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United States Department of Agriculture

Agricultural Research Service

Research Project: GENETIC AND BIOLOGICALLY-BASED MANAGEMENT OF VEGETABLE CROP DISEASES

Location: Vegetable Research

Title: Exploiting the small RNA deep sequencing technology for identification of viruses and viroids in plants

Authors
item LING, KAI-SHU
item LI, RUGANG
item WECHTER, WILLIAM
item Hernamdez, Alvaro -
item Fei, Zhangjun -

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: November 4, 2011
Publication Date: November 17, 2011
Citation: Ling, K., Li, R., Wechter, W.P., Hernamdez, A.G., Fei, Z. 2011. Exploiting the small RNA deep sequencing technology for identification of viruses and viroids in plants. Meeting Abstract. p 31.

Technical Abstract: Small RNAs (including miRNA and siRNA) are produced abundantly in plants and animals in regulating gene expression or in defense against virus or viroid infection. Analysis of a siRNA profile upon virus infection in plant may allow for de novo assembly of a virus genome. In the present study, four suspected virus-infected tomato samples collected in the U.S. and Mexico were used for small RNA library construction and sequencing. The bar-coded small RNA libraries were prepared and sequenced in one lane on an Illumina GAIIx. Each library generated between 5-7 million of sRNA reads. Using computer-assisted subtraction against the tomato genome, more than 90% of tomato-related sRNAs were removed. The highly enriched virus-like siRNAs pools were assembled with the Velvet program with or without reference Pepino mosaic virus (PepMV) and tomato-infecting pospiviroids. For a small genome of Potato spindle tuber viroid (PSTVd, 359 nt), a complete viroid genome was assembled by siRNAs. On the other hand, a near complete virus genome (98%) could be assembled for PepMV. A common mixed infection of two strains of PepMV (EU and US1), sharing only 82% of genome nucleotide sequence identity in three U.S. samples, was confirmed through Sanger sequencing of RT-PCR products. For the Mexican sample with an unknown etiology, a novel potyvirus with only 70% sequence identify to other known viruses was identified and full genome completed through additional Sanger sequencing. The small RNA technology will likely become an efficient and powerful generic tool for complete virus and viroid analysis in plants.

Last Modified: 8/27/2014
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