Objective 1: Improve groundnut

productivity for marginal environments

from Sub-Saharan Africa

Report Year 2

EMBRAPA- UGA – ICRISAT – ISRA-Senegal – Malawi Nat.

Prog. – Tanzania Nat. Prog

Addis Ababa 8 May 2012

Activity 1 Confirm disease/drought resistance/tolerance sources –

Assess new diversity Milestone 1 Confirmation of disease resistance and drought tolerance of at least 10 new

genotypes

Milestone 2 Two new sources of disease resistance - new genetic material for breeding

(either CSSL, AB line, synthetics)

Milestone 3 Chromosome segments from wild donor for disease and/or drought (CSSL

Ad xAi – Fleur11)

Milestone 4 Pre-breeding material incorporating wild favourable alleles for future MARS.

Milestone 5 8 available synthetics tested for disease resistance; 1–2 AB-QTL populations

with one synthetic amphidiploids.

Milestone 6 Database of phenotyping data for drought /

knowledge of key traits for drought adaptation.

Milestone 7 A sub-set of 10 tolerant lines for TLII breeding / PVS trials.

60 lines for drought

ICG 12879

ICGV 02189

55-437

ICG 3140

ICG 4729

ICG 3584

ICGV 02038

ICGV 02266

ICGV 96466

ICGV 97183

ICG 4750

ICG 11088

47-10

ICG 14482

ICG 2772

ICG 5663

JL24

ICG 1834

ICG 12625

ICG 8106

ICGV 99001

Trial on-going in

Senegal / India /

Niger

Repeat in Niger /

Senegal

Confirmed tolerant

Rust: ICGV 02194, ICG 11426,

ICGV 01276, ICGV 02286, and

ICG 02446

Rosette: ICG 14705, ICG 13099,

ICG 9449, and ICG 15405

ELS in ESA; ICG 6022, ICG

405, ICG 14466, ICG 6057, ICG

9449 and ICG 12509

ELS in WCA: ICG 6703; ICG

10036, ICG 10384 ICG 9449, ICG

12509, and ICG 11219

Confirmed sensitive

New sources of resis-

tance to disease

On-going PVS trials in Mali

Arrows indicates Re-watering

Profile of water use from flowering to maturity

Sensitive

Tolerant

Tolerant lines develop a smaller canopy

Work on going to assess variation / test with modeling (obj 5)

Ratnakumar & Vadez 2011 - FPB

Rainy

season

R² = 0.03

0

2

4

6

8

10

12

0.00 0.50 1.00 1.50 2.00 2.50 3.00

Po

d y

ield

- W

S

Transpiration Efficiency

R² = 0.65

-2

0

2

4

6

8

10

12

14

16

0.00 0.50 1.00 1.50 2.00 2.50 3.00

Po

d Y

ield

- W

S

Transpiration Efficiency

Post Rainy

season

Relationship between TE and yield

TE effect depends on season

Fleur11(AABB)

AB-QTL (BC2F3, BC3F2)

A. ipaensis (BB) x A. duranensis (AA)

F1 (AB) sterile

colchicine

AiAd (AABB) fertile x

F1

BC4F1

CSSL (BC4F3)

Genetic map

Marker assisted AB-QTL population

development

AB-QTL Analysis

142 families (BC2F3 &

BC3F2)

Two water regimes

27 traits recorded: – Days to flowering

– Plant architecture

– Pod morphology

– Seed morphology

– Yield components

– Stress tolerance indices

Foncéka et al. 2012

a01 b01 a02 b02 a03 b03 a04 b04 a05 b05

a06 b06 a07 b07 a08 b08 a09 b09 a10 b10

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Pod number

Pod weight

Pod number

Seed number

Stress tol.

indices

Pod width

Seed size

Pod length

Seed length Pod width

Chromosome segments from wild donor involved in drought adaptation

Activity 1 – milestone 3

Chromosome segments from wild donor involved in drought adaptation

Activity 1 – milestone 3

CSSL Population AiAd x Fleur11: phenotyping

– Two years characterization in

Senegal

(2011 & 2012 ongoing)

• Yield and yield components

• Morphology and phenology

• Two water regimes / 6 rep/trt

Mapping of new QTLs and validation of

previously identified QTLs

CSSL Population AiAd x Fleur11: distribution

• Seeds multiplication /distribution:

– Icrisat Niger

– Icrisat Malawi

– Icrisat India

– Embrapa (ongoing)

• Ongoing multiplication in Senegal / India

Chromosome segments from wild donor involved in disease resistance

Activity 1 – milestone 2

Development of new AB-QTL populations Activity 1 – milestone 5

• Amphidiploids from ICRISAT

• Succesful germination: • ISATGR 278-18

• ISATGR 52B

• ISATGR 206

• Successful hybridization with

Fleur11 • ISATGR 278-18: 22 BC1 plants produced.

BC2 development ongoing.

• ISATGR 206: Hybridation ongoing

ICRISAT identity number Cross

ISATGR 1212 A. duranensis x A. ipaensis AAxBB

ISATGR 278-18 A. duranensis x A. batizocoi AAxBB

ISATGR 10B A. magna x A. valida BBxBB

ISATGR 9A A. batizocoi x A. cardenasii BBxAA

ISATGR 5B A. magna x A. batizocoi BBxBB

ISATGR 265-5A A. kempff mercadoi x A. hoehnei AAxAA

ISATGR 90B A. kempff mercadoi x A. stenosperma AAxAA

ISATGR 34B A. batizocoi x A. duranensis BBxAA

ISATGR 206 A. duranensis x A. valida AAxBB

ISATGR 52B A. valida x A. duranensis BBxAA

Activity 2 Develop SNP markers for cultivated groundnut

Milestone 8 One suitable method to identify SNPs.

Milestone 9 Genome-wide resource of SNPs useable towards a broadly applicable

SNP, from an exploration of at least 8+8 contrasting genotypes.

Simple and flexible genotyping-by-sequencing

requiring minimal infrastructure

1. Digest total genomic DNA of

individual samples with pre-selected

restriction enzyme

2. Ligate to sample-specific adaptors

that include bar codes

3. Pool, then size-select all at once

4. Sequence pool -- map reads, call

SNPs, infer genotypes

Steps 1-3 require minimal infrastructure (gel

electrophoresis, waterbath, UV illuminator,

refrigeration)

Flexible to low polymorphism rates (via sequencing

depth)

Presently piloting in diploid and polyploid genomes

with and without reference sequences

Activity 3 Map disease resistance QTLs

Anchor these QTL to the physical map

Milestone 10 4 populations phenotyped (1 per disease) for disease resistance

Milestone 11 QTLs for resistance to major groundnut diseases - QTL flanking

markers deposited in MBP database.

Milestone 12 25 additional SSR markers in strategic regions of the groundnut

genome containing disease resistance QTLs

Major QTL for LLS

Major QTL for rust

Common QTLs for LLS and rust

Consensus map with QTLs for LLS and rust resistance

Molecular Breed 2012, DOI 10.1007/s11032-011-9661-z.

Stable QTLs identified for LLS and rust resistance

Major QTLs detected in two or more than two out of 6 environments

Molecular Breed 2012, DOI 10.1007/s11032-011-9661-z.

QTLs Linkage

group

Marker interval No. of

environments

R² (%)

Late leaf spot (LLS) resistance

QTLR4-LLS01 AhXII GM1573-pPGPseq8D09 6 10.27-62.34

QTLR4-LLS02 AhXV GM2009-GM1536 2 12.49-67.98

QTLR4-LLS03 AhXV GM1536-GM2301/GM2079 2 10.83-17.37

QTLR4-LLS05 AhV IPAHM356-GM1577 4 10.81-15.34

QTLR4-LLS09 AhXVIII TC11F12-TC2G05 3 3.39-8.5

QTLR5-LLS01 AhXV GM2009-GM1536 2 7.58-49.64

QTLR5-LLS02 AhVIII GM2504-GM2746 2 9.79-22.46

Rust resistance

QTLR4-Rust01 AhXV GM2009-GM1536 6 10.68-82.27

QTLR4-Rust02 AhXV GM1536-GM2301/GM2079 6 12.43-62.35

QTLR4-Rust03 AhXV IPAHM103-GM1954 6 23.12-82.96

QTLR4-Rust05 AhII TC1B02-pPGSseq18E07 2 2.54-3.29

QTLR5-Rust01 AhXV GM2009-GM1536 7 17.57-66.05

QTLR5-Rust02 AhXV IPAHM103-GM1954 6 17.42-78.96

QTLR5-Rust03 AhV RN16F05-GM1988 2 15.07-29.02

Runner-886 Amphidiploid

Starting point

A. hypogaea IAC-Runner-886

susceptible agronomically adapted

Synthetic amphidiploid

resistant unadapted phenotype

end of season LS damage more resistant (not as resistant as new synthetics)

pod types

INTROGRESSION OF DISEASE RESISTANCES BY BACKCROSSING

We can see introgression of resistance in BC1F2

A. hypogaea cv IAC Runner-886

BC-111

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Aix

Ad

BC

-5

BC

-3

BC

-13

6

BC

-13

8

BC

-20

3

BC

-36

BC

-33

BC

-15

6

BC

-6

BC

-77

BC

-15

7

BC

-15

BC

-30

BC

-13

BC

-17

0

BC

-11

1

BC

-14

5

Productivity (g)

%DLA *10

Productivity x resistance

0.00

2.00

4.00

6.00

8.00

10.00

BC

-14

6

BC

-12

5

BC

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BC

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5

BC

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3

Aix

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5

BC

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4

BC

-6

BC

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8

BC

-16

BC

-12

2

BC

-15

2

BC

-15

BC

-70

BC

-13

BC

-37

BC

-33

BC

-17

0

%DLA

Resistance

0.00

50.00

100.00

150.00

Aix

Ad

BC

-19

5

BC

-13

6

BC

-85

BC

-36

BC

-15

0

BC

-6

BC

-16

BC

-15

BC

-19

6

BC

-17

0

BC

-13

5

BC

-14

6

Productivity Productivity

Field evaluations of BC1F2s

Resistance is not inversely related to productivity – low linkage drag

Map construction

RIL (F5/F6) map

Pop. development

Phenotyping

EMBRAPA and UnB,

Brazil

Genotyping

Kazusa, Japan

1228 SSR markers

Also contributions from

University of Georgia

ICRISAT

Diploid AA and BB maps

of similar density

also produced

Runner-886 x AiAd

AABB

S. PIC value range

Genomic SSRs Genic SSRs Total markers No. Number (%) Number (%) Number (%) 1 0.10-0.20 51 19.6 284 37.4 335 32.8 2 0.21-0.30 55 21.2 196 25.8 251 24.6 3 0.31-0.40 35 13.5 146 19.2 181 17.7 4 0.41-0.50 29 11.2 62 8.2 91 8.9 5 0.51-0.60 24 9.2 44 5.8 68 6.7 6 0.61-0.70 39 15.0 15 1.9 52 5.3 7 0.71-0.80 21 8.1 11 1.4 32 3.1 8 0.81-0.90 6 2.3 2 0.3 8 0.8 Total markers 260 760 1020

Identification of highly informative SSR markers

• Availability of sequence information for 946 novel polymorphic SSR markers

• Identification of 199 highly informative SSRs with PIC >0.50

Parental screening of parental genotypes for disease resistance

Parental screening for Malawi : Six populations Niamey : One population

Populations Markers screened

Poly. markers

Disease resistance

Locations

ICGV 93437 X ICGV 94114 510 36 Rust resistance Malawi

ICGV 93437 X ICGVSM 95342 510 30 Rust resistance Malawi

CG7 X ICGVSM 90704 510 63 GRD Malawi

CHALIMBANA X ICGVSM 90704 510 9 GRD Malawi

ICGV 93437 X ICGV 95714 510 24 ELS Malawi

ROBUT 33-1 X ICGV 95714 510 31 ELS Malawi

ICGV 86124 X ICG 7878 510 62 ELS Niamey

GRD : Groundnut rosette disease ELS : Early leaf spot

Resistance

Source

Adapted

parent

No. of Progenies Locations

ICGV-SM

94114

JL 24 295 Malawi

ICGV-SM

95342

ICGV 93437 188 Malawi and

Tanzania

Resistance Source Adapted parent No. of Progenies Location

ICGV-SM 95714 ICGV 93437 139 Malawi

ICGV-SM 95714 Robut-33-1 187 Malawi

Resistance Source Adapted parent No. of Progenies F

Generation

ICGV-SM 90704 CG 7 242 F6

ICGV-SM 90704 Chalimbana 320 F6

Rust

ELS

Rosette

DNA extraction and SSR genotyping

of parental genotypes from Mali

• DNA extracted from fresh leaves of 5 parental lines in 5 populations developed for resistance to ELS

– Fleur 11 x ICG 7878 (1polymorhic SSR)

– 55-437 x ICG7878 (6 polymorphic SSRs

– JL24 x ICG 7878 (1 polymorphic SSR)

– 47-10 x ICG 7877 (10 polymorphic SSRs)

– ICG 86124 x ICG 7878 (16 polymorphic SSRs

• To be advanced to F5:6 in 2012 crop season for further phenotyping and genotyping

Activity 4 Use MABC to introgress disease resistance QTL

New breeding population for drought and disease towards MARS Milestone 14 4 populations advanced to RILs - Multi-location disease phenotyping.

Milestone 15 8 MABC materials introgressed with rust and rosette disease resistance QTLs in four

FMPV backgrounds and transferred to TLII breeders in Year 4

Milestone 16 4 breeding products from MABC with enhanced attributes available for testing and

selection by NARS.

Milestone 17 5 advanced backcross breeding materials with disease resistance tested in

PVS trials.

Milestone 18 PVS trials of 10–20 new sources of disease resistance and drought tolerance

(TLII) Milestone 19 At least 4 new crosses (one per disease) developed and QTL identified for each disease

between FMPV lines and new sources of disease resistance.

Milestone 20 At least 4 new crosses developed between advanced BC lines in FMPV background and

new sources of drought tolerance from Activity 4, Phase I.

Milestone 21 Relevance of F4-family phenotyping for drought-related traits tested in at least one

segregating population.

Milestone 22 Polymorphism information content (PIC) of lines involved in crossing available

Details of different

linkage maps

TAG 24

x

ICGV 86031

(RIL-1)

ICGS 76

x

CSMG 84-1

(RIL-2)

ICGS 44

x

ICGS 76

(RIL-3)

TAG 24

x

GPBD 4

(RIL-4)

TG 26

x

GPBD 4

(RIL-5)

Marker loci mapped 191 119 83 188 181

Linkage groups 22 18 16 20 20

Marker loci/LG 2-19 2-14 2-10 2-17 2-15

Avg. marker loci/LG 8 7 5 9 8

Total map distance (cM) 1785 888 2203 1922 1964

Avg. distance/LG (cM) 81.15 59.2 110.1 96.1 85.4

Avg. inter-locus distance

(cM)

9.54 11.88 15.47 10.23 9.9

Five genetic maps maps for 4x groundnut

TAG 2009, 118:729-739; TAG 2010, 121:971-984; Field Crops Res 2011, 122:49-59;

TAG 2011 122:1119-1132; Mol Breeding 2012, DOI 10.1007/s11032-011-9661-z;

Mol Breeding 2012, DOI 10.1007/s11032-011-9660-0.

Maps Linkage groups

Polymorphic loci

Mapped loci

Map length (cM)

Map density (cM)

References

RIL-1 22 211 191 1785.4 9.35 Varshney et al. 2009b; Ravi et al. 2011

RIL-2 20 128 119 2208.2 18.56 Gautami et al. 2012

RIL-3 15 87 82 831.4 10.14 Gautami et al. 2012

RIL-4 20 209 188 1922.4 10.23 Khedikar et al. 2010; Sujay et al. 2012

RIL-5 21 209 181 1963 10.85 Sarvamangala et al. 2011; Sujay et al. 2012

RIL-6 19 146 132 793.1 6.01 Hong et al. 2010

RIL-7 21 124 109 503.1 4.62 Hong et al. 2010

RIL-8 13 64 46 357.4 7.76 Hong et al. 2010

RIL-9 26 261 233 1304.9 5.6 Qin et al. 2012

RIL-10 22 183 173 917.45 5.3 Qin et al. 2012

BC1F1-11 21 339 332 847.4 2.53 Foncéka et al. 2009

Consensus map 20 - 897 3,863.6 4.42 -

Eleven 4X- genetic maps used for consensus map

RIL-1: TAG 24 x ICGV 86031; RIL-2: ICGS 76 x CSMG 84-1; RIL-3: ICGS 44 x ICGS 76; RIL-4: TAG 24 x ICGV 86031; RIL-5: TG 26 x GPBD 4; RIL-6: Yueyou 13 × Zhenzhuhei; RIL-7: Yueyou 13 × Fu 95-5; RIL-8: Yueyou 13 × J11; RIL-8: Tifrunner × GT-C20; RIL-10: SunOleic 97R × NC94022; RIL-11: Fleur11 × AiAd (synthetic amphidiploid)

PLoS

ONE 2

012, su

bmitte

d

LG_AhI

LG_AhII

LG_AhIII

LG_AhIV

LG_AhV

LG_AhVI

LG_AhVII

LG_AhVIII

LG_AhIX

LG_AhX

LG_AhXI

LG_AhXII

LG_AhXIII

LG_AhXIV

LG_AhXV

LG_AhXVI

LG_AhXVII

LG_AhXVIII

LG_AhXIX

LG_AhXX

Marker loci mapped 897

Total map distance (cM) 3863.6

Map density (cM) 4.42

High density reference consensus genetic map

PLoS

ONE 2

012, su

bmitte

d

Consensus QTL map for drought tolerance traits

Cluster 6

Cluster 13

Cluster 11

Cluster 1

Cluster 12

Cluster 2

Cluster 7

Cluster 14

Cluster 16

Cluster 4

Cluster 5

Cluster 8

Cluster 9

Cluster 10

Cluster 3

Cluster 15

Mol B

reed 2

012,

DOI 1

0.1

007/s

11032-011-9660-0.

How yield and traits QTL co-map

in cultivated groundnut ?

QTL cluster for:

Yield

Transpiration

Growth

How yield and traits QTL co-map

in cultivated groundnut ?

QTL cluster for:

Leaf expansion

Leaf area

Leaf conductance

22 crosses made / 12 currently advanced. F2 seeds harvested.

Development of new crosses for drought

S.No Female Parent Male Parent

Number of surviving F1 plants

during 2011-12 postrainy

season 1 ICGS 44 JL 24 41 2 55-437 JL 24 35 3 ICGV 91114 ICG 1834 63 4 ICGV 44 ICG 1834 45 5 ICGV 97182 ICG 1834 65 6 Fluer 11 ICG 1834 32 7 ICGV 01232 ICG 1834 61 8 ICG 434 ICG 1834 65 9 ICGV 91114 ICG 13787 28 10 ICGV 02189 ICG 4598 55 11 55-437 ICG 2772 45

12 55-437 ICGV 99001 65

QTLs/markers for rust

A major QTL for rust in groundnut (PVE upto 82.96%)

Environments:

ER-I Rainy 2004,

ER-II Rainy 2005,

ER-III Post-rainy 2007,

ER-IV Rainy 2007,

ER-V Rainy 2008.

Stages:

LR-I: scored at 70 DAS,

LR-II: scored at 80 DAS,

LR-III: scored at 90 DAS,

LR-IV: scored at 105 DAS,

LR-V: scored at 113 DAS,

LR-VI: scored at 120 DAS,

IP: incubation period,

LP: latent period,

IT: infection type.

TAG 2010, 121:971-984; Molecular Breed 2012, DOI 10.1007/s11032-011-9661-z

Marker-assisted selection (MAS) for rust resistance

Selection based on disease reaction, agronomic and yield performance

Com

plete

d RP

ICGV 91114, JL 24, TAG 24

Recurrent Parent (RP)

BC1F1

×

GPBD 4

Backcrossing

Selfing

Selfing

Backcrossing

Post Rainy 2008-09

Rainy 2010

Post Rainy 2010-11

Rainy 2011

Rainy 2009

Post Rainy 2009-10 FG selection-heterozygote

FG selection-homozygote

FG selection-homozygote FG selection-heterozygote

Screened for rust

RP

RP

Selfing

Backcrossing

FG selection-heterozygote

Donor Parent (DP)

×

×

×

F1

BC2F1

BC2F2

BC3F1

BC2F3

BC3F2

Screening of introgression lines for rust resistance

A review on Arachis genomics published in reputed journal “ Biotechnology Advances” in collaboration with several GCP partners

• This article provides current updates on accelerated development of genomic resources such as development of molecular markers, genetic and physical maps, generation of expressed sequenced tags (ESTs), development of mutant resources, and functional genomics platforms

• These developments leading to identification of QTLs and discovery of genes associated with tolerance/resistance to abiotic and biotic stresses and agronomic traits

• Translation of information developed through use of genomics tools for improvement of disease resistance and oil quality traits in groundnut

Activity 5 Strengthen capacity of NARS partners

Milestone

23

At least 2 NARS partners empowered to breed groundnut varieties with multiple

attributes.

Milestone

24

3 scientists and 3 technicians trained in drought phenotyping and in the

logistics of fast advancement of breeding populations.

Milestone

25

1 scientist and 1 technician from Malawian and Malian national programmes

trained in DNA extraction and use of simple markers.

Activity 5: Capacity building

• Mamary Traore, Msc student under TLII in Mali trained in phenotyping for foliar disease resistance

• 2 PhD students at WACCI (Usman Alhassan- Nigeria and Aboudoulay Adama- Niger) in groundnut breeding supported by TLI to participate in the 2-day training in IB filed book and data management (TLI)

• Groundnut breeders from Burkina Faso (1), Ghana (1), Mali (1), Niger (2), Nigeria (1) and Senegal (1), supported by TLII to attend the IB Field Book and data management training

Activity 6 Management and storage of data

Milestone 26 Marker genotyping data for groundnut reference collection

- At least 4 populations stored in public database and

linked to the MBP.

Milestone 27 Phenotyping data of reference collection over 6 sites (India, Niger,

Mali, Senegal, Tanzania and Malawi) and 6 years;

RIL and MABC lines analysed and data stored in public database -

Data from TLII trials added to this database.

•Most data delivered or close to be

Data provided to Bioinformatics unit

In summary

Major progresses with the genomic tools (maps, markers)

Progress continues on drought adaptation mechanisms

QTLs for disease resistance getting harnessed:

QTL introgression of rust / LLS resistance

BC work with AiAd

Synthetic x cultivated populations

AB-QTL populations

Wild relatives have a big stake there

Thank you