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United States Department of Agriculture

Agricultural Research Service

Research Project: Whitefly and Bottle Gourd Genome Sequencing and Transcriptome Analysis to Identify Target Genes That Are Useful for Interfering Rna 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

1. Conduct genome sequencing of cassava-feeding whitefly (super-abundant whitefly). 2. Perform global transcriptome profiling on the super-abundant whitefly upon infection by Cassava brown streak virus (CBSV) and African cassava mosaic virus (ACMV)and identify genes of interest. 3. Comparative analysis of siRNA and miRNA profiles of whiteflies upon feeding on virus infected tomato and cassava plants and identification of 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. 5. Initiate a new genome sequencing project for bottle gourd, a popular watermelon rootstock. 6. Perform transcriptome profiling analysis on bottle gourd in different tissue organs (leaf, root, stem, flower, fruit and seed) at various developmental stages, as well as comparative expression of genes in resistance and susceptible plants upon virus-infection.

Collect super-abundant whitefly in a cassava plantation to ensure genetic homogeneity by a collaborator in Tanzania and ship to ARS for isolation of male population for DNA extraction. Provide high quality DNA to BTI for library preparations for Illumina sequencing. Through contract services for library preparation and conduct sequencing using Illumina sequencing platforms. Conduct reference-guided genome assembly using B biotype of Bemisia tabaci genome as a reference, annotation and analysis. Using established whitefly (Bemisia tabaci) colonies on collar green or broccoli, over 2,000 will be transferred on CBSV and ACMV -infected and health cassava 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 cassava 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) at BTI. Another half volume of RNA preparation will be used for sRNA construction at US Vegetable Laboratory. 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 cassava 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 virus transmission in cassava. In a second genome sequencing project to bottle gourd, a virus-resistant bottle gourd line (USVL1-8) will be used for this purpose. Seeds will be germinated in greenhouse and leaf tissues will be collected and shipped to BTI for DNA isolation, library preparation and Illumina sequencing. In transcriptome analysis, tissues from different organs (leaf, stem, root, flower, fruit and seed) will be collected from both virus resistant (USVL1-8) and susceptible line (USVL10) upon infection by Papaya ringspot virus. Total plant RNA will be prepared and shipped to BTI for RNA-seq library preparation, sequencing and analysis. Differentially expressed genes will be identified and candidate resistant genes will be characterized on their functions using RNAi approaches.

Last Modified: 11/28/2015
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