Yam: Recent Advances in Genetic and Genomic Resources

Ranjana Bhattacharjee, Michael Abberton, Antonio Lopez-Montes, P. Lava Kumar

and Robert Asiedu

Ø  Completing the Whole Genome Sequencing of six important cultivated Dioscorea spp.

- D. rotundata (IITA-IBRC-JIRCAS) - D. alata (IITA-TGAC) - D. dumetorum (underway, IITA-AOCC) - D. cayenensis - D. esculenta - D. bulbifera Ø  Development of genomic resources Ø  Development of mapping populations for different target traits Ø  Development of training populations Ø  Genotyping-by-Sequencing: - understanding population structure, genetic diversity, varietal identification, etc. - linkage mapping and identification of QTLs - GWAS and GS Ø  Metabolomics and high-throuput phenotyping Ø  Application of transcriptomics, if applicable

Targets for Genomics Assisted Breeding

Ø  Speeding the Breeding Cycle - Vine propagation technique (high multiplication ratio and cuts down time of producing seed yam) Ø  Whole Genome sequencing - D. rotundata (IITA-IBRC-JIRCAS) - D. alata (IITA-TGAC) - D. dumetorum (underway, IITA-AOCC) Ø  Genotyping-by-Sequencing - 810 D. rotundata genotypes (core collection: 477; breeding lines: 300; market varieties: 33) - Mapping population for anthracnose disease in D. alata for linkage mapping and identification of QTL (s) Ø  Metabolomics - 49 genotypes from five Dioscorea spp. - GC-MS method on leaf and tuber samples (both showed similar compositional trends while different portions showed a gradiance, although it is minimal).

Different species: > 450 species, of which 10 are cultivated.

Variability within each species: Opportunities for crop improvement

Food security

Resilience Sustainability Income generation

Inter and intra-species

hybrids

D. alata

D. dumetorum

D. bulbifera

D. rotundata D. alata

D. esculenta

D. trifida

D. dumetorum

Heterogeneity within varieties of each species

Different tuber portions used for metabolite profiling

Achievements in Yam Breeding and Genomics

Focus:Screeningforanthracnosedisease;developmentofaddi8onalmarkers;construc8onoflinkagemap;QTLmappingforanthracnosedisease;denovosequencingofparentallines;GBSofparentsandprogenies(Dioscoreaalata)

USAID-Linkage Project

TDa 95/00328

TDa 95 -310

Ø  >40,000 EST-sequences from the two mapping population parents: a total of 1152 EST-SSRs identified Ø  de novo sequencing of parents: 18,584 SSRs

identified in TDa95/00328 and 15,952 SSRs identified in TDa95-310

Ø  20 linkage groups identified corresponding to the basic chromosome number of D. alata: a total of 301 polymorphic EST-SSRs mapped covering a total length of 3229.7 Cm

Ø  Three putative QTL (s) identified for anthracnose disease: one QTL was consistent at position interval of 51.4-155.5 cM

Saski et al (2015). Development of genomic and molecular resources for water yam. Published in PLOS One journal Bhattacharjee et al (2015). SSR-based genetic linkage map and identification of QTL (s) for anthracnose disease in Dioscorea alata (under review)

Estimated genome size 1GbNumber of samples 96Index depth 48 individuals per lanePhenotype Anthracnose disease trait

Sequencing type Illumina HiSeq 2500 (1-2 lanes)

Read type 1x100bp Single end

Averages from 96 Samples [using TASSEL (UNEAK) pipeline] •  Unique Tag Counts:

83,018 •  Raw Reads: 3,156,223 •  Aligned Reads:

2,535,879 •  Coverage per unique

tag: 30 0

500

1000

1500

2000

2500

3000

>=50% >=60% >=70% >=80% >=90%

Figure. Shared SNP distribution contained in at least 50% of the individuals

USAID-Linkage Project: GBS analysis

P2

P1

Focus:Genotypingbysequencing(GBS);high-throughputphenotyping(metabolomics);morphologicalcharacteriza8on;breedingapplica8ons(inter-andintra-specificcrosses)Ø  GBSof810D.rotundatagenotypes(corecollec5on=470landraces;breedinglines=307

genotypes;varie5esfrommarkets=33)completedØ  Differentbioinforma5cspipelinetested:ahighpropor5onoffilteredreadsalignedtothe

referenceD.rotundatagenomeusingbow5e2soKwareØ  AcustomizedRscriptiden5fied3068polymorphiclociofwhich55.9%werebi-allelicand

44.1%mul5-allelic.Ø  Morphologicalcharacteriza5on(aboveandundergroundtraits)of810genotypes(inthree

replica5ons:head,middleandtailpor5ons)completedfortwoyears

CRPRTB Complementary Project

Genotyping by sequencing (GBS) reveals the complex genetics of D. rotundata. This sequence alignment shows the heterozygous sequence of the reference genome and three alleles found in D. rotundata germplasm. The number of alleles per sample ranged between 1 and 3, indicating that some clones are polyploids.

Totalreadsprocessed:438,685,435Readswithadapters:340,851,682(77.7%)ReadswriXen(passingfilters):438,685,435(100.0%)Totalbasepairsprocessed:44,307,228,935bpQuality-trimmed:2,290,709,390bp(5.2%)TotalwriXen(filtered):25,613,093,559bp(57.8%)Shorterthan64bp:273,540,127(62.4%)Notassignedtobarcode:12,892,837(2.9%)Totalreadspassingallfilters:152,252,471(34.7%)

Ø 49accessionsfromIITAbreedingprogramØ 5species(D.alata,D.bulbifera,D.cayenensis,D.dumetorum,D.rotundata)Ø Tubermaterial(Head,middleandtail;selectedleafmaterial)

CRPRTB Complementary Project: Metabolomics

Ø  No significant difference in metabolite composition between tuber and leaf sampling Ø  Slight gradation in metabolite composition across different portions Ø  Only few metabolites correlate in pathways: due to postharvest deterioration

Inter- and Intra-specific Crosses TraitofInterest

No.offemale

No.ofmale Crosses Progenies

NUE 6 6

TDr04-219

TDrAlumaco 24TDr00/00362 29TDr95/01932 629TDr99/02562 23

TDr89/02157 TDr95/01932 16

TDr89/02665

TDrAlumaco 23TDr00/00362 2TDr97/00777 16TDr95/01932 4

TDr97/00917 TDr95/01932 37TDa00/00194 TDa02/00012 341TDa98/01166 TDa02/00012 4

Conven5onalkaryotypingofDioscoreaspp.

Partnerships

Ø RTBteamØ CornellUniversityØ RoyalHollowayUniversityofLondonØ TheGenomeAnalysisCenterØ AfricanOrphanCropsConsor5umØ IwateBiotechnologyResearchCenterØ JIRCASØ CIRADØ Na5onalprogramswithinAfrica,AsiaandthePacificØ Universi5es:MastersandPhDstudents