repeats in chickpea (Cicer arietinum L.). Theor. Appl. Genet. 90:90–96.

Shaukat A., A. Baksh, M. Wahid, A. Rashid, and M. A. Zahid. 2003. Evaluation of chickpea germplasm for semi arid zones of Balochistan. 36 (2):113–116.

Singh, D. P., and B. B. singh. 1992. Inheritance of morphological characters in chickpea (Cicer arietinum L.). Indian. J. Genet. 52 (1):55-57.

Singh, K. B., 1997. Chickpea (Cicer arietinum L.). Field crop Res. 53:161-170.

Singh, K.B. 1987. Chickpea breeding. In: Saxena MC, Singh KB (eds) The Chickpea. CAB International, Willingford, UK.

Singh, O., and O.P. Rupela. 1998. A new gene that controls root nodulation in chickpea. Crop Sci. 38:360-362.

Singh, O., H. A. Van Rheenen, and O. P. rupela. 1992. Inheritance of a new nonnodulation gene in chickpea. Crop Sci. 32:41-43.

Singh, R. K., and B. D. Chaudhry. 1979. Biometrical methods in quantitative genetic analysis. 2nd Ed. Kalyani Pub. Ludhiana and New Delhi India: 303.

Singh, R., P. Sharma, R. K. Varshney, S. K. Sharma, and N. K. Singh. 2008. Chickpea Improvement role of wild species and genetic markers. Biotech. Genet. Eng. Rev. 25:267-314.

Slatkin, M. 1995. A measure of population subdivision based on microsatellite allele frequencies. Genetics 139:453-462.

Smith, S. E., K. B. Sing, and R. S. Malhotra.1991. Morphological and agronomic variation in North Africa and Arabian alfalfa. Crop Sci., 31:1150-1163.

Smithson, J. B., J. A. Thompson, and R. J. Summerfield. 1985. Chickpea (Cicer arietinum L.). In: R.J. Summerfield and E.H. Roberts (eds.). Grain legume crops. Collins, London, UK: 312-390.

123

Smouse, P. E., R. Peakall. 1999. Spatial autocorrelation analysis of individual multiallele and multilocus genetic structure. Heredity 82:561–573.

Sneath, P., and R. Sokal. 1973. Numerical Taxonomy. W. H. Freeman, San Francisco: 887.

Stallknecht, G., K. M. Gilberston, G. R. Carlson, J. L. Eckhoff, G. D. Kushnak, J. R. Sims, M. P. Wescott, and D. M. Wichman. 1995. Production of chickpeas in Montana. Montana Agr. Res. 12:46–50.

Stanton, M. A., J. M. Steward, H. E. Percival, and J. F. Wendel. 1994. Morphological diversity and relationships in the A-genome cotton,Gossypium arborum and Gossypium herbaceum. Crop Sci. 34:519-527

Steel, R. G. D., J. H. Torrie, and D. A. Dickey. 1980. Principles and Procedures of Statistics: A Biometrical Approach. McGraw-Hill, New York. Taylor, C.B. 1997. Comprehending cosuppression. Plant Cell. 9:1245–1249.

Stephen, K. B., E. A. Slinkard, and L. F. Walley. 2002. Evaluation of Rhizobial Inoculation Methods for Chickpea. Agron. J. 94:851-859.

Struss, D., and J. Plieske. 1998. The use of microsatellite markers for detection of genetic diversity in barley populations. Theo. & Appl. Gene. 19 (1-2):308-315

Suiter, K. A., J. F. Wendel, and J. Stephens. 1983. LIKAGE-1: a PASCAL compute program for the detection and analysis of genetic link age. Heredity 74:203-204

Tamura, K., J. Dudley, M. Nei, and S, Kumar. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24:1596–1599

Tar’an. , T. Warkentin, A. Tullu, and A. Vandenberg.2007. Genetic relationships among Chickpea (Cicer arietinum L.) genotypes based on the SSRs at the quantitative trait Loci for resistance to Ascochyta Blight. Eur. J. Plt. Path. 119 (1):39-51

Tautz, D., M. Trick, and G. Dover. 1986: Cryptic simplicity in DNA is a major source of genetic variation. Nature 322:652-653.

Tekeoglu, M., D. Santra, V. J. Sant, P. N. Rajesh, G. Kahl, and F. J. Muehlbauer. 2000. A linkage map of the chickpea (Cicer arietinum L.) genome based on recombinant inbred lines from a C arietinum x C. reiculantum cross; localization of resistance genes for fusarium wilt races 4 and 5. Theo. Appl. Genet. 101:1155-63.

124

Tellawi, A., N. Haddad, and B. Hattar. 2007. Effect of several Rhizobium strains on nodulation, nitrogen uptake and yield of chickpeas (Cicer arietinum L.). J. plt. Nut. & Soil Sci. 149 (3):314-322

Thies, J. E., P. W. Singleton, and B. B. Bohlool. 1991. Influence of the size of indigenous rhizobial population on establishment and symbiotic performance of introduced rhizobia on field grown legumes. Appl. Environ. Microbiol. 57:19-28

Thomas, E. D., G. Reid, G. Palmer, and R. I. Buzzell. 1983. Analysis of genetic linkage in the soybean. J. Heridity 74 (6):457-460.

Thomas, M., K. Devis, W. Foster, and D. A. Phillips. 1985. Nodulation mutants in chickpea. Crop sci. 25:345-348.

Thomas, M., K. Devis, W. Foster, and D.A.Phillips.1986.Inheritance and Expression of three genes controlling root nodule formation in chickpea. Crop Sci. 26:719-723.

Tufenkci, S., M. Erman, and F. Sonmez. 2006. Effect of nitrogen application and rhizobium inoculation on the yield and nutrient uptake in lucerne (Medicago sativa). New Zealand J. of Agri. Res. 49 (1):101-105

Udupa, S. M., and M. Baum. 2003. Genetic dissection of pathotypespecific resistance to ascochyta blight disease in chickpea (Cicer arietinum L.) using microsatellite markers. Theor. Appl. Genet. 106:1196–1202

Udupa, S. M., and M. Baum. 2001. High mutation rate and mutational bias at (TAA)n microsatellite loci of chickpea (Cicer arietinum L.). Mol. Genet. Genomics 265:1097–1103.

Udupa, S. M., L. D. Robertson, F. Weigand, M. Baum, and G. Kahl .1999. Allelic variation at (TAA)n microsatellite loci in a world collection of chickpea (Cicer arietinum L.) germplasm. Mol. Gen. Genet. Mar. 261 (2):354-63

Upadhyaya H. D., O. Rodomiro, B. Paula J, and S. Sube. 2002. Phenotypic diversity for morphological and agronomic characteristics in chickpea core collection. Euphytica 123: 333-342.

Upadhyaya, H. D., J. B. Smithson, M. P. Haware, and J. Kumar. 1983. Resistance to wilt in chickpea. II. Further evidence for two genes for resistance to race 1. Euphytica 32: 749-754.

Upadhyaya, H. D., P. M. Gaur, C. L. L. Gowda, S. Chandra, S. M. Udupa, B. J. Furman, and M. Baum. 2003. Completing genotyping of composite set of chickpea, available at www.icarda.org/generationcp/cp-1-chickpea.htm.

Upadhyaya, H. D., S. L. Dwivedi, M. Baum, R. K. Varshney, S. M. Udupa, C. L. L. Gowda, D. A. Hoisington, and S. Singh. 2008. Genetic structure,

125

diversity, and allelic richness in composite collection and reference set in chickpea (Cicer arietinum L.). BMC Plt. Biol. 8:106-117

Upadhyaya, H. D. 2003. Geographical patterns of variation for morphological and agronomic characteristics in the chickpea germplasm collection. Euphytica 132 (3):343-352

Van der Maesen, L. J. G. 1987. Origin, history and taxonomy of chickpea. In: M. C. Sexena and K. B. Singh (eds.). The chickpea. CAB. International Cambrian News Ltd, Aberystwyth, UK: 11-34.

Van der Measen, L. J. G. 1972. Cicer L. A monograph on the genus with special reference to the chickpea (Cicer arietinum L.), its ecology and cultivation. PhD diss. Agricultural University Wageningen, Wageningen, Netherlands.

Videira, L. B., G. N. Pastorine, and P. A. Balatto. 2001. Incompatibility may not be the rule in sinorhizobium fredii-soygean interaction. Soil Bio. & biochem. 33:837-840

Virmani, S. S., K. B. Singh, K. Singh, and R. S. Malhotra. 1983. Evaluation of mungbean germplasm. Indian j. Genet. 43:54-58.

Wang, L, Guan R, Liu Z, Chang R, Qiu L (2006) Genetic diversity of Chinese cultivated soybean revealed by SSR markers. Crop Sci 46:1032–1038.

Weber, J., and P. E. May. 1989. Abundant class of human DNA polymorphism, which can be typed using the polymerase chain reaction. Am. J. Genet. 44:388-396.

Weir, B. 1996. Genetic Data Analysis II. Sinauer Associates, Sunderland, MA.

Wikipedia. 2010. Rhizobia. Available at http://en.wikipedia. org/ wiki/Rhizobia

Williams, P. C., R. S. Bhatty, S. S. Deshpande, L. A. Hussein, and G. P. Savage. 1994. Improving nutritional quality of cool season food legumes. p. 113-129. In: F.J. Muehlbauer and W.J. Kaiser (eds.), Expanding the Production and Use of Cool Season Food Legumes. Kluwer Academic Publishers, Dordrecht, the Netherlands.

Wills, A. B., and P. Smith. 1972 Genetics of B. oleracea. Ann. Rep., Scottish Hortic. Res. Inst., 41-42.

Wills, A. B., and P. Smith. 1973. Genetics and cytology of B.oleracea. Ann. Rep., Scottish Hortic. Res. Inst., 43-44.

Wills, A. B., and P. Smith. 1974. Brassicas: genetics and cytology of B.oleracea. Ann. Rep., Scottish Hortic. Res. Inst., 46-47

Winter, P. 1999. Development and use of molecular markers for chickpea improvement. In: Udupa SM, Weigand F (eds) DNA markers and

126

breeding for resistance to ascochyta blight in chickpea. Proc Symposium on “Application of DNA finger printing for crop improvement: marker assisted selection of chickpea for sustainable agriculture in the dry area”, ICARDA, Aleppo, Syria: 153-174.

Winter, P., T. Pfaff, S. M. Udupa, B. Hüttel, P. C. Sharma, S. Sahi, R. A. Espinoza, F. Weigand, F. J. Muehlbauer, and G.Kahl. 1999. Characterization and mapping of sequence tagged microsatellite sites in the chickpea (Cicer arietinum L.) genome. Mol. Gen. Genet. 262:90–101.

Winter, P., A. M. Banko-Iseppon, B. Huttel, M. Ratnaparkh, A. Tullu, G. Sonnante, T. Pfaff, M. Tekeoglu, D.K. Santra, V.J. Sant, P.N. Rajesh, G. Kahl, and F.J. Muehlbauer. 2000. A linkage map of chickpea (Cicer arietinum L.) genome based on recombinant inbred lines from a C. arietinum x C reticulatum cross:localization of resistance gene for fusarium wilt races 4 and 5. Theor Appl Genet. 101:1155-1163.

Winter, P., T. Pfaff, S. M. Udupa, B. Huettel, P. C. Sharma, S. Sahi, R. Arreguin-Espinoza, F. Weigand, F.J. Muehlbauer, and G. Kahl. 1999. Characterization and mapping of sequence-tagged microsatellite sites in the chickpea (Cicer arietinum L.) genome. Mol. Gen. Genet. 262:90–101

Yadav, S. S., J. Kumar, N. C. Turner, J. Berger, R. Redden, D. McNeil, M. Materne, E. J. Knights, and P. N. Bahl. 2004. Breeding for improved productivity, multiple resistance and wide adaptation in chickpea (Cicer arietinum L.). Plt. Genetic Res. 2:81-187.

Yates, F. (1934). Contingency table involving small numbers and the χ2 test. Supplement to the Journal of the Royal Statistical Society, 1(2):217-235

Yiwu, C., and R. L. Nelson. 2005. Relationship between Origin and Genetic Diversity in Chinese Soybean Germplasm. Crop Sci 45:1645-1652.

Zenglu, L., and R. L. Nelson. 2002. RAPD marker diversity among Cultivated and Wild Soybean Genotypes from Four Chinese Provinces. Crop Sci. 42:1737-1744.

127

APPENDICES

ANALYSIS OF VARIANCE TABLES

Table A1: Days to 50% flowering

Source of Degree of Sum of Mean F.value Probability variance freedom square square

Replication 2 50.90 25.45 5.83 0.0041 Genotype 46 8585.74 186.64 42.53 0.0001** Error 92 401.75 4.36 Coefficient of Variation: 1. 9%

Table A2: Days to Maturity

Source of Degree of Sum of Mean F.value Probability

variance freedom square square

Replication 2 24.55 12.27 2.13 0.12 Genotype 46 1766.55 38.40 6.67 0.0001** Error 92 529.44 5.75

Coefficient of Variation: 1.4%

Table A3: Plant height



Replication 2 1.80 0.900 0.06 0.94 Genotype 46 11962.46 260.05 60.42 0.0001** Error 92 1337.53 14.53 Coefficient of Variation: 5.7 %

128

Table A4: Leaf lets leaf-1


Replication 2 1.1489362 0.5744681 0.68 0.5112 Genotype 46 396.49 8.61 10.14 0.0001 Error 92 78.1843972 0.8498304 Coefficient of Variation: 6.67%

Table A5: Leaf area


Replication 2 0.91 0.45 0.20 0.81 Genotype 46 418.48 9.09 4.07 0.0001 Error 92 205.51 2.23 Coefficient of Variation: 21%

Table A7: Seed yield plant-1


Replication 2 7.29 3.64 0.55 0.57 Genotype 46 3254.87 70.75 10.74 0.0001 Error 92 606.26 6.58 Coefficient of Variation: 24.2 %

129

Table A6: 100 Seed weight


Replication 2 3.50 1.75 0.29 0.74 Genotype 46 4049.16 88.02 120.17 0.0001 Error 92 546.44 5.93 Coefficient of Variation: 23%

Table A8: Biological yield plant-1


Replication 2 21.64 10.82 0.80 0.45 Genotype 46 16530.73 359.36 447.09 0.0001 Error 92 1242.56 13.50 Coefficient of Variation: 12 %

Table A9: Number of nodules plant-1


Genotype 42 573,20 13.64 4.38 0.0001 Error 86 268.12 3.11 Coefficient of Variation: 22.54%

130

Table A9: Rhizobium effect on number of nodules plant-1



A 1 648.37 648.37 295.45 0.0001

B 42 1202.94 28.64 13.05 0.0001

A*B 42 336.64 8.01 3.65 0.0001

Error 172 337.46 2.19

Coefficient of Variation: 15.73%

Table A10: Rhizobium effect on seed yield plant-1



A 1 1104.01 1104.01 92.81 0.0001

B 42 8466.90 201.59 16.95 0.0001

A*B 42 1206.32 28.72 2.41 0.0001

Error 172 2046.06 11.89

Coefficient of Variation: 26.5.%

genetic and molecular analyses of nodulation in chickpea...

Documents