1. 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 June 2012

2. Cassava (Manihot esculenta Crantz.)
Germplasm Collection at IITA

3. 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

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

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. Thank You