GRM 2013: Molecular Breeding and Selection Strategies to Combine and Validate QTLs for Improving WUE and Heat Tolerance of Wheat in India -- KV Prabhu

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

Source: IWMI
Water Stress Indicator Map

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

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

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

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

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

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*
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).

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

Drought tolerance from Parent B
HI 1500
Yield traits from Parent
DBW43
z
1A 1B 2B 2D 3A 3B 3D1D 2A
Best possible individual by modeling
6A 6B 7A 7D7B
5A

FAMILY. 1
FAMILY. 4FAMILY. 3
FAMILY 2
x x
2nd Recombination
x
MARS for Accumulating Favourable Genes
F1F1

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)

Breeding CrossGCPWheatCITeam,India

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
F1F1 F1
F2
F2
F2F2
.1432Fam.1 54 7…6
Selection of
homozygous
plant
BDW 43(P2)
Double cross Double cross

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)

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

LOCATIONS
DELHI
LUDHIANA
POWARKHEDA
PUNE
TRAITS
CANOPY TEMPERATURE
CHLOROPHYLL CONTENT
YIELD
BIOMASS
HARVEST INDEX

**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 43GCPWheatCITeam,India

HI 1500 X DBW 43

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 43

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 CONDITIONSGCPWheatCITeam,India

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

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

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

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 (+/+)

GCPWheatCITeam,India 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 populationsGCPWheatCITeam,India

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

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

Xbarc 186 linked Canopy Temperature by olivares el al 2008
Segregation pattern in bulked families
HI 1500 203 bp
DBW 43 211 bp

HI 1500 DBW 43
Barc 151 Segregation pattern in
Families
F1 type
HI 1500 217 bp
DBW 43 231 bp

Segregation pattern Xbarc 68 linked to Chlorophyll content
DBW 43 145 bp
HI 1500 137 bp

WMC 487 linked to 1000 grain weight by son et al 2008
A1 = HI1500 216
A2 =DBW 43 216 and 222bp

Cfd 73 segregation pattern in F4 families
A1 = HI1500 264bp
A2 =DBW 43 256bp

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

A1 = HI1500 264bp
A2 =DBW 43 256bp
Identification of new QTLs (Cfd 73) in F4
families

A1 = HI1500
A2 =DBW 43
Identification of new QTLs (Xbarc 327) in F4
families

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

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

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

HD2733 X C306
F1 X HD 2733
Kadam et al Funct Integr Genomics (2012)
HD2733 C 306
HD2733 C 306
Kadam et al Funct Integr Genomics (2012)
xbarc20
gwm 368

Foreground selection :QTL for Stay green under drought on 7D
HD 2733 (Recurrent parent) X HI 1500 (Donor parent )
F1 X HD 2733
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

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

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 F1HD2733 HD2733 HD2733× × ×
BC1 BC1 BC1HD2733 HD2733 HD2733× × ×
BC2 BC2BC2HD2733 HD2733 HD2733
BC2F1 BC2F1
BC2F2 BC2F2
BC2F2
Double cross F1
NIL for Lr19
NIL for Lr24 NIL for Yr15
BC3 BC3
BC3
×××
IndianAgriculturalResearchInstitute

IndianAgriculturalResearchInstitute
 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 TeamGCPWheatCITeam,India

Thank you
GCP
and “Special” Thanks to
Prof. R. Trethowan
Dr. Xavier Delanay

GRM 2013: Molecular Breeding and Selection Strategies to Combine and Validate QTLs for Improving WUE and Heat Tolerance of Wheat in India -- KV Prabhu

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GRM 2013: Molecular Breeding and Selection Strategies to Combine and Validate QTLs for Improving WUE and Heat Tolerance of Wheat in India -- KV Prabhu