Genetic diversity in indigenous food legumes of Pakistan through molecular markers – challenges and opportunities

Abdul GHAFOORPlant Genetic Resources InstituteNARC, Islamabad, PAKISTANghafoor59pk@yahoo.com

PGR collection/acquisition, evaluation, conservation, utilizationField evaluation

Monogenic traits, polygenic traits, G-E interactionGene evaluation

Biotic, abiotic. Climate change Biochemical/molecular

Seed proteins, 2-D electrophoresis, isozymesDNA markers; AFLP, ALP, AP-PCR, AS-PCR, CAPS, DAF, ISA, RAPD, RFLP, SAP, SCAR, SSCP, SSLP, SSLP, SSR, STS, STMS: emerging more

Dissemination and utilization

Understanding PGR diversity

Indigenous food legumes

VignaLathyrisCicerLensVicia

GlycinePhaseolusArachis

Challenges

Food securityThe emerging challenges: can we feed the hunger planet?

Molecular markers, how could assist?Enhancing productivity

VerticalHorizontal

Legumes (one of the largest family for human consumption)

Food, feed, fibre, shelter: how to exploit?

Crop Total Evaluated

Multiplied

Molecular markers

Cereals 11,757 60% 95% <20%

Legumes 10,089 60% 95% <20%

Oilseeds 4,236 40% 60% <10%

Vegetables 2,012 40% 60% Nil

Others 3,580 Nil 15% <5%

Germplasm status

Molecular markersMolecular markersUsed in five major areas;

Genetic diversity within and between crop speciesFingerprinting; varietal identificationPlant domestication in relation to genetic resources conservation and breedingGenome relationshipsTool in crop improvement

Marker assisted breeding/QTL Genetic transformation

Intra-specific diversity in seed proteins of Pisum sativum

MM

SD

S-7

0

MM

SD

S-7

0

88 P

038-

10-1

8 [A

ustr

alia

]

88 P

050-

6 -9

[Aus

tral

ia]

88 P

090-

5-21

[A

ustr

alia

]

Pk -

1041

3 [P

akis

tan]

88 P

090-

5-26

[A

ustr

a lia

]

Pk -

1056

2 [P

akis

tan]

Spr

ing

Pea

s [A

ustr

alia

]

DM

R-4

[In

dia]

DM

R- 7

[In

dia]

DM

R- 2

0 [I

ndia

]

Pk -

1047

7 [P

akis

tan]

88 P

007-

2 -1[

Aus

tral

ia]

88 P

001-

4 -9

[Aus

tral

ia]

Pk -

1060

4 [P

akis

tan]

Pk -

1064

2 [P

akis

tan]

Pk -

1064

6 [P

akis

tan]

88 P

090-

5-10

[A

ustr

a lia

]

88 P

090-

5-22

[A

ustr

alia

]

Pk -

1062

6 [P

akis

tan]

PS

75/8

7 [R

oman

ia]

PS

157/

87 [

Rom

ania

]

Pk -

1063

9 [P

akis

tan]

Ale

ppo

1 [S

yria

]

LS

-169

0 [A

ustr

alia

]

Gre

en F

east

66.0.kd

45.0 kd

45.0 kd

45.0 kd

45.0 kd

Lin

kage D

ista

nce

0

1

2

3

4

5

D

MR

-7

D

MR

-4

D

MR

-20

88P

001

PK

10638

M

AT

EO

R

S

L-3

2

AU

S1

690

GR

EE

N F

E

PK

10643

PK

10642

PK

10641

PK

10639

PK

10636

PK

10632

PK

10631

PK

10630

PK

10562

PK

10628

PK

10475

PK

10609

88P

090-4

88P

007

S

P-3

88P

090-2

88P

050

88P

038

PK

10645

PK

10646

PK

10644

88P

101

PK

10637

PK

10622

PK

10617

PK

10634

PK

10616

PK

10613

PK

10611

PK

10610

PK

10605

PK

10604

PK

10603

PK

10601

PK

10479

PK

10626

PK

10600

PK

10478

PK

10477

PK

10627

PK

10476

PK

10633

PK

10629

PK

10413

PK

10620

PK

10625

PK

10615

PK

10607

P

75/8

7

88P

090-3

88P

090-1

P157/8

1

PK

10566

PK

10621

PK

10619

PK

10618

PK

10567

PK

10506

PK

10303

Coefficient0.68

0.760.83

0.910.98

001Afg 048Pak 050Pak 049Pak 058Pak 059Pak 047Pak 034Pak 035Pak 037Pak 036Pak 038Pak 007Chi 002Alb 004Bra 056Pak 005Can 041Pak 023Leb 073Uni 009Den 085Var 053Pak 054Pak 055Pak 065Swe 010Den 078Var 051Pak 012Eth 074USA 014Ger 042Pak 052Pak 043Pak 083Var 084Var 026Net 028Net 029Net 060Par 061Par 016Gui 033Nor 006Can 022Ivo 031Net 030Net 027Net 024Mex 019Ira 082Var 066Syr 077Ven 080Var 081Var 003Arg 013Eth 070Tur 011Eth 015Gre 025Nep 018Ind 075USA 020Ira 086Var 044Pak 076USS 046Pak 062Per 091Jap 092Jap 094Jap 093Jap 017Ind 067Syr 068Tur 079Var 008Cze 045Pak 071Uni 021Ita 069Tur 064Swe 039Pak 040Pak 063Per 072Uni

Diversity: a case in Pisum sativumIndigenous germplasm

Map explaining diversity pattern of Pisum sativum L. based on qualitative traits, quantitative traits and total seed protein profiles. The symbols represents quantitative traits, qualitative traits and represents total seed protein profiles. One third shaded symbols indicates low diversity, half shaded medium diversity and full shaded symbols indicate high diversity for specific technique.

Syria

UK Turkey

Pakistan

Peru

France

Sweden

Germany

Czech Republic

Netherlands

3 4 B

66.0.kDa

24.0 kD

2 B

6 B7 B8 B

9 10 11 B

Major Bands Minor Bands

13 B14 B

16 B17 B19 B20 B

21 22 B

24 B

1064

4 IC

AR

DA

1129

1 P

eru

1064

5 IC

AR

DA

1068

1 In

dia

1839

1 P

akis

tan

1834

2 E

thio

pia

1063

7 IC

AR

DA

1129

4 P

eru

1830

8 E

thio

pia

1955

4 P

akis

tan

01B

05B

12B

15B

18B

23B

Linkage of a RAPD marker with er1 gene in Pisum sativum L.

Among 43 RADP primers, 21 were polymorphic and one was linked with er1.Falloner and 11760-3ER selected under heavy infestation with Erysiphe pisi through artificial inoculation under greenhouse. F1 plants (Fallon/11760-3) indicated dominance of the susceptible

allele, F2 plants segregated in 3:1 ratio (susceptible: resistant).

The RAPD maker OPB18 (5’-CCACAGCAGT-3’) was linked to the er1 gene with 83% probability, and was located at a distance of 11.2 cM from the er1 gene.

Nisar & Ghafoor, RJG, 47: 300-304 (2011)

DNA markers reported for MAS for powdery mildew in pea

Marker Distance (cM) from er

Marker type Reference er gene

Sc-OPO-181200 0.0 RAPD/SCAR Tiwari et al. 1998 er1OPL-6 2.0 RAPD Tiwari et al. 1998 er1OPD 10650 2.1 RAPD/SCAR Timmerman et al. 1994 er1Sc-OPD10650 3.4 SCAR Janila and Sharma 2004 erSc-OPE-161600 4.0 RAPD/SCAR Tiwari et al. 1998 er1P236 9.8 RFLP Dirlewanger et al. 1994 erPSMPSAD60 10.4 SSR Ek et al. 2005 er1PSMPSAA374e 11.6 SSR Ek et al. 2005 er1PSMPA5 14.9 SSR Ek et al. 2005 er1ScX171400 2.6 SCAR Katoch et al. 2010 er2SCAB1874 2.8 SCAR Fonddevilla et al. 2008 Er3BC210 8.2 RADP/SCAR Tonguc and Weeden (2010) er1

OPB18430 11.2 RAPD Nisar and Ghafoor 2011 er1

Ghafoor & McPhee. Euphytica, 186: 593-607 (2012)

Powdery mildew resistant (right) and susceptible (left) lines of Pisum sativum. Powdery mildew symptoms on leaves (lower left) and on pods (lower right)

Powdery mildew resistant and lodging tolerantadvanced line derived from the hybrid PS99102238/11760-3 using OPB18.

Diversity (Inter and inrta-accessions) in indigenous legumes based on molecular markers

Crop Intra-accession (Protein/DNA) Inter-acc

Chickpea Low/High (< 10% accessions) Low/High

Mungbean Low (< 15% accessions) Low

Blackgram Low/High (< 10% accessions) Low/High

Cowpea Low (< 10% accessions) Low

Lentil Low/High (< 15% accessions) Medium/High

SoybeanPea

Low (<10% accessions)Medium/High (< 30 % accessions)

LowHigh/High

Sample size varies from 10 to 20 samples per accession

Journal of Genetics & Breeding 54, 125-131 (2000)Acta Biologica Cracoviensia 47, 69-75 (2005)Field Crops Research 69, 183-190 (2001)Journal of Genetics and Breeding 57, 5-14 (2003)Pakistan Journal of Botany, 37(1): 71-77 (2005)Russian Journal of Genetics, 47, 19-25 (2011)Pakistan Journal of Botany, 44(2): 473-478.

Genetic diversity in indigenous legumes– a case study

V. mungo P. sativum Characterization Low variation (484) High variation (450) Evaluation High variation (484) High vartiation (298) Seed proteins Low variation (111) High variation (390) 2-D electrophoresis High variation (10) NA Geographic pattern Low linkage (88) High linkage (90) DNA markers High variation (40) High variatio (47) Germplasm Needed more Acquisition needed

Genetic diversity/fingerprinting

Crop QLT QNT Protein RAPD/SSR

Reference

Len culinaris High High Medium High Sultana et al., 2005, Sultana & Ghafoor 2008

Vigna mungo Low High Low High Ghafoor et al., 2001; 2002; 2012

Pisum sativum High High High High Ghafoor et al., 2005; Javaid et al., 2002; Nisar et al., 2011

Vigna radiata High High Low High

Vigna unguiculata High High Low High Iqbal et al., 2003

79.781.2

87.4

73

65

70

75

80

85

90

4.704.62

4.45

5.30

4.0

4.2

4.4

4.6

4.8

5.0

5.2

5.4

48.8

7

48.8

4

51.6

2

47.3

6

47.1

4

74.8

5

75.8

5

38.9

7

67.7

8

71.6

5

0

20

40

60

80

12.28 12.25

22.3823.56

0

5

10

15

20

25

Day

s to

mat

urit

yB

iolo

gica

l yie

ld g

-110

0-se

ed w

eigh

t g-1

Gra

in y

ield

g-1

B3a B5a

B9a B12b

B3b B10aB5a B9a B10b

B3b B5a

–

––

–

– – – – –

– –

++

+

+

+ + +

+

+ +

Markers in relation with quantitative traits in blackgram

Ghafoor et al. 2005

0

5

10

15

20

25

30

0

5

10

15

20

25

30C

lust

er I

(Y

ear

I)

Clu

ster

II

(Yea

r I)

Clu

ster

III

(Y

ear

I)

Clu

ster

I (

Yea

r II

I)

Clu

ster

II

(Yea

r II

I)

Clu

ster

III

(Y

ear

III)

Clu

ster

I (

Yea

r II

)

Clu

ster

II

(Yea

r II

)

Clu

ster

III

(Y

ear

II)

Agronomic scores based on three years evaluation (left) and RAPD markers (right)

Clu

ster

I

Clu

ster

V

Clu

ster

VI

Clu

ster

VII

Clu

ster

II

Clu

ster

III

Clu

ster

IV

Agr

onom

ic s

core

Agr

onom

ic s

core

Clusters on agriculturally important traits had more breeding value than DNA markers

Photoperiod insensitive Vigna mungo suitable for dual season planting developed through conventional breeding coupled with diversity analysis for DNA markers (Ghafoor et al., Crop Science, 45:1659.

Identification of Resistant Sources

Crop Pathogen Resistant Sources

Pisum sativum Powdery mildew; Pea seed borne mosaic virus

25;15

Vigna mungo MYMV 145

Vigna unguiculata Cowpea Aphid borne mosaic virus

3

Phaseolus vulgaris Bean common mosaic virus 3

Cicer arietinum Ascochyta blight 2

Inter-specific variation rather than intra-specific for protein markers. Clusters on agriculturally important traits had more breeding value than protein and DNA markers. DNA markers resolved higher levels of genetic diversity as compared to proteins in most of legumes.Identification of related and non-related species, seed proteins more powerful technique [Euphytica, 2002; PJB, 2013]. DNA markers in combination with protein give better evidence for phylo-genetic relationship.RAPD – not enough for fingerprinting!

Molecular markers for Molecular markers for genetic diversity genetic diversity

Molecular markers have the potential for target breeding [Euphytica, 2012].Selection in early generations and many rounds of selection in a year.Complex traits are the most difficult to handle.More reliable quantitative data and markers are pre-requisite for MAS.Markers are cross specific – common mapping populations.

Molecular markers for MASMolecular markers for MAS

Molecular markers have potential scope for diversity and target plant breeding – the induced evolution.

Molecular markers are not equal and none is ideal, for MAS and mapping, select the best one/s.

Enhance scope of markers and PGR strengthening.

Molecular markers preferable to morphological markers for mapping, frequency and polymorphism high.

Use the known markers for the trait/s of interest for extreme parents – free access to literature.

Integrated approach involving breeders, pathologists and molecular geneticists.

SummarySummary

OpportunitiesGenetic diversity has linearity with crop improvement:

need to have more legumes genetic resourcesBasic genetic information linked to progress!Biotic and abiotic stresses resistant germplasmEnhancing food legumes production

Vertical Horizontal New crops; Phaseolus, Vicia, Lathyris?Cropping systems

Cereal-legumesCotton-legumes

Sandwich cropRice-wheat > 1.5 mh

Opportunities

Investigated diversity in indigenous legumes in relation to geographic origin – medium to high

Developed powdery mildew, lodging tolerant pea genotypes using MAS – ready for evaluation

Developed pea for dry seed – new avenue Developed dual season blackgram cultivar –

expected enhanced productivityExploiting newly developed blackgram and pea

genotypesG-E interactionUtilization in crop breeding programMAS and mapping

Molecular markers have potential scope for investigation of diversity (>80%) and for target plant breeding (<10%) – the induced evolution.

Molecular markers are not equal and none is ideal for particular objective/s, select the best one/s based on objectives – some are better for some purposes than others.

Protein markers resolve taxonomic discrepancies efficiently, and DNA markers are preferred over proteins and morphological markers for mapping – frequency and polymorphism high.

DNA markers are not universal, need conformation or development!

Interdisciplinary approach for target oriented task – involving participatory crop breeding.

Take home message

Plant germplasm is a resource for Plant germplasm is a resource for mankind’s future . . . mankind’s future . . .

… if it is collected and conserved!

… if we understand it and know how to evaluate and use it!

Inter-agency partnerships are strengthening our national ability to manage, understand, and use germplasm.

Develop local DNA markers technology for sustainable use..

Collard et al. 2005. An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement. Euphytica 142:169-196.

Francia et al. 2005. Marker assisted selection in crop plants. Plant Cell, Tissue and Organ Culture 82:317-342.

Gepts & Hancock. 2006. The future of Plant Breeding. Crop Science 46:1630-1634.

Mago et al. 2005. Development of PCR markers for the selection of wheat stem rust resistance genes Sr24 and Sr26 in diverse wheat germplasm. Theoretical and Applied Genetics 111:496-504.

McCouch et al. 1988. Molecular mapping of rice chromosomes. Theoretical and Applied Genetics 76:815-829. Mohan et al. 1997. Genome mapping, molecular markers and masrker-assisted selection in crop plants. Molecular Breeding 87:87-103.

Sharma et al. 2002. Application of biotechnology for crop improvement: prospects and constraints. Plant Science 381-395.

Young. 1996. QTL mapping and quantitative disease resistance in plants. Annual Review of Phytopathology 34:479-501.

Suggested reading/acknowledgements Suggested reading/acknowledgements

AcknowledgementsFarmers for collection of genetic resources

Postgraduate students (R & D)

CRDI for screening against diseases

PGRI for executing experimentations under lab and field

Thank you