This document reports on the progress of a project with the objective of improving groundnut productivity in marginal environments in Sub-Saharan Africa. It summarizes the results from Year 2, including confirming drought and disease resistance in new genotypes, identifying two new sources of disease resistance, developing pre-breeding material incorporating wild alleles for drought adaptation, and developing new AB-QTL populations for drought and disease resistance. It also describes ongoing work to map drought tolerance traits, develop SNP markers, map disease resistance QTLs, and introgress disease resistance into adapted varieties through backcrossing.

1. 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

2. Confirm disease/drought resistance/tolerance sources –
Activity 1
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.

3. 60 lines for drought New sources of resis-
Confirmed tolerant Confirmed sensitive tance to disease
ICG 12879 47-10
Rust: ICGV 02194, ICG 11426,
ICGV 02189 ICG 14482
ICGV 01276, ICGV 02286, and
55-437 ICG 2772
ICG 02446
ICG 3140 ICG 5663
ICG 4729 JL24
Rosette: ICG 14705, ICG 13099,
ICG 3584 ICG 1834
ICG 9449, and ICG 15405
ICGV 02038 ICG 12625
ICGV 02266 ICG 8106
ELS in ESA; ICG 6022, ICG
ICGV 96466 ICGV 99001
405, ICG 14466, ICG 6057, ICG
ICGV 97183 Trial on-going in 9449 and ICG 12509
ICG 4750 Senegal / India /
ICG 11088 Niger
ELS in WCA: ICG 6703; ICG
Repeat in Niger /
Senegal 10036, ICG 10384 ICG 9449, ICG
12509, and ICG 11219
On-going PVS trials in Mali

4. Profile of water use from flowering to maturity
Sensitive
Tolerant
Arrows indicates Re-watering
Tolerant lines develop a smaller canopy
Work on going to assess variation / test with modeling (obj 5)
Ratnakumar & Vadez 2011 - FPB

5. Relationship between TE and yield
16
14
R² = 0.65 Post Rainy
Pod Yield - WS
12
10 season
8
6
4
2
0
0.00 0.50 1.00 1.50 2.00 2.50 3.00
-2
Transpiration Efficiency
12
R² = 0.03 Rainy
Pod yield - WS
10
8
season
6
4
2
0
0.00 0.50 1.00 1.50 2.00 2.50 3.00
Transpiration Efficiency
TE effect depends on season

6. Marker assisted AB-QTL population
development
A. ipaensis (BB) x A. duranensis (AA)
F1 (AB) sterile
colchicine
Fleur11(AABB) x AiAd (AABB) fertile
F1
Genetic map
AB-QTL (BC2F3, BC3F2)
BC4F1
CSSL (BC4F3)

7. 


–
–
–
–
–
–
BC3F2)
Foncéka et al. 2012
Pod width
Seed size
Pod morphology
Days to flowering
Plant architecture
Seed morphology
Yield components
AB-QTL Analysis
27 traits recorded:
Two water regimes
142 families (BC2F3 &
a01
a06
Stress tolerance indices
Pn_ww
Pw_ww
b06
Shw_ww
GH_ww
Activity 1 – milestone 3
Hw_wl
b01
Pb_wl
Pb_ww
Pmat_wl
Pwi_wl
a02
Pwi_ww HI_wl
Ss_ww HI_ww
STI-Tb Hw_wl
Swi_wl Hw_ww
Tb_wl Pb_wl
Pb_ww
Pod weight
Pc_ww
Pod number
a07
STI-Hw
GH_ww
Hsw_wl
b02
Hsw_ww
Pb_wl Hpw_wl
Pb_ww Hpw_ww
Pc_ww Hsw_wl
Pc_ww Hsw_ww
PH_ww Pb_wl
Pl_ww Pwi_wl
Pwi_wl Pwi_ww
Pwi_ww Ss_wl
Sl_wl Ss_ww
Sl_ww STI-Hpw
Ss_wl STI-Hsw
Ss_ww Swi_wl
STI-Hsw Swi_ww
STI-Pn
STI-Sn
Swi_wl
Swi_wl
a03
Swi_ww GH_ww
Swi_ww
b03
b07
Pmat_ww
DFL
a04
a08
Pb_ww PH_ww
Pc_ww
Pl_wl
b04
Pwi_ww GH_ww
Sl_ww PH_ww
Pod length
involved in drought adaptation
b08
Seed length
a05
PH_ww
Hw_wl
Pn_ww
Sn_ww
a09
Pb_ww STI-Hw
Pc_ww STI-Pn
Pl_wl STI-Pw
Sl_ww STI-Sw
STI-Tb
Chromosome segments from wild donor
Tb_wl
b09
Tb_ww
b05
indices
a10
GH_ww
Pc_ww
Hpw_wl
Pwi_wl
Stress tol.
Pod width
Hpw_ww
Pwi_ww
Pwi_wl
Pod number
Pwi_ww
b11
Seed number
STI-Hpw
b10
Pb_ww
GH_ww Sw_ww
Pc_ww
PH_ww Swi_wl
Pmat_wl
Swi_ww

8. 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

9. Chromosome segments from wild donor
involved in disease resistance
Activity 1 – milestone 2
CSSL Population AiAd x Fleur11: distribution
• Seeds multiplication /distribution:
– Icrisat Niger
– Icrisat Malawi
– Icrisat India
– Embrapa (ongoing)
• Ongoing multiplication in Senegal / India

10. Development of new AB-QTL populations
Activity 1 – milestone 5
• Amphidiploids from ICRISAT
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
• 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

11. Activity 2 Develop SNP markers for cultivated groundnut
Milestone 8 One suitable method to identify SNPs.
Genome-wide resource of SNPs useable towards a broadly applicable
Milestone 9
SNP, from an exploration of at least 8+8 contrasting genotypes.

12. 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

13. Activity 3 Map disease resistance QTLs
Anchor these QTL to the physical map
Milestone 10 4 populations phenotyped (1 per disease) for disease resistance
QTLs for resistance to major groundnut diseases - QTL flanking
Milestone 11
markers deposited in MBP database.
25 additional SSR markers in strategic regions of the groundnut
Milestone 12
genome containing disease resistance QTLs

14. Consensus map with QTLs for
LLS and rust resistance
Common QTLs
for LLS and
rust
Major QTL Major QTL
for LLS for rust
Molecular Breed 2012, DOI 10.1007/s11032-011-9661-z.

15. Stable QTLs identified for
LLS and rust resistance
Major QTLs detected in two or more than two out of 6 environments
QTLs Linkage Marker interval No. of R² (%)
group environments
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
Molecular Breed 2012, DOI 10.1007/s11032-011-9661-z.

16. INTROGRESSION OF DISEASE RESISTANCES BY BACKCROSSING
Starting point
A. hypogaea IAC-Runner-886
susceptible agronomically adapted
Synthetic amphidiploid
resistant unadapted phenotype
Runner-886 Amphidiploid
end of season LS damage pod types more resistant
(not as resistant as new synthetics)

17. We can see introgression of resistance in BC1F2
A. hypogaea
cv IAC Runner-886
BC-111

18. Field evaluations of BC1F2s
Resistance
%DLA
10.00
8.00
6.00 Productivity x resistance
4.00
2.00 90.00
0.00 80.00
AixAd
BC-6
BC-92
BC-174
BC-16
BC-15
BC-70
BC-13
BC-37
BC-33
BC-146
BC-125
BC-135
BC-203
BC-145
BC-138
BC-122
BC-152
BC-170
70.00
60.00
50.00
40.00 Productivity (g)
Productivity 30.00 %DLA *10
Productivity 20.00
150.00 10.00
100.00 0.00
BC-6
BC-30
BC-5
BC-3
BC-36
BC-33
BC-77
BC-15
BC-13
BC-157
BC-136
BC-138
BC-203
BC-156
BC-170
BC-111
BC-145
AixAd
50.00
0.00
AixAd
BC-6
BC-195
BC-85
BC-36
BC-16
BC-15
BC-136
BC-150
BC-196
BC-170
BC-135
BC-146
Resistance is not inversely related to productivity – low linkage drag

19. 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 Runner-886 x AiAd
ICRISAT AABB
Diploid AA and BB maps
of similar density
also produced

20. Identification of highly
informative SSR markers
S. PIC value Genomic SSRs Genic SSRs Total markers
No. range 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
• Availability of sequence information for 946 novel polymorphic SSR markers
• Identification of 199 highly informative SSRs with PIC >0.50

21. Parental screening of parental
genotypes for disease resistance
Parental screening for
Malawi : Six populations
Niamey : One population
Populations Markers Poly. Disease Locations
screened markers resistance
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

22. Rust
Resistance Adapted No. of Progenies Locations
Source parent
ICGV-SM JL 24 295 Malawi
94114
ICGV-SM ICGV 93437 188 Malawi and
95342 Tanzania
ELS
Resistance Source Adapted parent No. of Progenies Location
ICGV-SM 95714 ICGV 93437 139 Malawi
ICGV-SM 95714 Robut-33-1 187 Malawi
Rosette
Resistance Source Adapted parent No. of Progenies F
Generation
ICGV-SM 90704 CG 7 242 F6
ICGV-SM 90704 Chalimbana 320 F6

23. 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

24. Use MABC to introgress disease resistance QTL
Activity 4
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

25. Five genetic maps
maps for 4x groundnut
Details of different TAG 24 ICGS 76 ICGS 44 TAG 24 TG 26
linkage maps x x x x x
ICGV 86031 CSMG 84-1 ICGS 76 GPBD 4 GPBD 4
(RIL-1) (RIL-2) (RIL-3) (RIL-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 9.54 11.88 15.47 10.23 9.9
(cM)
TAG 2009, 118:729-739; TAG 2011 122:1119-1132;
TAG 2010, 121:971-984; Mol Breeding 2012, DOI 10.1007/s11032-011-9661-z;
Field Crops Res 2011, 122:49-59; Mol Breeding 2012, DOI 10.1007/s11032-011-9660-0.

26. Eleven 4X- genetic maps
used for consensus map
Maps Linkage Polymorphic Mapped Map Map References
groups loci loci length density
(cM) (cM)
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
PLoS ONE 2012, submitted
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 -
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)

27. High density reference
consensus genetic map
LG_AhXX
LG_AhXV
PLoS ONE 2012, submitted
LG_AhXVI
LG_AhXVII
LG_AhXIX
LG_AhIX LG_AhXIV
LG_AhII LG_AhXVIII
LG_AhVI
LG_AhXI
LG_AhI
LG_AhVIII
LG_AhVII
LG_AhXII
LG_AhXIII
LG_AhIII
LG_AhIV LG_AhX
LG_AhV Marker loci mapped 897
Total map distance (cM) 3863.6
Map density (cM) 4.42

28. Consensus QTL map for
drought tolerance traits
Mol Breed 2012, DOI 10.1007/s11032-011-9660-0.
Cluster 4
Cluster 2
Cluster 5
Cluster 3
Cluster 1 Cluster 9
Cluster 10
Cluster 7
Cluster 8
Cluster 6
Cluster 13
Cluster 14
Cluster 11
Cluster 12
Cluster 15 Cluster 16

29. How yield and traits QTL co-map
in cultivated groundnut ?
QTL cluster for:
Yield
Transpiration
Growth

30. How yield and traits QTL co-map
in cultivated groundnut ?
QTL cluster for:
Leaf expansion
Leaf area
Leaf conductance

31. Development of new crosses for drought
Number of surviving F1 plants
during 2011-12 postrainy
S.No Female Parent Male Parent 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
22 crosses made / 12 currently advanced. F2 seeds harvested.

32. 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

33. Marker-assisted selection
(MAS) for rust resistance
ICGV 91114,
Post Rainy 2008-09
JL 24, TAG 24 × GPBD 4
Recurrent Parent (RP) Donor Parent (DP)
Rainy 2009 RP × F1
Backcrossing
Post Rainy 2009-10 RP × BC1F1
Completed
FG selection-heterozygote
Backcrossing
Rainy 2010 RP × BC2F1 FG selection-heterozygote
Backcrossing Selfing
Post Rainy FG selection- BC3F1 BC2F2 FG selection-homozygote
2010-11 heterozygote
Selfing Selfing
FG selection-
Rainy 2011 BC3F2 BC2F3 Screened for rust
homozygote
Selection based on disease reaction, agronomic and yield performance

34. Screening of introgression
lines for rust resistance

35. 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

36. Strengthen capacity of NARS partners
Activity 5
Milestone At least 2 NARS partners empowered to breed groundnut varieties with multiple
23 attributes.
Milestone 3 scientists and 3 technicians trained in drought phenotyping and in the
24 logistics of fast advancement of breeding populations.
Milestone 1 scientist and 1 technician from Malawian and Malian national programmes
25 trained in DNA extraction and use of simple markers.

37. 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

38. 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.

39. Data provided to Bioinformatics unit
•Most data delivered or close to be

40. 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

41. Thank you