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United States Department of Agriculture

Agricultural Research Service

Research Project: Whitefly Genome Sequencing and Transcriptome Analysis to Identify Target Genes That Are Useful for Interfering Rna (Rnai) Technology

Location: Vegetable Research

Project Number: 6080-22000-025-11
Project Type: Specific Cooperative Agreement

Start Date: Sep 01, 2013
End Date: Mar 31, 2016

Objective:
1. Conduct genome sequencing of whitefly (B. tabaci, B. biotype). 2. Perform global transcriptome profiling on B. tabaci upon infection by TYLCV and identify genes of interest. 3. Comparative analysis of siRNA and miRNA profiles upon TYLCV infection on whiteflies and identify candidate genes that may be useful for RNAi technology. 4. Develop and evaluate RNAi technology using selected functional genes with key biological functions on whiteflies.

Approach:
Establish an isogenic line of whitefly (B. tabaci, B biotype) and isolation of male population for DNA extraction. Provide high quality DNA for library preparations for Illumina and PacBio sequencing. Through contract services for library preparation and conduct sequencing using Illumina and PacBio sequencing platforms. Conduct de novo genome assembly, annotation and analysis. Using established whitefly (Bemisia tabaci) colonies on collar green or broccoli, over 2,000 will be transferred on TYLCV-infected and health tomato plants, respectively. After feeding on the test plants for specific time intervals at 24 hr, 48 hr and 72 hr, 500 whiteflies will be aspirated at each time point and immediately frozen at -80C till use. We plan to perform 3 biological replications for each set of whiteflies. In total, the number of whiteflies needed is 12, 000, including 6,000 viruliferous and 6,000 feeding on health tomato plants. In each subsample, total RNA will be prepared using TRIzol method. A half volume of RNA preparation will be used for RNA-seq library construction (paired-end). Another half volume of RNA preparation will be used for sRNA construction. Sequencing of RNA-seq and sRNA will be multiplexed and conducted on HiSeq at Cornell genomics service center. Bioinformatics analysis on RNA-seq and sRNA (miRNA) will be performed at BTI and those genes highly regulated upon TYLCV infection will be selected. Once whitefly functional genes or their predicted precursor sequences are identified, dsRNA will be designed and synthesized. These dsRNAs in varied concentration will be added on artificial diet or in nutrient solution for tomato cuttings for whitefly feeding. The effect of RNAi on whiteflies will be evaluated in replicated experiments for their mortality rate relative to controls. Upon initial screening, those promising dsRNAs will be tested again in replicated tests to evaluate the effects on whitefly survivability and impact on TYLCV transmission.

Last Modified: 10/25/2014
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