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