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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
b11
Pn
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pw
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pw
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L
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Pb
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Pc_
ww
Pm
at_
wl
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
-92
BC
-13
5
BC
-20
3
Aix
Ad
BC
-14
5
BC
-17
4
BC
-6
BC
-13
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