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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR CHARACTERIZATION OF 'HONEYSWEET' SMALL RNAS SUPPORTING A EUROPEAN 'HONEYSWEET' DEREGULATION DOSSIER

Location: Appalachian Fruit Research Laboratory: Innovative Fruit Production, Improvement and Protection

2012 Annual Report


1a.Objectives (from AD-416):
The objectives of this project will be to: .
1)collect data on siRNA expression needed for the successful development, submission, and shepherding of a deregulation dossier for 'HoneySweet' plum in Europe;.
2)organize a workshop entitled, "The deregulation of genetically engineered Plum pox virus resistant 'HoneySweet' plum in the U.S. and the potential impacts on regulatory approval and plum cultivation in Europe"; and.
3)support the exchange of information necessary for the successful development, submission, and shepherding of a deregulation dossier for 'HoneySweet' plum in Europe.


1b.Approach (from AD-416):
'HoneySweet' siRNA will be purified and sequenced using next generation sequencing technologies to identify the specific species of siRNAs produced. Candidate siRNAs will be confirmed via conventional molecular approaches and assessed for potential animal toxicity via bioinformatic analyses. A visiting scientist from the Crop Research Institute, Drnovska, Czech Republic, will conduct the research at the Appalachian Fruit Research Station. This person will be educated and trained in 'HoneySweet' molecular characterization and U.S. regulatory policy to serve as a technical liaison between the U.S. and EU partner laboratories. Meetings will be organized with the specific purpose of developing a dossier on 'HoneySweet' plum for the submission to the European Food Safety Association by the Crop Research Institute, Drnovska, Czez Republic (a European public institution), and meetings at regular intervals to monitor the progress of that dossier and to address roadblocks that may develop in the progress of the dossier.


3.Progress Report:

This project has three principle goals: 1) Identify the specific siRNA species that are produced in 'HoneySweet' plums. 2) Perform bioinformatic analyses to assess the potential of animal toxicity. 3) Educate a scientist from the host petitioning country (Czech Republic) in the U.S. biotech regulatory system as well as 'HoneySweet' molecular biology so they can serve as an expert liaison between the U.S. and EU.

This project was significantly delayed due to difficulties encountered with collecting European field samples exposed to PPV and having them shipped to the U.S. for analyses. Leaf and fruit samples of 'HoneySweet' and a control non-transgenic cultivar, 'Stanley', were finally obtained during Fall 2011. RNA extractions have been completed for all samples in triplicate and sent to a service provider for sRNAseq and RNAseq. RNAseq has been completed and analysis is underway. After trimming and filtering the sequences, a total of 894,000,000 reads (about 24,000,000 reads per sample) were obtained from 36 libraries. Preliminary results show that the precise transgene derived transcripts and expression levels can be readily ascertained. The data also confirms a near lack of detectable PPV in 'HoneySweet' leaf and fruit tissues even when carrying grafted non-transgenic limbs heavily infected with the virus. sRNAseq samples are currently being processed for sequencing. Because a suitable candidate could not be found from the Czech Republic, a visiting scientist from a cooperating EU country (Poland) was hired starting in November 2011 for a period of one year. This scientist is currently performing the RNAseq and sRNAseq experiments as well as bioinformatic data analysis. Upon completion of the analysis, the visiting scientist will prepare the data for the EFSA deregulation petition and engage with the host petitioning country in the preparation and submission of the dossier. A meeting was held in the Czech Republic in October 2011 to coordinate data collection and dossier development. The meeting included scientists from over seven different countries as well as representatives from the Ministries of Agriculture and Environment from the Czech Republic. This was a highly successful meeting that outlined a clear path forward for dossier submission to EFSA.


Last Modified: 9/21/2014
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