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SSR Analysis of the USDA-ARS Cacao collection

SSR Analysis of the USDA-ARS Cacao collection. Brian M. Irish USDA-ARS Tropical Agriculture Research Station Mayaguez, Puerto Rico. Management of Tropical/Subtropical Genetic Resources and Associated Information. Horticulturist/Plant pathologist

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SSR Analysis of the USDA-ARS Cacao collection

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  1. SSR Analysis of the USDA-ARS Cacao collection Brian M. Irish USDA-ARS Tropical Agriculture Research Station Mayaguez, Puerto Rico

  2. Management of Tropical/Subtropical Genetic Resources and Associated Information • Horticulturist/Plant pathologist • Bananas, plantains, cacao, sapote, nispero, Annona spp., Garcinia spp., bamboo, mango, quenepa (Spanish lime) • Acquire, propagate (disease-free), maintain, characterize and distribute • Genetic diversity • Characterize • Phenotypic, Molecular • All information is incorporated in our GRIN database • All accessions are propagated clonally by means of divisions, cuttings, air-layers or grafting • 2 field/1 lab technicians, 1 Information technologist, 1 gardener, 3 part time lab/field technicians, 1 graduate/1 undergraduate students

  3. Cacao or Cocoa • Industry (billion dollar industry in the U.S.) • Cosmetics, soaps • Chocolate (bars, powder) • Large food processors: ADM/Barry Callebaut • Confectionary industry • Mars, Hershey's, Cadbury, Nestle and many others • Other industries that are directly related in chocolate making • Sugar, dairy, nuts • Replicated and characterized collection with freely available germplasm

  4. Cacao collection at TARS • Theobroma cacao • 154 accessions/clones (924) • In the process of introducing ~50-100 accessions • Breeding/selection programs at CATIE, EET-Pichilingue, CRU, U. Reading, Brazil and USDA • Evaluation • Number, weight, color, size of pods, number and weight of seed, chocolate quality, disease and insect resistance • Due to the nature of how the crop is propagated and the length of time it has been maintained, errors in propagation have accumulated (world) • A set of 15 microsatellite markers have been used to fingerprint cacao worldwide.

  5. Accession -AC T 1/1. From 9/6/07-9/14/07 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9

  6. April 8th, 2008 6 1 2 5 3 4

  7. Microsatellites (SSRs) • Short repetitive sequences in Eukaryotes in non coding regions of the genome • Highly variable and inherited in Mendelian fashion • Developed at CIRAD and USDA • On different chromosomes (10) and separate linkage groups • High allelic diversity in cacao genotypes • mTcCIR – Microsatellite Theobroma cacao CIRAD

  8. Table 3.Characteristics and summary statistics for the 15 International set of microsatellite primers utilized for fingerprinting the USDA-ARS Tropical Agriculture Research Station cacao (Theobroma cacao) collection.

  9. Clone #101-TARS 23Heterozygous and consistent for all 6 plants

  10. Clone #42-EET 53Heterozygous/homozygous and inconsistent across the 6 plants

  11. Analysis (continued) • Mislabeling • Homonymous or ‘intra-plant’ error • Synonymous or ‘duplicate’ error • Non-match error • Synonymous multi-locus fingerprint profiles condensed into a single profile • 139 unique profiles used for subsequent analysis

  12. Accessions grouped into four main clusters • Based on geographic and common genetic background • Trinitarios/Criollos • Lower A. Forasteros • Upper A. Forasteros • Hybrids

  13. 0.00 0.68 0.34 Genetic Distance Coefficient CC10 B EET353[ECU] B EET381[ECU] P10[MEX] A P22[MEX] P43[MEX] RIM105[MEX] RIM13[MEX] A RIM15[MEX] RIM2[MEX] RIM34[MEX] RIM41[MEX] RIM48[MEX] RIM52[MEX] RIM6[MEX] RIM75[MEX] RIM78[MEX] RIM10[MEX] SGU69 ICS39 POUND7[POU] B SIC72 B SC49[COL] GS46 UF668 ICS40 GS7 ICS29 UF601 Mislabeling: e.g., RIM group • RIM identical (SSRs) • Yellow/green highlighted • (A/B) ending • More than one genotype • e.g., RIM13 [MEX] • Several clones don’t belong in this group • CC10, EET 353, SGU69

  14. RIM 2 [MEX] RIM 6 [MEX] RIM 10 [MEX] RIM 13 [MEX] A RIM 2 [MEX] RIM 15 [MEX] RIM 34 [MEX] RIM 41 [MEX] RIM 48 [MEX] RIM 52 [MEX] RIM 75 [MEX] RIM 105 [MEX] P 10 [MEX] P 22 [MEX] P 43 [MEX] RIM 13 [MEX] B RIM 30 [MEX] EET 381 EET 353 SGU 69

  15. Table 2. Fourteen synonymous groups (including 49 accessions) within the USDA-ARS Mayaguez cacao collection identified by microsatellite DNA analysis. Accessions in the same synonymous set shared identical multi-locus microsatellite profiles.

  16. Table 1. Name, source and results of identification verification (RIV) of cacao accessions maintained at the USDA-ARS Tropical Agriculture Research Station in Mayaguez, PR.

  17. Summary • Multilocus profiles, dendrogram, assignment test and reference genotypes help sort out mistakes • Allelic diversity proportional to CATIEs and not very different for major alleles • Genetic redundancy does exist • Shannon’s diversity index • Rationalization of collection is an option, but only after field characterization • Agronomic traits

  18. THANK YOU. QUESTIONS?

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