Ranjana Bhattacharjee Genetic Resources Center, IITA – Ibadan, Nigeria

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Ranjana Bhattacharjee Genetic Resources Center, IITA – Ibadan, Nigeria DNA Fingerprinting of Cassava Germplasm Collection at IITA Using SSR Markers Ranjana Bhattacharjee Genetic Resources Center, IITA – Ibadan, Nigeria Second Scientific Conference of the Global Cassava Partnership for the 21st Century - GCP21-II Kampala, Uganda 18-22 June 2012

Cassava (Manihot esculenta Crantz.) Germplasm Collection at IITA

Cassava (Manihot esculenta Crantz.) Germplasm Collection CNTRY_NAME No of Accessions Angola 2 Australia 1 Benin 334 Brazil 25 Burkina Faso 7 Cameroon 184 Cape Verde 17 Central African Republic Chad 5 Congo 51 Gabon 3 Gambia, The 6 Ghana 287 Guinea 155 Ivory Coast 52 Kenya 10 Liberia 8 Madagascar Malawi 14 Mali Mozambique 15 Niger Nigeria 1932 Rwanda Sierra Leone 83 Tanzania, United Republic of Togo 130 Uganda 53 Zaire 90 Zambia

Sustainable Conservation/Maintenance In vitro bank •Reduced risk loss •Facilitated exchange Cryopreservation ‘Low cost’ Field bank (5% loss)

Constraints: Cassava Germplasm Collection 7 million germplasm in 1750 gene banks; 1-2 million are distinct (FAO, 1996) Mismatch (TME and TMe) and other nomenclature issues Presence of duplicates Mismatch between field bank and in vitro bank Maintenance cost and implication on distribution (and breeding) Low genetic diversity GCDT Funds: Identification of genetically unique accessions in cassava using SSR markers

Identification of Duplicates: Methodology Entire Collection: Pairwise comparison of accessions using passport data to identify synonyms or mismatch Yes No Potential Duplicates Second source of data: DNA fingerprinting data Second source of data: DNA fingerprinting data No No Yes Yes Potential Duplicates Duplicates confirmed No Yes No Third source of data: Field trial data Third source of data: Field trial data No No Yes Yes Duplicates Confirmed Duplicates Confirmed

Identification of Duplicates: Methodology Question: Differentiate two accessions as Unique Number of accessions studied: 2418 (mainly landraces) Number of SSRs used: 25 System used: PAGE (10 SSRs) and Capillary Electrophoresis (ABI3130) (15 SSRs) Data scored as presence (1) or absence (0) for each allele per marker per accession (PAGE) The average number of alleles per locus was 4.1, ranging from 7 alleles (SSR51) to 3 alleles (AGC129, AGC163, CAG61 and AAG 54) Genetic distances (GD) were calculated among pairs of accessions following Modified Roger’s distance method Two accessions were defined as “Absolute Duplicates”, if GD = 0 between them Two accessions were defined as “Potential Duplicates”, if GD = 0 and 0.15 High-throughput DNA Extraction (192 – samples per day) High-throughput PAGE gels

Statistical Cut-off: Genetic Distance With the increase in the number of markers, P (MRD <= 0.15) diminishes: if more markers are added, the probability of differentiating accessions increases

Identification of Duplicates: Methodology 893 (36.9%) duplicates (absolute and potential) identified Further fingerprinting with additional 15 SSR markers using capillary electrophoresis (multiplexed) (ABI3130) Data scored in base pairs based on actual peaks observed (GeneScan and GeneMapper) The average number of alleles per locus was 4.7, which ranged from 8 alleles (AT47) to 3 alleles (TCT140, TTC59 and AT101) Based on genetic distance, 347 (14.7% of entire collection) absolute duplicates were identified Rationalization: Further Fingerprinting with SNPs, conduct field trials for morphological evaluation, conservation in long-term storage

No duplicates in the core collection Development of Core Collection Characterization data: 2418 accessions (evaluated in two locations) Ibadan: 1766 accessions (3 checks); Ubiaja: 1890 accessions (1 check) 1503 accessions common (Ibadan = 263; Ubiaja = 387) 40 traits: 8 continuous and 32 discrete Analysis of 1503 accessions (Gower’s genetic distance): Basic core of 301 accessions (10,000 bootstrapping) Analysis of 263 accessions: 51 (Ibadan); 387 accessions: 76 (Ubiaja) Final core collection: 428 accessions No duplicates in the core collection Bhattacharjee et al., 2012, Plant Genetic Resources

Thank You