1a.Objectives (from AD-416)
Develop a set of standardized genetic markers for coca DNA that will make it possible to conclusively identify the different coca species in Colombia.
1b.Approach (from AD-416)
These coca leaf samples will be used for DNA and RNA extractions, as well as DNA and cDNA sequencing. Selected genes with unique regions from the resulting DNA and cDNA sequences will be used for identifying varietal and species differences. The project will look for short sequence repeats (SSRs) and single nucleotide polymorphism (SNPs) markers. Once identified these marker regions will be validated and screened for their ability to show varietal differences within the coca samples.
This interagency agreement provides funds to develop molecular markers and conduct the genetic diversity analysis of Erythroxylum species. Through this project new plant varieties were acquired and placed into the Beltsville collection. Short sequence repeats or SSR markers were developed resulting in 15 polymorphic primers that can separate both E.c.c. and E.n. varieties of Erythroxylum into separate groupings. Additional primers are being developed to add more differentiation power to the SSR analysis. One hundred and fifty single nucleotide polymorphic or SNP primers have been identified from more than 60,000 SNP variations identified from Solexa RNA sequencing of 10 libraries of E. c. c. and E. n. varieties. These primers will be screened and validated for their ability to show differences within specific populations of coca plants. This information will be used by researchers and drug control specialists to understand the genetic diversity that exists within this plant species.