Molecular breeding & genetic engineering for drought tolerance ...

Molecular breeding & genetic engineering for drought tolerance What scope ? Vincent Vadez ICRISAT

ICRISAT in short International Crops Research Institute for the Semi-Arid Tropics

5 mandate crops: Chickpea, pigeonpea, groundnut Pearl millet, sorghum 1.3 Billions people in drought-prone areas (India/Africa) Drought is a major research focus for ICRISAT

Drought research at ICRISAT

Today’s presentation

MAS (pearl millet, sorghum) - PNHI and staygreen QTL identification (chickpea) -Roots QTL identification + Transgenics (groundnut) - Transpiration Efficiency (TE)

MAS for pearl millet Pearl millet line-source irrigation experiment, summer drought nursery, ICRISAT-Patancheru

MAS for pearl millet

LG 2

“H 77 alle le” S tress perform ance

A major terminal drought tolerance QTL on LG2

Panicle no. 2 B iom ass yield 2 S tover yield 2

M 322

M 321 M 592 M 443 M 356

D rought tolerance

S tress perform ance

M 394

M 214

“PR LT allele”

Grain yield 3 10 0-grain m ass 3 H arvest index 3 Panicle grain no. 1 Leaf rolling 1

per se

Grain yield 2 1 00-grain m ass 3 H arvest index 3 B iom ass yield 1

M 738

Grain yield (g m-2)

40 cM

450 430

H 77/833-2 PRLT 2/89-33

410 390 370

ICMR 01029

350 330 310 290 270 250 100%

92%

83%

75%

66%

58%

49%

41%

32%

Percentage of non-stress irrigation


Near-isogenic pearl millet hybrids differing in terminal drought tolerance 843A x ICMR 01029

843A x H77 (=HHB 67) : Smaller percentage of florets setting grains : Poorer grain filling


Moisture at 90-105 cm

Neutron probe observations in the field Grain filling 35 H77/833-2

30

PRLT2/89-3 25

ICMR 01029

20

Dates of sampling

Tolerant material deplete more moisture at deeper layers

MAS for sorghum Staygreen

Senescent

% Green leaf area

ISIAP Dorado IDSG 02034 IDSG 02368 120 100 80 60 40 20 0

Maintenance of a green leaf area Under terminal drought

1 2 3 4 5 6 7 8 9 Weeks from flowering

MAS for sorghum Evaluation in the field under terminal drought 1.000

Relative 100 Grain Mass

0.950 0.900 0.850 0.800 0.750 0.700

R2 = 0.72

0.650 0.600 0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

Relative %GLA

Roots – staygreen contribute to better grain filling

MAS for sorghum ISIAP Range in IL ILs > ILs < Dorado Dorado Dorado Harvest index (%)

40.6

28.3 − 44.4

2

11

Grain yield (g plant-1)

54.2

34.5 − 57.4

0

13

Stover yield (g plant-1)

65.8

41.4 − 77.0

1

5

NIRS-predicted NDM and digestibility values of stover Entry name

NDM, % of R 16 mean

Digestibility, % of R 16 mean

RSG 03061

147%

108%

RSG 03123

137%

118%

Need to advance more generations of backcrosses

MAS for sorghum 6 QTLs related to the stay green character

Staygreen consensus QTL of B35 LgA

LgB

LgC

LgD

LgE

LgF

LgG

LgH

LgI

LgJ

Stg1 Stg4

Stg2

Stg2 LgC

Linkage drag (dwarfing genes Grain setting and quality)

Stg3 StgA

Xtxp59 Xtxp231

StgB Xtxp120

Z locus (mesocarp

R locus (pericarp pigmentation)

Xtxp31

thickness)

Need to further saturate the staygreen QTL genome portion

MAS for sorghum

R16 Dorado

90 -1 20 cm 15 018 0 cm 21 024 0 cm 27 030 0 cm

B35

cm

30 25 20 15 10 5 0

30 -6 0

% of root in shallow layer

Evaluation in large 2.0m PVC cylinders under drought

Roots are involved in staygreen QTL(s)

E36-1

Staygreen

Before

Chickpea - Roots

Chickpea - Roots Screening of the minicore collection for root depth

160

Depth (cm)

140 120 100 80 60 40 20 0

Screening procedure easier (1.2 m PVC tubes)

Seed yield

Chickpea - Roots

r = 0.77 RLD at 60cm depth

A good relation between deeper rooting and seed yield An essential question to answer: Roots for what environment??

Chickpea - Roots ICC283

Annigeri ICC4958

ICC8261

Root Depth(m )

1.10

1.00

0.90

0.80

0.70 234 Genotypes tested

One QTL from “old” population Annigeri x ICC4958 New RIL pops from more varied parents About 350 SSR markers available: Need more!!

Groundnut - Transpiration efficiency

Two approaches:

RILs

DREB1A transgenics

Groundnut - Transpiration efficiency Phenotyping of RIL for transpiration efficiency (TE) in groundnut RILs


Frequency of RILs (number)

RILs 50 ICGV 86031

40 30 20

TAG 24

10 0 2.65 2.75 2.85 2.95 3.05 3.15 3.25 3.35 3.45 3.55 Transpiration efficiency (g kg-1)

Good segregation between RILs

Polymorphic markers

: 35 TI A A A B B B

Segregation data on ABI (9 markers)

T I

T I

13E09

: 463

2012

Already tested

2C11

RILs Available SSR markers : 900

18E7


T I

T= TAG24, I= ICGV 86031

A clear need to develop more markers Develop synthetic groundnut from wild ancestors

Groundnut - Transpiration efficiency DREB1A

Testing Transgenics

P2 Glasshouses

Groundnut - Transpiration efficiency DREB constructs used for Agrobacterium-mediated transformation

DREB1A

Groundnut Variety Used: JL 24 • Total no. of transgenic events: 30 • Generation: T3 to T5

Groundnut - Transpiration efficiency Groundnut Dry-Down set-up DREB1A

Groundnut - Transpiration efficiency DREB1A Wild type JL24

RD19 RD12 RD20 RD2 RD11

Dendrogram based on soil moisture value where Tr declines and number of days to deplete soil water

Groundnut - Transpiration efficiency DREB1A TE under well watered and drought stress in 5 DREB1A events and JL24

JL 24 RD 19 RD 12 RD 20 RD 2 RD 11

TE - WW

TE - DS

2.05 4.31 5.13 3.19 4.09 4.96

4.29 4.99 4.63 4.52 6.12 5.59

Most DREB1A have larger TE than JL24 under WW Two events with higher TE under both water regimes


TE (g DW/kg water)

DREB1ARange of values in TE in a RIL population and in DREB1A groundnut 7 6 5 4 3 2 1 0

Min Max

RIL

DREB Well Watered

DREB drought

Larger range of variation for TE in DREB1A

Conclusion MAS (pearl millet and sorghum) Need to saturate QTL areas to increase the efficiency Pyramid with other QTLs Get rid of linkge drag Root-Chickpea: More markers, QTL identification, Start marker-assisted breeding TE-Groundnut: More markers Re-synthesize the cultigen from its ancestors Transgenic approach New traits

Thank you