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Regional planning meet Harare : Genomics- ESA by Damaris Achieng odeny

  1. GENOMICS-ESA Damaris Achieng Odeny Scientist – Biotechnology Rajneesh Paliwal Post-doctoral Research Fellow Cereal Genomics With support from Samuel Manthi and Vincent Njung’e
  2. 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
  3. 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. 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
  5. 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 and kabate locations in Kenya  DNA extracted from 95 (57 desi and 28 kabuli) lines and generated  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
  6. 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 Flowering 50% (DF), number of leaf let/leaf (NLL), pos length (PL), primary branch (PB), secondary branch (SB), days to maturity (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
  7. 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
  8. 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
  9. Activities Planned - 2015 Cereals 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)
  10. 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. kigeziensis SP1 SP2 (E. coracana subsp. africana) E. coracana subsp. coracana E. coracana subsp. coracana Cultivated accessions
  11. 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)
  12. Results: Whole Genome Sequencing of Finger Millet
  13. Development of early generation mapping population for snapping trait in finger millet Paliwal R, Ojulong H, Odeny DA HOPE2  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 molecular characterization  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-814 and are ready to be sentt for GBS sequencing at Cornell University  Each of the populations will be advanced as RILs
  14. 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
  15. 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 been extracted from 400 diverse finger millet accessions  Samples will be sent for GBS sequencing at Cornell University  Subsequent association studies
  16. Sorghum
  17. Characterization Of Sorghum Parental Lines for Hybrid Development Ringo 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_GP1 B_GP13 B_GP4 R_GP100 B_GP3 B_GP33 B_GP19 B_GP18 B_GP25 B_GP16 B_GP12 B_GP9 B_GP30 B_GP31 B_GP32 B_GP6 B_GP14 R_GP99 B_GP26 B_GP27 B_GP11 B_GP17 R_GP105 B_GP20 B_GP35 R_GP61 R_GP102 R_GP103 R_GP88 R_GP89 R_GP98 B_GP21 B_GP22 R_GP95 R_GP80 R_GP68 R_GP77 R_GP71 R_GP75 R_GP70 R_GP74 R_GP97 B_GP29 R_GP36 R_GP37 R_GP40 R_GP46 R_GP41 R_GP47 R_GP38 R_GP39 R_GP43 R_GP44 R_GP45 R_GP63 R_GP60 R_GP72 R_GP81 R_GP82 R_GP79 R_GP86 R_GP59 R_GP76 R_GP101 R_GP55 R_GP65 R_GP66 R_GP104 R_GP92 R_GP91 R_GP58 R_GP62 R_GP56 R_GP57 R_GP85 R_GP93 R_GP94 R_GP48 R_GP50 R_GP53 R_GP49 R_GP54 R_GP51 R_GP52 R_GP84 R_GP87 R_GP96 R_GP90 R_GP67 R_GP106 R_GP64 R_GP78 B-LinesR lines
  18. 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
  19. 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 17 accessions) screened  Presence of volatile induction trait was found to be very rare in improved maize (Fig.2) and absent in sorghum diverse lines NS NS NS NS NS 0 1 2 3 4 CML442 CKSBL10027 CML395 SCDuma PH4 Meanno.eggs parasitized/plant Eggparasitism
  20. 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 GWAS using GLM approach Chromosome Number of SNPs detected 1 4 2 12 3 16 4 11 5 7 6 5 7 3 8 2 9 4 10 7 Total SNPs 71
  21. 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
  22. 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)
  23. Genetic characterization for disease resistance (anthracnose & leaf blight) in sorghum Paliwal 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 at Alupe, in western Kenya  30K SNPs were generated using 14.5 GB raw GBS sequenced zipped (> 40 GB) data of 95 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 disease response.
  24. 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
  25. 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)
  26. Thank You
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