SEMINAR –IIDOUBLE HAPLOIDS (DH)

Presenter: Shilpa v malaghan.

Class : Sr. MSc(Agri).

Agriculture collage Raichur.

Date: 7/12/2012.

Contents

Introduction

Doubled Haploid : An individual with the doubled

chromosome number of the haploid

(Gupta., 2006)

What is a doubled haploid plant?

Each cell contains 2 sets of genetic

information which are

(but not exactly) identical most.

For example, one gene set may carry a

gene for disease resistance when the

other set does not.

Doubled haploid plant has

cells containing 2 gene sets

which are exactly identical.

If one gene set has the disease

resistance gene the other gene

also having resistance.

History

• Blakeslee et al. (1922) - Datura stramonium

• Guha and Maheswari (1964) - Anther culture technique for the production of haploids in the laboratory

• Niizeki and Oono (1968) - Production of rice haplpoids

• wide crossing

Kasha and Kao, (1970) - Barley

Burk et al., (1979) - Tobacco

Doubled haploid methodologies have now been applied to over 250 species

- Forster and Thomas, 2007

1) Haploid production

- In vitro versus in vivo

- Maternal versus Paternal

2) Haploid identification

- Markers: Morphological or molecular

- Cytological / flowcytometry

3) Genome doubling

- Colchicine

- Other

Technologies of DH production

Identification of haploid kernels using an R1--‐nj marker system

Haploid identification

Figure . Flow cytometric analysis of the ploidy level. The x-axis of the histogram represents the intensity of DNA fluorescence in relative units; the y-axis represents the number of nuclei counted per histogram channel. (A) A representative peak set for diploid or doubled haploid material. (B) Peaks corresponding to a typical haploid individual.

Payam et al., 2007

Mechanisms of genome doubling

Genome doubling

(Seguí-Simarro et al., 2008)

Genome doubling methods.

Spontaneous Chromosome doubling

Artificial Chromosome doubling

Spontaneous Chromosome doublingLianquan et al., 2011

Anti-microtubule drugs

Colchicine

Oryzalin

Amiprophosmethyl(APM)

Trifluralin

Pronamide

Stages of application of anti mitotic in DH production

Anther treatment

Microspore treatment

Haploid embryo treatment

Young haploid seedling treatment

Young haploid Root tip treatment

Fig. The interaction between the colchicine concentration in the pre-treatment medium and the duration of colchicine pre-treatment on the

ELSs production in the ETH-M82 genotype. Payam et al., 2007

Fig; Haploid embryos in cotyledonary stage (a and b) normal regenerated plant (c) normal doubled

haploid plant (d)

Haploid embryos treated with colchicine and inoculated in colchicine free NLN-13 medium for regeneration

Payam et al., 2011

Table : Effects of colchicine concentration and duration treatment on regeneration and recovery of doubled

haploid plants of oilseed rape.

Colchicinetreatment

concentration(mg/L)

Colchicinetreatment

duration (h)

Number ofregenerated

plants

Numberof doubled

haploid plants

0 (control) 0 53 0

125 12 49 10

24 37 9

36 18 8

250 12 41 13

24 42 27

36 15 9

500 12 17 7

24 14 8

36 9 6

1000 12 0 0

24 0 0

36 0 0

Table :percentage rates of doubled haploid (DH) plants

derived from individual treatments

Genotype Total no.of plants

tested

mean Colchicine in vivo

Colchicine In vitro

OryzalinIn vitro

TrifluralinIn vitro

SL-3/04 560 55.53 71.09 76.62 86.02 80.24

OP-41/1 615 39.34 55.65 77.29 69.80 88.07

SL-2/04 534 31.88 39.94 68.38 43.78 88.67

Mean 1709 42.25 55.56 74.10 66.53 85.66

Miroslav et al., 2008

Diploidization frequency of individual treatments

Miroslav et al., 2008

Genetics of doubled haploid populations

• Only two types of genotypes – pair of alleles – A ,a

- Frequency – ½ AA and ½ aa

- Diploid – ¼ AA,1/2Aa,1/4aa

• Probability of getting desired genotype is (1/2)n

- Diploid – (¼)n

Kunzel et al., 2000

• Selfing in autogymous spp.

• Vegetative propagation

• Bud pollination

• Late or early pollination methods

Breeding Using Doubled Haploid System

Conventional Breeding System

Difference between DH method and conventional method.

Particulars DH method Conventional method

Time required for developing pure line

1 year or 1 crop season

3-5 year

Time required for cultivar development

2-3 year 7-8 year

Fixation of heterosis possible Not possible

Expenditures More than Conventional method

Less than DH method

Identification of recessive mutation

Very easy Difficult

Singh., 2007

Applications of DHs in Plant Breeding

Mapping Quantitative Trait Loci (QTL) Backcross breeding Bulked segregant analysis (BSA)Hybrid sorting Genetic maps Genetic studies Elite crossing Cultivar developmentFixation of heterosis

QTL MAPPING

Type of Population

Strength Weakness

F2:3- Speed of production- d and a estimates

- Heterogeneous families

RIL - Homogeneous families- Power of QTL detection

- Slow production

DH - Speed of producing homogeneous families- Power of QTL detection

- Laborious production process- Lower recombination (>RIL)

BC - Speed of production - Heterogeneous families

QTLs controlling six traits determining root morphology and distribution in a IR6429 X Azucena doubled-haploid rice population

Yadav et al., 1997

Conti….

Comparison between QTLs identified in IR64 x

Azucena and Co39 x Moroberekan

RFLP linkage map showing chromosomal locations of QTLs for the three traits

Liu et al., 2006

Cont….

Liu et al., 2006

Phenotypic performance of parents and the DH population for biomass yield (BY), straw yield (SY) and grain yield(GY) in

two growth seasons

Liu et al., 2006

Backcrossing or Gene Pyramiding

Thomas et al., 2003

Trait / Gene Stacking

X

Line 1 Line 2

or

Selfing (F2) DH Induction

Goal: Fixation of target alleles

No. of genes F2 DH

1 0.25 0.5

2 0.0625 0.25

4 0.004 0.0625

8 0.00002 0.004

16 0.00000000002 0.00002

Probability for Fixation of Target Genes

Resistant genotype their pedigree, resistance genes and linked molecular markers Kay et al., 2005

strategy I: Scheme of pyramiding BaYMD resistance genes rym4, rym9 and rym11 by two haploidy steps. Kay et al., 2005

Strategy II: pyramiding of in one haploid step Kay et al., 2005

Total DH lines lines

Target gene No DH lines

107 DH(Strategy 1)

rym4 rym9 rym11 20 DH

187 DH(Strategy 2)

rym4 rym9 rym11 27 DH

DH- plants carrying all possible two gene combinations

Kay et al., 2005

Pyramiding the stem rust resistance genes Sr24, Sr26, and SrR in Westonia background

Mago, et al., 2011

pyramiding the stem rust resistance genes Sr24, Sr26, Sr31 and SrR in Pavon background

Mago, et al., 2011

Doubled haploid lines with multiple stem rust resistant genes

Gene combinations Backgroundcultivar

No. of putativehaploids received

No. of haploidswith genes

No. of DHswith genes

Sr24–Sr26–SrR Westonia 11 3 0

Sr24–Sr26 Westonia 34 2 2

Sr24–SrR Westonia 30 2 1

Sr26–SrR Westonia 31 4 3

Sr24–Sr31–SrR Pavon 37 10 3

Sr24-Sr26-Sr31 Pavon 20 4 2

Sr24–Sr31 Pavon 40 4 3

Sr24–SrR Pavon 40 2 2

Sr26–Sr31 Pavon 25 2 2

Bulked segregant analysis (BSA)

• BSA is dependent on accurate phenotyping and the DH population has particular advantage in that they are true breeding and can be tested repeatedly.

• DH populations are commonly used in bulked segregant analysis, which is a popular method in marker assisted breeding. This method has been applied to rapeseed and barley.

A linkage map of RAPD markers in a DH population drivedfrom 'Quantum‘ X 'China A' cross.

Samizadeh et al., 2007

PCR profiles produced by RAPD analysis in B. napus with PL 18 (A) and PL 2 (B) primers.

Samizadeh et al., 2007

Analysis of variance for pod length markerSamizadeh et al., 2007

Marker MS R2 Mean ± SE

Long Short

PL2 2202.4 14.60 105.5 ± 22.01 92.10 ±10.03

PL9 712.42 4.70 101.6 ± 20.33 94.10 ±13.27

PL 14 863.62 6.00 102.2 ± 20.22 93.90 ±13.56

PL 17 522.39 3.40 101.7 ± 18.98 95.14 ± 16.12

PL 18 1468.3 9.70 105.2 ± 19.46 93.94 ±12.24

PL 26 1027.4 7.80 100.6 ± 18.98 89.94 ± 8.19

PL 2 × PL 18 1129.6 22.40 112.2 ± 21.86 89.23 ± 5.23

Hybrid sorting

• Hybrid sorting - Selection of superior plants among hapliodsderived from F1 through anther culture

• Selection of recombinant superior gametse

• Superior over pedigree & bulk method

- frequency of superior gametes – higher than the corresponding F2 generations

- Reduces the time required to release a variety

• Successful in china & Japan

Varieties bred through hybrid sorting

crop Varieties developed Attributes

Rice Tanfong 1, xin xion,

late keng 76,

shanyou 63

Good quality, high

yield

Wheat Yunhua 1, yunhua 2 Rust resistance,cold

resistance,lodging

resistant

Tobacco Tanyu 1, Tanyu 2,

Tanyu 3

Disease resistant,

Mild smoking

SELECTION OF SALT TOLERANCE GENOTYPES FROM DOUBLED HAPLOIDS IN RICE

Dang et al., 2004

Table : Relative response of anther culture lines and the tolerance checks at salinity of 6dS/m

and 15dS/m.

Rice anther culture to obtain DHs with multiple resistanceBambang et al., 2010

• Released varieties: Way Rarem, Jatiluhar.

• Accessions tolerant to aluminum toxicity( Al): Dupa, Krowal

• Accessions tolerant to shade: Dodakan, ITA-247

Results

• 5 lines – tolerant to Al toxicity, and shade and tolerant to 4 races of blast.

• 11 lines – tolerant to Al toxicity, and tolerant to 4 races of blast.

• 1 lines – tolerant to shade and tolerant to 4 races of blast.

• 2 lines – sensitive to Al toxicity, and shade but tolerant to 4 races of blast.

Linkage maps of F2 populations derived from the cross between two temperate japonica cultivars

‘Koshihikari’ and ‘Akihikari’

Masumi Yamagishi et al.,2010

Linkage maps of DH populations derived from the cross between two temperate japonica cultivars ‘Koshihikari’ and ‘Akihikari’

Masumi Yamagishi, et al

Genetic studies - mutation genetics

• Genetic ratios and mutation rates can be read directly from haploid populations.

• A small doubled haploid (DH) population was used to demonstrate that a dwarfing gene in barley is located chromosome 5.

• In another study the segregation of a range of markers has been analyzed in barley.

Means ± standard errors and ranges for erucic acid (% of total fatty acids) in seeds of the M2 and M3 generations of six doubled haploid

mutant lines of Brassica carinata Barro et al.,2002

Line Plants analysed Generation

M1 M2

Control 10 42.8 ± 0.7 43.5 ± 0.5

40.2–44.4 41.4–44.7

BC2.6.1 8 48.5 ± 0.3 50.6 ± 0.4

47.8–48.7 49.7–51.5

BC2.8.1 6 48.3 ± 0.4 49.6 ± 0.2

48.1–48.9 49.4–49.9

BC3.3.2 8 48.1–48.9 50.3 ± 0.6

47.8–49.8 49.2–51.1

BC5.2.2 10 48.7 ± 0.4 49.7 ± 0.3

48.3–50.1 49.1–50.7

BC6.19 8 49.5 ± 0.6 48.5 ± 0.2

48.8–50.2 48.0–48.8

BC1.5 10 48.7 ± 0.6 48.7 ± 0.1

48.4–49.6 48.5–48.9

Dendrogram showing the relationship among 102 DH wheat based on gliadins bands.

Ojaghi and Akhundova., 2010

Based on morphology.

Figure . Dendrogram showing the relationship among 102 doubled haploid wheat based on RAPD

markers

Doubled haploid varieties

Crop Varieties Country

Rice Xin-Xin, Hua-Hau-Zao china

Wheat Jing Hua 1,3,5 china

Barley Mingo Canada

Chickpea Quantum, Q2, Duplo, Mingo

Cabbage Orange queen Japan

Brocolli Three man Japan

Tobacco Dan-yu1,2,3 China

Hot pepper Haihua 19 China

Sweet pepper Haihua 29 China

Raina ,1997

Elite crossing:• Traditional breeding methods are slow and take

10–15 years for cultivar development.

• Another disadvantage is inefficiency of selection in

early generations because of heterozygosity.

• These two disadvantages can be over come by DHs,

and more elite crosses can be evaluated and

selected within less time.

• Improved genetic gain can be achieved with DHs .

Obtained DH lines from spring and winter wheat hybrids

Grauda et al.,2010

Application of Doubled haploid in Cultivar development

Singh., 2007Name of crop No of cultivar released Developed by

Barley 115 Anther culture

Rape seed 47 Anther culture

Wheat 21 Anther culture

Melon 9 irradiation

Capsicum annum 8 Anther culture

Rice 8 Anther culture

Asparagus 7 irradiation

Tobacco 6 Anther culture

Egg plant 5 Anther culture

Rao, 2006

Double haploid rice varieties in India.

AICRIP and PH-43

has performed well (2000- 05)

CRAC 2224 -1041 (early duration )

PHB71, PA6201, DRRH1, KRH2, Pusa RH 10,

CRHR 4 and Rajlaxmi

2000 DH lines developed

G.J.N.Rao 2006

Rao, 2006

Speed in Line Development

Founder line 1 x Founder line 2

F1Selfing

Inbred line

DH

Inbred line

Advantages

of Doubled Haploid Techniques

• No risk of herterozygosity - Based on gamete selection

• Develop immediate homozygosity, shorten the time to cultivar release -additive & additive x additive variances

• Provide greater efficiency of selection in plant breeding

cytogenetics

• Production of aneuploids & determine the basic chromosome number

• Improve the precision of genetic and mapping studies

• Accelerate gene pyramiding

• Improve efficacy and efficiency in screening for resistance

Some Drawbacks

with Doubled Haploid Production

GENERAL:

– More expensive: expertise, facilities

– Restriction on number of crosses

• SPECIFIC:

– Mutagenic treatment

– Genotype dependent haploid induction

– Low haploid regeneration frequency

Video clip