2010 Annual Report 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. 3.Progress Report This interagency agreement provides funds to develop molecular markers and conduct the genetic diversity analysis of Erythroxylum species. Through this project 14 new plant varieties were acquired and placed into the Beltsville collection. Ten of those varieties survived the move and are growing in the greenhouse. SPCL has dried leave material for all 14 varieties. SSR markers were developed resulting in 18 polymorphic primers and 28 monomorphic primers for E. c. c. and 24 polymorphic and 20 monomorphic for E. n. The polymorphic primers are being tested for their cross reactivity. All confirmed polymorphic primers will be tested on the 14 new varieties from Colombia. Ten varieties of E. coca and E. novo were subjected to RNA sequencing by Solexa to develop SNP markers. Sequencing was completed in June and the bioinformatics selection of these markers has started. Once identified the all molecular markers will be validated and screened for their ability to show differences within specific populations of coca plants. This project was monitored by e-mail and phone calls between the principal investigator and the principal collaborators listed for this project.