GENOMICS-ESA
Damaris Achieng OdenyScientist – Biotechnology
Rajneesh PaliwalPost-doctoral Research Fellow
Cereal Genomics
With support from Samuel Manthi and Vincent Njung’e
Activities Planned - 2015
Legumes (Odeny)
• Association mapping for drought tolerance in chickpea
• Marker analysis and polymorphism study on parental lines of RIL population for ELS, GRD and drought tolerance in groundnut
Robut 33-1 ICGV-SM 95714
Origin India Malawi
Botanical class Virginia Valencia
Maturity (days) Early (115) Early (90-100)
Yield (Kgha-1) 1,200 – 1,500 1,700 – 2,000
Dormancy Long None
Disease Reaction Susceptible Resistant
Sex in cross Female Male
Table 1: Characteristics of Parental Lines
3
X
F1
F8 - 113 Recombinant Inbred Lines (RILs)
Robut 33-1 ICGV-SM 95714
QTL Mapping for ELS in groundnut
Funds: USAID (I-FINITE) Tembo E.1, Charlie H.3, Njoroge S.M.C3, Okori P, Mweetwa A.M.1, Tembo L.1, Kaimoyo E.1, Monyo E.S.2, Paliwal R.2 and Odeny D.A
4
QTLs Detected - Summary
QTL Category Linkage
group
Position
(cM)
Left Marker Right Marker LOD
score
PVE (%) Additive
Effect
1 Major 2 81 GM1585 GNB284 15.74 37.91 25.65
2 Minor 2 130 GM951 Ah1TC4G02 3.51 7.98 -11.15
3 Minor 9 45 AHGS0798 GNB357 5.52 12.31 12.93
Season of 2014
QTL Category Linkage
group
Position
(cM)
Left Marker Right Marker LOD score PVE (%) Additive
Effect
1 Major 2 81 GM1585 GNB284 22.97 52.4 15.8
2 Minor 2 185 Ah1TC4G02 GM2206 16.3 7.98 -7.9
3 Minor 5 173 pPGPseq2C11 GM2284 5.2 8.1 9.2
4 Minor 8 106 GNB608 GM1996 6.4 13.2 -7.2
Season of 2015_16
Association mapping in chickpea (CRP-funded)
Tesfamichael SM, NVPR Ganga Rao, Githiri SM, Paliwal R and Odeny DA
100 (60 desi & 40 kabuli) diverse chickpea lines
Characterized for agronomic traits (2013 and 2014) at Juja andkabate locations in Kenya
DNA extracted from 95 (57 desi and 28 kabuli) lines andgenerated
Generated >45 Gb of GBS sequencing data from which we identified 923 SNPs markers
After filtering, 358 SNPs used for association studies for 11 agronomic traits
GWAS Results
Table 1: The SNP_192816019_chr8 is showing association with chickpea yield traits 1) Hundred seed weight (HSW),2) Seed yield/hac (SY) and 3) Biomass yield/hac (BM) and also with other 8 agronomical traits Days to Flowering50% (DF), number of leaf let/leaf (NLL), pos length (PL), primary branch (PB), secondary branch (SB), days tomaturity (DM), pod filing period (PFP), pod/plant (PP).
GLM MLM
Traits Marker/Position Chromosome marker_Rsq P.value marker_Rsq P.value
J1-PFP S8_192816019 8 0.28886 7.90E-07 0.28987 3.02E-05
J2-PFP S8_192816019 8 0.24367 1.35E-05 0.2458 2.47E-04
K1-PFP S8_192816019 8 0.16883 3.63E-04 0.16883 7.15E-04
PFP-Mean S8_192816019 8 0.24454 1.02E-05 0.24737 1.49E-04
J1-PP1 S8_192816019 8 0.17177 2.22E-04 0.17585 6.91E-04
K1-PP1 S8_192816019 8 0.14714 8.61E-04 0.14351 0.00131
K2-PP1 S8_192816019 8 0.11045 0.00459 0.11552 0.00886
PP1-Mean S8_192816019 8 0.17459 1.79E-04 0.17467 0.00177
J1-BM S8_192816019 8 0.16088 5.17E-04 0.16249 0.00135
J2-BM S8_192816019 8 0.14631 7.51E-04 0.1746 0.00133
K1-BM S8_192816019 8 0.12306 0.00216 0.14274 0.00277
K2-BM S8_192816019 8 0.14415 9.71E-04 0.14595 0.00204
BM-Mean S8_192816019 8 0.15551 5.41E-04 0.15511 0.00188
J1-SY S8_192816019 8 0.16568 3.56E-04 0.1692 0.00118
J2-SY S8_192816019 8 0.1406 0.00107 0.14832 0.00384
K1-SY S8_192816019 8 0.14402 8.20E-04 0.16231 0.0013
K2-SY S8_192816019 8 0.14058 0.00103 0.13469 0.00271
SY-Mean S8_192816019 8 0.15922 4.30E-04 0.16353 0.00149
J1-HSW S8_192816019 8 0.29624 5.05E-07 0.30251 3.13E-05
J2-HSW S8_192816019 8 0.27925 8.33E-07 0.29942 3.98E-05
K1-HSW S8_192816019 8 0.27061 2.22E-06 0.27087 9.16E-05
K2-HSW S8_192816019 8 0.26001 2.81E-06 0.2649 6.06E-05
HSW-Mean S8_192816019 8 0.299 4.27E-07 0.30693 2.59E-05
GWAS Results
GLM MLM
Traits Marker/Position Chromosome marker_Rsq P.value marker_Rsq P.value
J1-DF S8_192816019 8 0.3069 3.32E-07 0.30718 1.79E-05
J2-DF S8_192816019 8 0.27664 1.89E-06 0.28091 2.87E-05
K1-DF S8_192816019 8 0.22773 2.78E-05 0.2303 1.15E-04
DF-Mean S8_192816019 8 0.28061 1.68E-06 0.28178 2.75E-05
J1-NLLP1 S8_192816019 8 0.13573 0.00265 0.1287 0.00404
K2-NLLP1 S8_192816019 8 0.13223 0.003 0.12731 0.00405
LL1-Mean S8_192816019 8 0.11384 0.00601 0.11035 0.00782
J1-PL S8_192816019 8 0.20815 6.40E-05 0.2233 3.70E-04
J2-PL S8_192816019 8 0.2159 3.82E-05 0.23865 1.49E-04
K1-PL S8_192816019 8 0.16623 4.95E-04 0.1719 0.00252
K2-PL S8_192816019 8 0.21025 5.68E-05 0.2179 2.87E-04
PL-Mean S8_192816019 8 0.2108 5.54E-05 0.22888 2.68E-04
J1-PB S8_192816019 8 0.14694 0.00137 0.12555 0.00327
J2-PB S8_192816019 8 0.14908 9.20E-04 0.15432 0.00395
K1-PB S8_192816019 8 0.17848 2.04E-04 0.17325 0.00125
K2-PB S8_192816019 8 0.14057 0.00141 0.14208 0.00271
PB-Mean S8_192816019 8 0.19609 1.19E-04 0.18871 8.98E-04
J1-SB S8_192816019 8 0.20194 8.41E-05 0.19159 2.11E-04
J2-SB S8_192816019 8 0.13549 0.00174 0.14577 0.00396
K2-SB S8_192816019 8 0.11096 0.00548 0.10353 0.00882
SB-Mean S8_192816019 8 0.20248 9.10E-05 0.19924 4.58E-04
J1-DM S8_192816019 8 0.13969 0.0016 0.14436 0.00286
J2-DM S8_192816019 8 0.17096 2.89E-04 0.17472 6.61E-04
K1-DM S8_192816019 8 0.12228 0.0035 0.1201 0.00404
DM-Mean S8_192816019 8 0.1306 0.00184 0.14441 0.00267
Other Legume activities
• QTL mapping for resistance to rust disease in groundnut– Funded by USAID-CGIAR linkages (Tuskegee University-
ICRISAT/AWARD)
– Chapwa Kasoma of Zambia
• Assessment of genetic variation for pod borer resistance among elite pigeonpea accessions in Kenya – CRP-GL in collaboration with University of Eldoret
– Juliana Cheboi
• Characterization of selected rhizobia isolates from Ethiopia– In collaboration with Chris Ojiewo and UC-Davis
– Samuel Manthi
Activities Planned - 2015Cereals
Finger millet
• Molecular characterization of available germplasm and development of genomic tools for genetic characterization and varietal development of finger millet (Odeny)
• Development of MAS for Striga resistance in finger millet (Odeny)
• Development of early generation mapping population for snapping trait in fingermillet (Paliwal)
• Characterization for different agronomic traits in ESA fingermillet germplasm collection (Paliwal)
• Genetic and nutritional profiling of fingermillet germplasm collection in ESA (Paliwal)
Sorghum
• Molecular characterization for important agronomic traits in ESA sorghum germplasm (Paliwal)
• Characterization of sorghum/maize germplasm for semio-chemical response to stem borer infestation (Paliwal)
• Genetic characterization for disease resistance in sorghum (Paliwal)
• Genotyping 105 sorghum germplasm across Kenya, Ethiopia and Sudan for their response to Striga(Odeny)
Molecular characterization of available germplasm and development of genomic tools for genetic characterization and varietal development of finger millet
• Funded by Bio-Innovate/ABCF/BecA-ILRI
• Generated NGS data from genotypes KNE796 and KNE755
• Developed SNP markers and converted a representative set into KASPAR/Sequenom assays
• Tested developed markers across cultivated and wild germplasm
RESULTS Davis Gimode
Wild accessions Cultivated accessions
E.
kig
ezi
ensis
SP1 SP2
(E. coracana subsp. africana)
E. coracana subsp. coracana
E. coracana subsp. coracana
Cultivated accessions
Whole Genome Sequencing of Finger Millet
• In partnership with…………….Katrien Devos (UGA, USA), F Stomeo (BecA-ILRI), S de Villiers (PU, Kenya), Allen van Deynze (UC Davis), K Tesfaye (UAA, Ethiopia), M Dida (UM, Kenya), E Mneney (MARI, Tanzania), E Bongkam-Rudloff (SLU, Sweden), Mark Wamalwa (BecA-ILRI), Yao Nasser (BecA-ILRI), Song Bo (BGI, China), Davis Gimode, Erick Owuor
• Funded by Bio-Innovate/African Orphan Crops Consortium (AOCC)/ABCF
Activities
• Generated the whole genome sequence of KNE796
• Undertook whole genome de novo sequence assembly
• Generated comprehensive transcriptome data and genome annotation
• Anchored and refined genome assembly with dense genetic map(s)
Results: Whole Genome Sequencing of Finger Millet
Development of early generation mapping population for snapping trait in finger millet Paliwal R, Ojulong H, Odeny DAHOPE2
Two F2 mapping population (U15 x Ex-Alupe-green and KNE-814 x Ex-Alupe-purple)previously generated by H Ojulong
250 F2 progeny lines per cross were tagged alongside parents for molecularcharacterization
Molecular characterization could not be initiated due to lack of funding in year 2015
DNA has been extracted from 230 F2 segregated lines of the cross U15 and KNE-814and are ready to be sentt for GBS sequencing at Cornell University
Each of the populations will be advanced as RILs
GWAS for Striga Resistance
• In collaboration with KALRO-Kakamega/University of Eldoret
• 95 breeding lines screened in two environments
– Locations: Alupe (Busia) and Kibos (Kisumu)
– Data collected over 2 seasons (long and short rains)
• Genotyping-by-sequencing data generated
– Association mapping going on
Genetic and nutritional profiling of finger millet germplasm collection in ESA
Paliwal R and Ojulong H
Approximately 500 diverse finger millet accessions previously characterized for nutritional traits including calcium and iron in two seasons/environments
Molecular characterization could not be initiated due to lack of funding in year 2015
Molecular characterization has been initiated this year and DNA has beenextracted from 400 diverse finger millet accessions
Samples will be sent for GBS sequencing at Cornell University
Subsequent association studies
Sorghum
Characterization Of Sorghum Parental Lines for Hybrid DevelopmentRingo J, Paliwal R, Odeny DA
Justin Ringo, MARI, TZ
• Undertaken in collaboration with University of Eldoret and MARI, TZ
• 120 parental lines (B-lines and R lines) characterized using genotyping-by-sequencing (GBS)
• GBS outsourced: Cornell University
• Data analysis done locally
B_GP1B_GP13B_GP4
R_GP100B_GP3
B_GP33B_GP19B_GP18
B_GP25B_GP16
B_GP12B_GP9B_GP30
B_GP31B_GP32
B_GP6B_GP14R_GP99
B_GP26B_GP27B_GP11B_GP17
R_GP105B_GP20
B_GP35R_GP61
R_GP102R_GP103R_GP88R_GP89R_GP98
B_GP21B_GP22
R_GP95R_GP80
R_GP68R_GP77
R_GP71R_GP75
R_GP70R_GP74R_GP97
B_GP29R_GP36R_GP37R_GP40R_GP46R_GP41
R_GP47R_GP38R_GP39R_GP43
R_GP44R_GP45
R_GP63R_GP60R_GP72
R_GP81R_GP82
R_GP79R_GP86R_GP59
R_GP76R_GP101
R_GP55R_GP65R_GP66
R_GP104R_GP92
R_GP91R_GP58R_GP62
R_GP56R_GP57R_GP85
R_GP93R_GP94
R_GP48R_GP50R_GP53R_GP49R_GP54R_GP51
R_GP52R_GP84R_GP87R_GP96
R_GP90R_GP67
R_GP106R_GP64
R_GP78
B-LinesR lines
Results: GWAS Analysis
• The detected associated SNPs confirm earlier reports for locus Dw2-chr6, Dw3-chr7 and Dw1-chr9 (Higgins et al. 2014)
• Associated SNPs for grain color were identified on chr1, chr3, chr4, chr6 and chr10
Characterization and genotyping of maize & sorghum germplasm for semio-
chemical response to stem borer infestation
Funded by BBSRC/BMGF/SCPRID
Paliwal R, Midega C (ICIPE), Khan Z (ICIPE), Odeny DA, Bruce T (Rothamsted Interntational)
1118 maize (representative of 104 diverse accessions) and 204 sorghum (representative of 17accessions) screened
Presence of volatile induction trait was found to be very rare in improved maize (Fig.2) andabsent in sorghum diverse lines
NS
NS
NS NS NS
0
1
2
3
4
CML442 CKSBL10027 CML395 SC Duma PH4
Me
an
no
.eg
gs
pa
rasit
ize
d/p
lan
t
Egg parasitism
GWAS Results
Generated 0.2M SNPs 12K filtered SNPs used for molecular study In total, 71 SNPs were found significantly associated genome-wide
Fig. 3: Associated SNPs with the semiochemical responses traits in piolt GWASusing GLM approach
Chromosome Number of SNPs detected
1 42 123 164 115 76 57 38 29 4
10 7Total SNPs 71
Genotyping 105 sorghum germplasm across Kenya, Ethiopia and Sudan for their response to Striga
Clet Masiga
• In collaboration with HM Abdalla (CRP-DC funded/HOPE)
• Plant material
• Reference sorghum line BTX623
• 10 lines with a sorghum association panel number already
sequenced by Paterson (UGA)
• Various sorghum cultivars from ECA
• Four striga resistant lines released by Abdalla et al 2014
• E-Tian, Ji2731, Keller cultivars
• 44 sorghum genotypes already re-sequenced
• Evaluated for 2 seasons, 2 environments (Uganda, Kenya)
Screened on Striga-free and Striga sick plots
Genotyped-by-sequencing all genotypes at Cornell University
Discovery: GWAS for Striga resistance
Generated >30 Gb of sequencing from which we identified 78.3K SNP points from 95 sorghum lines;
Retained 27.2K SNPs after filtering, which was used for principal component (PC) analysis and subsequently for Genome Wide Association Studies (GLM and MLM)
Genetic characterization for disease resistance (anthracnose & leaf
blight) in sorghumPaliwal R, Manyasa E, Ojulong H, Otwani DA
100 diverse sorghum accessions were screened for leaf blight (LB), leaf anthracnose(LA) and overall leaf disease (OLD) under long and short rainy seasons in 2014-15 atAlupe, in western Kenya
30K SNPs were generated using 14.5 GB raw GBS sequenced zipped (> 40 GB) data of95 diverse sorghum lines
Out of 30K SNP, 18K SNPS were used for GWAS
Results• Significant association was observed between 38 SNP markers distributed over 8
chromosomes (chr) with leaf diseases• SNP_58251473_chr 10 was found to be associated with leaf blight, anthracnose and
overall disease suggesting its potential use for indirect selection for overall diseaseresponse.
Introgression of Striga resistance QTLs
Musyimi S, Odeny DA, Manyasa E
Genotype Response to Striga Female/male plant
N13 R Male
Seredo S Female
Nakhadabo S Female
IESV92041 S Female
IESV92022 S Female
• Used Simple Sequence Repeat markers converted into High Resolution Melting for rapid screening
• Currently at BC1F1• 27 lines with <3 Striga resistant QTLs were found
Planned Activities 2016/7
• Finalizing whole genome sequencing (NSF/BREAD)
• Transcriptomic analysis of finger millet in response to Striga (CRP-DC)
• Improving finger millet productivity through exploitation of wild finger millet (GCDT)
• Genotyping of new F2 populations of groundnut for resistance to diseases and high oleic acid content (PMIL)
• Characterization and genotyping of maize & sorghum germplasm for semio-chemical response to stem borer infestation (SCPRID/BBSRC)
Thank You