2011 Annual Report 1a.Objectives (from AD-416) The objective for this project will be to obtain the necessary data for foreign regulatory packages for the Plum pox virus resistant plum cultivar 'HoneySweet', including sequencing the 'HoneySweet' genome, identifying the location and structure of all transgene insertion events, and conducting fruit compositional studies. The objectives include the maintenance and increase in communications with foreign collaborators and regulatory agencies, and the support of regulatory submissions by foreign collaborators based on data developed through this agreement. 1b.Approach (from AD-416) The 'HoneySweet' genome will be sequenced. Assembly will be performed using the peach genome as a template. Transgene insertion events will be identified via BLAST. Existing Prunus reference markers will be tested on 'HoneySweet' to refine the assembled physical map and determine the chromosome locations of transgene insertion events. Fruit compositional studies will be conducted and compared with controlled non-genetically engineered plums. Fruit of 'HoneySweet' and a number of non-GE controlled varieties will be collected in multiple years in a design suitable for statistical analyses of composition. Samples will be analyzed by a certified lab. Meetings between the U.S. and foreign collaborators will be for the purpose of sharing data and for the planning of regulatory package submissions, which will be supported through this agreement. 3.Progress Report The objective of this project is to develop a data package for the international deregulation of the Plum pox virus (PPV) resistant genetically engineered 'HoneySweet' plum. This project focuses on the sequencing of the entire PPV resistance transgene insert and the development of additional data on fruit compositional analyses in multiple seasons compared with an expanded range of non-transformed plums. In collaboration with Clemson University, Next Generation sequencing of the entire genome of 'HoneySweet' plum was undertaken. Enough sequence was determined to have covered all of the plum genome 20 times. Sequencing verified the insert structure as determined earlier through molecular techniques including PCR and Southern blotting. Transgene sequences are all closely linked and act as a single locus for resistance. Plum fruit for compositional analyses have been sampled from the Appalachian Fruit Research Station (AFRS) orchards in Kearneysville, WV. At the workshop titled, "Towards the EU Deregulation of 'HoneySweet', a Plum Pox Virus Resistant Plum", held in the Czech Republic on May 30-31, 2011, it was decided that the 'HoneySweet' dossier being prepared for regulatory approval of 'HoneySweet' plum cultivation in the EU will require plum fruit compositional studies on plums grown in the EU. Towards this end, fruit samples will be sent to the AFRS under an APHIS permit from collaborators in the EU. These, along with U.S. collected fruit samples, will be forwarded to a certified commercial laboratory for compositional analyses. The ADODR has monitored activities through emails, meetings, and calls.