1. Harnessing New TechnologiesFor Yam ImprovementRanjana Bhattacharjee, Antonio Lopez-Montes, MichaelT Abberton, P Lava Kumar and Robert AsieduRTB Annual Meeting, Entebbe, Uganda29 Sept 4 Oct, 2014

2. Outline Introduction Progress Outlook 3. Yams (Dioscorea spp.)Different species: >600 species, ofwhich 10 are cultivated. IITA yambreeding program works with fourvery important species.D. alata D. dumetorumD. bulbiferaD. rotundataVariability within each species:Opportunities for crop improvementD. rotundataD. alata 4. ObjectivesAssess genetic diversity and population structureusing genomic (GBS) information and phenotypiccharacterizationIdentification of diverse parents for crossing anddevelopment of mapping populations for traits ofinterest such as pests and diseases (anthracnose,viruses, nematodes), drought, nutrient use efficiency,flowering, earlinessEstablish good quality phenotypic data usingmetabolomicsEstablish inter- and intra-specific hybridizationamong and within different Dioscorea spp. for targettraits 5. GBS: Understanding evolutionNext-generation sequencing based genotyping, cytometry, and phenotyping forunderstanding diversity and evolution of guinea yamsGezhagehn et al, 2014 (TAG: DOI 10.1007/s00122-014-2339-2)D. burkillianaD. cayenensis (3x)D. togoensisD. rotundata (3x)D. prahensilis (2x)D. rotundata (2x)D. mangenotiana (3x) Genetic clustering was relatedto ploidy status Ploidy variation associatedwith incorporation of allelesfrom wild relatives to D.rotundata D. cayenensiscomplex Wild relatives are distinctlyclustered but there has beensome admixtures 6. GBS: Diversity and GWAS 810 D. rotundata genotypes selected Leaf tissues collected, lyophilized and keptfor future useGenotypes for GBS NumberVarieties from markets(Nigeria) 33Popular varieties 16IITA varieties 17TDr Core collection 470Nigeria 206Ghana 30Benin 33Cote d'Ivoire 26Burkina Faso 1Equitorial Guinea 1Togo 165Guinea 7Sierra Leone 1Yam Breeding 307Mapping populations 207Populations with targettraits 100 DNA extracted from 570 samples andshipped to Cornell University The GBS library preparation for Dioscoreaspp. has been standardized using Pst Irestriction enzyme The sequencing of the samples are underway 7. Yam: Phenotyping of GBS materials All 810 D. rotundata genotypes in two locationswith each tuber cut into three sections such ashead, middle and tail for planting Observations taken on above ground traits:- Days for germination- Number of vines per portion of the tuber- Pests and diseases (anthracnose, virus, andnematodes)- Leaf shape and area, SPAD, stomata density- Flowering and fruit bearing For below ground:- Tuber number and shape- Tuber weight Soil samples taken randomly from five differentspots in the field and sent for analysis(nematodes)GBS Field Evaluation 2014, IbadanMaleInflorescenceFemaleInflorescenceMonoecious Fruits 8. Yam: Phenotyping of GBS materialsLeaf shape and area; SPAD; Stomata DensityFlowering Fruit bearing Viruses Anthracnose 9. Yam: Genotyping (Diversity set) 384 D. alata germplasm from INRA,Guadeloupe; CIRAD, Guadeloupe; CTCRI,India and IITA, Nigeria were genotypedwith 34 SSR markers A total of 847 alleles were recordedacross 26 SSRs The mean number of alleles ranged from10.6 (CIRAD) to 7.5 (IITA)Comparaison of diversity parametersIndicators INRACIRAD CTCRI IITANAtAmAs1292179.0108325510.648821948.114901817.510IITAINRACIRADCTCRIGroup 1Group 1Group 2Group 3Group 3Group 4Group 4Group 5Group 5Group 6Group 7 10. Yam: Genotyping (SSRs) Elite parental clones and germplasmaccessions genotyped with 50 SSRs (25genomic and 25 EST-SSRs) Data analysis on 26 SSRs showed a total of241 alleles : diverse materials for breeding Analysis of phenotypic data from 15 years(yield and diseases) to asses the geneticgainMarkerMajor AlleleFrequencyAlleleNo Gene Diversity PICD88 0.8194 5 0.3175 0.3027D55 0.3333 9 0.7782 0.7462D5 0.3194 7 0.7816 0.7487D9 0.4444 10 0.7380 0.7104D83 0.9028 4 0.1817 0.1764D58 0.9306 4 0.1323 0.1293D71 0.5694 8 0.6385 0.6134D70 0.2500 13 0.8441 0.8272D10 0.9861 2 0.0274 0.0270D51 0.4167 6 0.7276 0.6881D73 0.4167 9 0.7261 0.6885D60 0.7361 4 0.4263 0.3913D32 0.8611 2 0.2392 0.2106D61 0.3889 6 0.7627 0.7313Dpr3D06 0.4444 9 0.7110 0.6717Dpr3F12 0.5000 7 0.6755 0.6353Dab2C05 0.4444 10 0.7492 0.7263Da1A01 0.5417 6 0.6535 0.6208Dab2C12 0.6111 5 0.5505 0.4929Dcay223 0.3889 12 0.7894 0.7688Dcay431 0.5000 8 0.6759 0.6367YM30 0.6528 4 0.5220 0.4767Dpr3b12 0.2917 18 0.8684 0.8588Dcay437 0.1667 33 0.9367 0.9337Dcay206 0.1667 24 0.9163 0.9107Dcay245 0.3056 16 0.8395 0.8241Mean 0.5150 9.3 0.6234 0.5980 11. Yam: Metabolomics 49 genotypes from 5 species (D. alata, D. bulbifera, D. cayenensis, D.dumetorum, D. rotundata) = tuber and leaf analysis Metabolomics: biochemical diversity; integrated with genomic data for mQTL10mg lyophilised tissuephase separationpolarstandard extraction800ul MeOH:H2O (1:1)1hr @ RT800ul CHCl3non-polarGC-MSLC-MS/MS (UP)LC-PDA/MSPolar = amino acids, sugars,phenolics, phosphates, derivativesNon-polar = sterols, fatty acids,tocopherols, derivatives 12. Analysis of non-polar extracts from tubersHigh-valuefatty acidsSterolsGlycerol-fattyacid estersDiscriminatingfatty acids &sterols TDd 3774 = much higher for many high-value fatty acids; Nutritionallyimportant and for bioavailablity of other compounds Non-polar extracts were not discriminatory for different species and thisis expected in starchy crops 13. D. rockiiD. membranaceaD. composita(1)D. composita(2)D. praehensilisD. minutifloraD. rotundataD. sansibarensis(2)D. antalyD. cochleari-apiculata(1)D. dumetorumD. cochleari-apiculata(4)D. cochleari - apiculata(3)D. sansibarensis Pax(1)D. bulbifera(2)D. cochleari - apiculata(2)D. pentaphyllaD. altissimaD. alataD. glabraD. bulbifera(1)D. preussi00.10.20.30.40.50.60.70.80.9DissimilarityDendrogramBasallineagesEast Africa Madagascar West AfricaD. rotundata+ CWRCompound- leafThailand & CentralAmericaEuropean / NorthAmericanRest of AsiaMexico & SouthAmericaSouthAfricaEarly-branching cladesLate-branching cladesPolar extracts: Lineages of Dioscorea spp. 14. Yam: Inter- specific hybridizationCrossesProgenyFemale MaleTDa 05/00015 TDr 99/02789 7TDb wild TDa 01/00039 2Agbanwobe TDc 03-5 1TDr 97/00205 TDc 04-71-2 1TDr 97/00917TDb Wild 3TDc 04-71-2 5TDc 04-71-2 14TDc 04-97-4 11TDr 95/18544 TDc 04-71-2 4TDr 04-219TDb 3690 1TDc 04-71-2 2TDc 04-97-4 36TDr 97/00632TDc 04-97-4 2TDc 03-5 7Male crossing block Female crossing blockObjective: Transfer of desirable traitsamong different species: high yield inD. alata to D. rotundata; aerial bulbilsto D. alata from D. bulbifera 15. Hybridization: Physical factors10th Jul. 17th Jul. 24th Jul. 31st Jul. 7th Aug. 14th Aug. 21st Aug. 28th Aug. 4th Sep. 11th Sep. 18th Sep. 25th Sep. 2nd Oct. 9th Oct. 16th Oct.TDr 87/00211**TDr 89/02157TDr 89/02475*TDr 89/02665*TDr 95/18531*TDr 95/18544*TDr 95/18988*TDr 95/19156*21st Aug. 28th Aug. 4th Sep. 11th Sep. 18th Sep. 25th Sep. 2nd Oct. 9th Oct.TDd 08-14-6TDd 08-14-30TDd 08-36-12TDd 08-37-5TDd 08-37-12TDd 08-37-24D. rotundataD. alata D. bulbifera D. cayenensis1. Planting date and flowering2. Effective pollination (time, magnified glass, needle) 16. Hybridization: Tissue culture Characterizing parents based on pollen production and viability Ovule culture: cross-pollinated ovaries collected based on different days of pollination9080706050403020100Meam pollen number (D. rotundata)aaa aabc c c ca a a aa6050403020100Mean pollen number (D. dumetorum)a1009080706050403020100Pollen viability (%)cdcd cddcdbcdacd 17. Hybridization: Anther and Ovule Culturea bPollen tube entry into stigma region of D. alata x D. alata. a)bright field; b) fluorescent. bar = 20 umPollen tube germinationon BK medium of TDr99/02789, bar = 20 mStigmaThe method has been successful but improving the efficiency is inprogress. 18. Yam: Intra- specific hybridizationTrait ofInterestNo. offemaleNo. ofmale Crosses ProgeniesAnthracnose4 4TDa00/00194TDa 87/01091 15TDa 02/00012 26TDa95/00328TDa 85/00250 33TDa 02/00012 26TDa 95 - 310 8TDa 87/01091 24TDa99/00240TDa 02/00012 203TDa 87/01091 70TDa 95 - 310 63TDa01/00041TDa 87/01091 47TDa 02/00012 4TDa 85/00250 14Highcarotenecontent4 4TDr97/00632TDr 95/01932 234TDr 99/02607 468TDr 89/02677 75TDr 99/02626 18TDr97/00793TDr 95/01932 320TDr 99/02607 42TDr 89/02677 45TDr97/00205TDr 99/02607 15TDr 89/02677 30TDr 99/02626 22TDr95/19158TDr 95/01932 13TDr 99/02607 70TDr 99/02626 6Droughttolerance1 2 TDr 04-219TDr 99/02789 148TDr 06-15 156 19. Outlook Integration of genomic data, phenotypic data, historical dataalong with metabolomics data to asses the genetic diversity andpopulation structure in the germplasm accessions as well asbreeding materials Genome-wide association analysis for important traits includingyield, diseases (viruses, anthracnose) and quality traits Development of genomic resources such as SNPs and SSRs Development of heterotic groups for targeted traits (based oninter- and intra-specific crosses GBS for other Dioscorea spp., specifically D. alata, D.cayenensis, D. dumetorum and D. bulbifera Database for yam 20. Partnerships CIRAD, France; CIRAD, Guadeloupe: Genetic diversity IBRC and JIRCAS, Japan: Whole genome sequencing of D.rotundata MAFF and MOFA, Japan: nutrient use efficiency and Tissueculture USDA-ARS, Stoneville: Anthracnose disease in D. alata Clemson University: GBS libraries in D. alata NARS partners: Breeding; sharing of genomic resources Cornell University, Cornell, USA: GBS analysis in D. rotundata Royal Holloway University of London, UK: Metabolomics 21. Acknowledgements CRP RTB W1, W2, and Complimentary Funding Graham Thiele; Luis Becerra Lopez-Lavalle; Peter Kulakow Technical staff of GRC, Yam Breeding and Bioscience Center,IITA Technical staff of Pathology and Virology, IITA