GCP Wheat CI Team • Indian Agricultural Research Institute, New Delhi • Punjab Agricultural University, Ludhiana • J L Nehru University of Agricultural Sciences, Powarkheda • Agharkar Research Institute, Pune • National Research Centre on Plant Biotechnology, New Delhi

India

kvinodprabhu@rediffmail.com

Molecular Breeding and Selection Strategies to Combine and Validate QTLs for Improving WUE and Heat

Tolerance of Wheat in India

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Generation Challenge Programme : GRM 2013, Lisbon September 27-30, 2013

Source: IWMI

Water Stress Indicator Map G

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Abiotic Stresses of Wheat in India • Wheat grown in 30 million hectares and over 85% of wheat is

irrigated

• But nearly 70% of irrigated area does not receive all 6 recommended irrigations

• Wheat suffers losses due to abiotic stresses such as heat (terminal, early, tillering stages), drought and salinity ranging from 10-40% (yield and reduced quality)

• Minor genes or QTLs based tolerance is likely to provide abiotic stress tolerance in wheat and very few varieties available for this purpose

• Options such as MARS, MABB and AM are expected to pick up/utilize tolerance imparting QTLs distributed along the complex wheat genome for their deployment in varietal backgrounds

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Wheat Breeding Strategies for Drought and Heat Tolerance

Development of varieties which adapt to high temperatures during early, mid and terminal stages (resilience)

Varieties suited to change in planting dates to avoid terminal heat stress

Varieties with high WUE, suited to low water availability

Varieties suited to altered crop rotations

Development of varieties for new agricultural areas resulting due to shift in climatic pattern

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Centres and Responsibilities •IARI, New Delhi MARS, MABB : Phenotyping, Genotyping •JNKVV, Powarkheda MARS: Phenotyping •PAU, Ludhiana MARS, MABB : Phenotyping, Genotyping •ARI, Pune MARS: Phenotyping •NRCPB, New Delhi Genotyping

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Traits targeted

Morphological traits 1. Days to flag leaf emergence 2. Days to 50% heading 3. Ground cover 4. Grain yield (g/plot) 5. Harvest index 6. Above ground biomass

(g/plot) 7. Test weight (g) 8. Grain filling duration

Physiological traits 1. Stay green duration(Using

NDVI) 2. Canopy temperature (CT)

using Infrared thermometer 3. Flag leaf area 4. Chlorophyll content 5. Chlorophyll florescence

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Per cent ground cover : Proportion of the Mean Grey Value to mean value if the image were completely white (255). (Grey value of a completely black image is 0 )

Digital estimate of per cent ground cover for early vigour evaluation

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VARIABLE AVERAGE MINIMUM MAXIMUM HERITABILITY GERM 88.664 40 95 0.59

DH 66.154 50 84 0.94 YLD 179.243 66 373 0.64 TKW 31.404 20 44 0.76 DM 102.822 80 125 0.81 CT 28.259 20.6 31.2 -

CTD 4.373 1.8 12.1 0.28 NDVI 0.747 0.58 0.88 0.33 SPAD 53.588 43.7 62.3 0.51

DH DM YLD TKW CT CTD NDVI DM 0.84** YLD -0.22** -0.11 TKW 0.20** 0.19** 0.20** CT -0.07 -0.16** -0.36** -0.09 CTD 0.08 0.19** 0.38** 0.04 -0.74** NDVI 0.39** 0.46** 0.24** -0.07 -0.34** 0.28** SPAD -0.04 -0.03 0.02 0.20** -0.06 -0.03 -0.12*

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Variability profile of the different populations: An example

Validation of markers linked to known QTLs

• More than two hundred microsatellite markers associated with physiological, phenological and agronomic traits were selected.

• These microsatellite markers associated with specific traits on the parents of our backcross generations to Introgress the QTLs (16 back cross BC1F1, now BC2F1 with 600-800 size generated).

•

• Polymorphic markers to target five physiological traits covering 17 chromosomes for identification of known QTls in our backgrounds.

•

• These include Canopy temperature QTLs on 15 Chromosomes (1A, 1B, 2A, 2B, 3A, 3B, 4A,4B, 5A, 5B, 6A, 6B, 6D, 7A,7B), NDVI on 3 chrs (2A, 2B and 4A), Chlorophyll on15 chrs (1A,1B,1D, 2A, 2B, 3A, 3B, 4B, 5A, 5B, 6A, 6B, 6D, 7A and 7B), WSC on 17 chrs (1A,1B,1D, 2A, 2B, 2D, 3A, 3B, 4A, 4B, 4D, 5A, 5B, 6B, 6D, 7A and 7B), stay-green on 3chrs (1A,3B and 7D).

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QTL references Chromosome no. Traits targeted Pinto et al. 2010 2B, 5A, 4A, 1B CT, Chl, NDVI,

yield Kumar et al. 2010 7B, 7D, 3B Stay-green trait

Mohammadi et al. 2008a and b

2D, 1B, 5B and 7B Yield and GFD under heat

Kumar et al. 2012 1D, 3B CT, Chl

Olivares et al. 2007

2A NDVI

Kadam et al. 2012 4B, 3D Yield under drought and root traits

Significant QTLs reported and utilized in our populations

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Drought tolerance from Parent B HI 1500

Yield traits from Parent DBW43

z

1A 1B 2B 2D 3A 3B 3D 1D

2A

Best possible individual by modeling

6A 6B 7A 7D 7B 5A

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FAMILY. 1 FAMILY. 4 FAMILY. 3

FAMILY 2

x x

2nd Recombination

x

MARS for Accumulating Favourable Genes

F1 F1

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SSRs markers linked to QTLs for Drought tolerance which are segregating population HI1500 X DBW43

GWM 484(2D) GWM 644(7B) GWM 156 (5A) GDM 132(6D) GWM 383(3D) GWM 165(4B) GWM 368(4B) GWM273(1B) GWM 582(1B) BARC 147(3B) GWM 566 (3B)

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Breeding Cross G

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HI 1500 (P1)

F1

F 4 And F5 generation

F2

Phenotyping in E1, E2 E3 and E4 in Rainfed and irrigated

Parental polymorphism 130 SSR markers

Genotyping and QTL validation

Selection of lines for intercrossing based on multilocation AMMI Analyses of phenotype (RF + RI) and QTLs genotyping

F1 F1 F1 F1

F2 F2

F2 F2

.14 3 2 Fam.1 5 4 7… 6

Selection of homozygous plant

BDW 43(P2)

Double cross Double cross

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Traits phenotyped (HI 1500 x DBW 43)

Morphological traits

1. Days to flag leaf emergence 2. Days to 50% heading 3. Ground cover 4. Grain yield (g/plot) 5. Harvest index 6. Above ground biomass

(g/plot) 7. Test weight (g)

Physiological traits 1. Stay green duration 2. Canopy temperature (CT)

using Infrared thermometer 3. Flag leaf area 4. Chlorophyll content 5. Leaf area index (0nly in 2011-

12) 6. Chlorophyll flouresence (0nly

in 2011-12) 7. NDVI index( only in 2011-12)

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Procedure used to select Individuals for intercrossing

• Final selection based on AMMI ranks on multilocation yield data (common individuals in top 30 ranks in all four location)

• AMMI analysis was done over the location for yield data. Rain fed and restricted irrigation data was combined as two treatments and for each location two replications were included

• Separate AMMI analysis was done separately for rain fed data and Four best lines were also included in selection.

• Four best performing lines from the least favorable environment were

also added • Homozygous lines for favourable QTLs are selected for intercrossing

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LOCATIONS DELHI LUDHIANA POWARKHEDA PUNE

TRAITS CANOPY TEMPERATURE CHLOROPHYLL CONTENT YIELD BIOMASS HARVEST INDEX

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**Yield in Rainfed and Restricted irrigation conditions **Yield Among locations and **Among lines **Canopy Temperature (CT) at vegetative and reproductive stages **Chlorophyll content at vegetative stage

HI 1500 X DBW 43 G

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HI 1500 X DBW 43 G

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Environment NE Em IPCAe[1] IPCAe[2] Delhi 1 639.4 21.09613 -14.15923 Ludhiana 2 709.9 -1.32147 20.07025 Powarkheda 3 349.1 - 22.06025 -13.91356 Pune 4 191.6 2.28559 8.00254

Number Environment Mean Score 1 2 3 4 1 Delhi 639.4 21.10 G28 G86 G34 G84 4 Pune 191.6 2.29 G87 G34 G149 G137 2 Ludhiana 709.9 -1.32 G109 G87 G14 G149 3 Powarkheda 349.1 -22.06 G76 G88 G93 G136

HI 1500 X DBW 43G

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Environment NE Em IPCAe[1] IPCAe[2] Delhi 1 499.5 25.85725 15.92357 Ludhiana 2 624.4 -25.47537 12.53966 Powarkheda 3 277.8 3.47501 -27.55992 Pune 4 114.0 -3.85689 -0.90331

Number Environment Mean Score 1 2 3 4 1 Delhi 499.5 25.86 G88 G133 G32 G84 3 Powarkheda 277.8 3.48 G121 G76 G93 G88 4 Pune 114.0 -3.86 G156 G76 G121 G136 2 Ludhiana 624.4 -25.48 G138 G151 G142 G73

FINAL SELECTIONS (RF + RI)

Entries: 19,26,43,44,76,84,87,98,105,137,143,146,155,156

AMMI for RAINFED CONDITIONS G

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S.No Population Families selected for intercrossing

1 IARI 1 19,26,43,44,76,84,87,98,105,137,143,146,155,156 2 IARI 2 68,111,46,7,72,107,101,68 3 PAU 1 51,5,43,113,105,95,49,23,104,84,48 4 PAU 2 8,11,74,97,80,28,44,112,153,118,52,4.

Families selected for intercrossing in Winter 2013

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Procedure followed for intercrossing

• In each family, 40 individuals were tagged as females and emasculated

• Selfed seeds of each female and male plant were

collected • Five spikes were pollinated in each cross • Crossed seed from each spike collected separately • Crossed seed were planted along with selfed male and

female plants and F2 + Selfed seed harvested

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ENT NO 20 26 32 34 38 59 87 88 98 110 136 137 146 156

20 X X X X X X X X X X X X X

26 X X X X X X X X X X X X

32 X X X X X X X X X X X

34 X X X X X X X X X X

38 X X X X X X X X X

59 X X X X X X X X

87 X X X X X X X

88 X X X X X X

98 X X X X X

110 X X X X

136 X X X

137 X X

146 X

156

INTERCROSSES MADE BETWWEN SELECTED FAMILIES DURING 2011-12 IN CROSS HI 1500 X DBW 43

In each cross 5 spikes are emasculated and from male and female plants selfed seeds were collected and planted along with crosses in lahoul spiti now in all above mentioned cross F2 seeds are available

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Few examples of cross made to accumulate QTLs Marker inretval Traits linked Family no 20 (POSITIVE FOR 2 QTLs) x Family no 59 ( POSITIVE FOR 6 QTLs)

Barc 186 (5A) CT Homo for DBW 43 (-/-) Homo for HI1500 (+/+)

Barc 147(3B) CT Homo for DBW 43 (-/-) Homo for HI1500 (+/+)

Gwm 273() CT Homo forHI1500 (+/+) Homo for DBW 43 (-/-)

Barc68-barc 101(3B) Fv/Fm, chl Homo for DBW 43 (-/-) Homo for HI1500 (+/+)

Barc 170 (4A) Fv/Fm Hetero (+/-) Homo for HI1500 (+/+)

Gwm 533() GFD Homo for DBW 43 (-/-) Homo for HI1500 (+/+)

Wmc 487 (6B) 1000 Gn wt Homo for DBW 43 (-/-) Homo for HI1500 (+/+)

Gwm 368-barc20 (4B) Yld (drought),root biomass Homo for HI1500 (+/+) Homo for DBW 43 (-/-)

Marker Traits linked

Family no 38 ( POSITIVE FOR 4 QTLs) x Family no 59 ( POSITIVE FOR 6 QTLs)

Barc 186 (5A) CT Homozygous for HI1500 (+/+) Homozygous for HI1500 (+/+)

Barc 147(3B) CT Homozygous for DBW 43(-/-) Homozygous for HI1500 (+/+)

Gwm 273 () CT Homozygous for HI1500 (+/+) Homozygous for DBW 43 (-/-)

Barc68-barc 101(3B) Fv/Fm, chl Homozygous for HI1500 (+/+) Homozygous for HI1500 (+/+)

Gwm 533(3B) GFD Homo for DBW 43 (-/-) Homo for HI1500 (+/+)

Barc 170 (4A) Fv/Fm Homozygous for DWB43(-/-) Homozygous for HI1500 (+/+)

Wmc 487 (6B) 1000 Gn wt Homo for DBW 43 (-/-) Homo for HI1500 (+/+)

Gwm 368-barc20 Yld (drought),root biomass Homozygous for HI1500 (+/+) Homozygous for DWB43 (-/-)

marker Traits linked

Family no 34 (POSITIVE FOR 2 QTLs) Family no 20 (POSITIVE FOR 2 QTLs) )

Barc 186 (5A) CT Homozygous for HI1500 (+/+) Homozygous for DBW 43 (-/-)

Barc 68-barc 101 (3B) Fv/Fm, chl Homozygous for HI1500 (+/+) Homozygous for DBW 43 (-/-)

Gwm 368-barc 20 (4B) Yld (drought),root biomass Heterozygous (+/-) Homozygous for HI1500 (+/+)

Barc 170(4A) Fv/Fm Heterozygous (+/-) Heterozygous (+/-)

Gwm 273() CT Homozygous for DBW 43 (-/-) Homo forHI1500 (+/+)

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When Richard Trethovan, our Coordinator gave us a surprise when he directly reached the field from Sydney to watch the intermating in operation!!

QTL references Marker interval Traits linked Pinto et al. 2010 Barc186 – Barc151

(5A)

Barc 147(3B)

CT

Kadam et al. 2012 Barc20-gwm368(4B) grain yield per plant, harvest index, and root biomass under drought.

Wang et al. 2009 gdm 132-cfd42 (6D) Barc113-gwm533(1A)

GFD

Kumar et al. 2012 Barc68-barc101(3B)

Xgwm369 (3A)

CT, Chl, Fv/Fm

Son et al. 2007

Wmc 487(6B) 1000 grain wt

Significant QTLs reported and utilized in our populations G

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HD 2733 X CROSS 1 -59 (5 Spikes ) CROSS 1-59X CROSS 4 P4 CROSS 1 P 85 X CROSS 4 P4

HD 2733 X CROSS 1- 20 (5 Spikes ) CROSS 1 -59 X CROSS 4 P5 CROSS 1 P 85X CROSS 4 P5

HD 2733 X CROSS 1 -156(5 Spikes ) CROSS 1 -59 X CROSS 4 P8 CROSS 1 P 85 X CROSS 4 P8

HD 3076 X CROSS 1-98 (5 Spikes ) CROSS 1 -59X CROSS 4 P11 CROSS 1 P 85 X CROSS 4 P11

HD 3076 X CROSS 1 -86 (5 Spikes ) CROSS 1 -59 X CROSS 4 P44 CROSS 1 P 85 X CROSS 4 P44

CROSS 1-38 X CROSS 4 P4 CROSS 1 P-59X CROSS 4 P55 CROSS 1 P 85 X CROSS 4 P55

CROSS 1-38X CROSS 4 P5 CROSS 1-59 X CROSS 4 P35 CROSS 1 P 85 X CROSS 4 P35

CROSS 1 -38 X CROSS 4 P8 CROSS 1 -59X CROSS 3 P5 CROSS 1 P 85 X CROSS 3 P5

CROSS 1-38 X CROSS 4 P11 CROSS 1 -59X CROSS 3 P51 CROSS 1 P 85 X CROSS 3 P51

CROSS 1 -38 X CROSS 4 P44 CROSS 1 -86X CROSS 3 P5 CROSS 1-87X CROSS 4 P35

CROSS 1-38 X CROSS 4 P55 CROSS 1 -86 X CROSS 3 P35 CROSS 1 -87X CROSS 3 P5

CROSS 1 -38 X CROSS 4 P35 CROSS 1-86 X CROSS 3 P51

CROSS 1 -87X CROSS 3 P51

CROSS 1-38 X CROSS 3 P5 CROSS 1 P 104 X CROSS 3 P5

CROSS 1 -38 X CROSS 3 P51 CROSS 1 P 104 X CROSS 3 P35

Crosses made among selected individuals of different populations of MARS these crosses were forwarded in lahaul spiti F2 seed available in each case

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Sl.No. Double cross(In each case 15 spikes were emasculated and pollinated)

1 (34 Χ136) Χ (59 Χ 146)

2 (146 Χ4) Χ (20 Χ 88)

3 (136 Χ 88) Χ (87 Χ 59)

4 (20 Χ 87) Χ (136 Χ 86)

5 (146 Χ 86) Χ (20 Χ 88)

6 (110 Χ 136) Χ (20 Χ 146)

7 (20 Χ 156) Χ (110 Χ 146)

8 (156 Χ 38) Χ (156 Χ 32)

9 (156 Χ32) Χ (156Χ 136)

10 (156Χ 38) Χ (34Χ 110)

List of double crosses made to accumulate favourable QTLs in lahaul spiti during 2012-13

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Xbarc 186 linked Canopy Temperature by olivares el al 2008

Segregation pattern in bulked families

HI 1500 203 bp

DBW 43 211 bp

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HI 1500 DBW 43

Barc 151 Segregation pattern in Families

F1 type HI 1500 217 bp

DBW 43 231 bp

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Segregation pattern Xbarc 68 linked to Chlorophyll content

DBW 43 145 bp

HI 1500 137 bp

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WMC 487 linked to 1000 grain weight by son et al 2008

A1 = HI1500 216 A2 =DBW 43 216 and 222bp G

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Cfd 73 segregation pattern in F4 families

A1 = HI1500 264bp A2 =DBW 43 256bp G

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Chromosome No. of Polymorphic markers

1A,1B,1D 58

2A,2B,2D 60

3A,3B,3D 48

4A,4B,4D 37

5A,5B,5D 46

6A,6B,6D 61

7A,7B,7D 110

Hunt for new QTLs segregating in the cross Polymorphic markers among parental genotypes

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A1 = HI1500 264bp A2 =DBW 43 256bp

Identification of new QTLs (Cfd 73) in F4 families

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A1 = HI1500 A2 =DBW 43

Identification of new QTLs (Xbarc 327) in F4 families

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Development of backcross populations

• In the last crop season crosses were developed among contrasting parents for heat and drought tolerance

• F1s from twenty three crosses were advanced in the off-season nursey at Lahaul-Spiti

• F1s were backcrossed with the high yielding recurrent parent to generate BC1F1 populations

• These Bc1F1s are advanced in the current crop- season to generate BC2F1 populations. DNA samples were drawn from the BC1F1 for foreground selection of the target trait and recovery of the background genome through background selection.

• A total of 850 microsatellite markers used and approximately hundred polymorphic markers were observed among them

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S.No Backcrosses PopSize BC1F1

Trait targeted Chr no.

1 HD 2733*1/C306 645 Yield, CT, Chl 4B,1D, 3B 2 HD 2733*1/HI 1500 516 NDVI, Stay-

green,Yield 2B, 7D

3 HD 2733*1/HD 2888 454 NDVI, CT, Yield 1D, 7B, 4A 4 HD 2733*1/HW 2004 706 NDVI, yield 2B, 7D 5 HD 2733*1/NI 5439 462 NDVI, CT, Yield 1D, 7B, 4A 6 HD 2733*1/WH730 725 Yield, CT 1B, 7B 7 GW 322*1/HD 2888 650 NDVI, CT 2A, 2B 8 GW 322*1/HD 2987 780 Stay-green, Yield,

GFD 7D, 4A, 6D

9 GW 322*1/HI 1500 540 Yield, stay-green, Chl content

4A, 7D, 4B

10 GW 322*1/NI 5439 352 Yield, Chl 4B, 3B 11 GW 322*1/RAJ 3765 392 CT, Yield 1B, 5B, 7B 12 GW 366*1/HD 2987 430 13 GW 366*1/WH 730 470 CT, Yield 1B, 5B, 7B 14 GW 366*1/RAJ 3765 540 CT, Yield 1B, 5B, 7B

Traits targeted for introgression through MABB G

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HD2733 X HD2888

F1 X HD 2733

BC1F1 (population size 454)

L 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

L 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

13 = HD2733(259bp) 14=HD2888(270bp)

13 = HD2733(300& 248 bp) 14=HD2888(306& 256bp)

Plants positive for both cfd55 & cfd79 selected (plant No. 3 and 22)

cfd55

cfd79

Kadam et al Funct Integr Genomics (2012)

Foreground selection for QTL for yield under drought on 3DS

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HD2733 X C306

F1 X HD 2733

BC1F1 (population size 645)

Kadam et al Funct Integr Genomics (2012)

HD2733 C 306 HD2733 C 306

Kadam et al Funct Integr Genomics (2012)

xbarc20 gwm 368

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Foreground selection :QTL for Stay green under drought on 7D

HD 2733 (Recurrent parent) X HI 1500 (Donor parent )

F1 X HD 2733

BC1F1 (population size 782)

L 1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19 20 21 22 23 24

Lane 13 = HD 2733 Lane 14=HI 1500

L 1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19 20 21 22 23 24

Kumar et. al Euphytica (2010)

gwm 111

gwm 437

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Generation advancement, intermating and backcrossing at Off-season (Lahaul Spiti, Kashmir) in summer

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An opportunity created GCP + DBT (Govt of India) for prolonged use through MAS products of

HD 2967, HD2733, GW 322, GW 366 (drought + rust resistant) HD2733 × HD2687+Lr19 HD2733 × HD2687+ Lr24 HD2733×HD2687 Yr15

F1 F1 F1 HD2733 HD2733 HD2733 × × ×

BC1 BC1 BC1 HD2733 HD2733 HD2733 × × ×

BC2 BC2 BC2 HD2733 HD2733 HD2733

BC2F1 BC2F1 BC2F2 BC2F2 BC2F2

Double cross F1

NIL for Lr19 NIL for Lr24 NIL for Yr15

BC3 BC3 BC3

× × ×

Indi

an A

gric

ultu

ral R

esea

rch

Inst

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Indi

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rch

Inst

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Evaluation of BC2F3 at all four locations for drought and heat

Intercross NILs: HD 2733 lines(DHT) X HD2733 (RR) MAS in F2-F4 (Foreground + carrier chromosome

background) Sometime in 2016/17

Reconstitution of Leading varieties introgressed with drought and heat

tolerance + Rust resistance

A joint output from GCP and DBT

This was a presentation on behalf of our GCP-Wheat Challenge Initiative Network on abiotic stress tolerance breeding GP Singh, Neelu Jain, T Ramya, Rajbir, PK Singh, C Pandey, SC Mishra, PC Mishra, NK Singh, TR Sharma, Praveen Chhuneja, VS Sohu, GS Mavi, KV Prabhu + 16 Research Staff*

Delhi Team X 4 = India GCP Team G

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Thank you

GCP and “Special” Thanks to

Prof. R. Trethowan Dr. Xavier Delanay

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