|SEO, EUN-YOUNG - Chungnam National University|
|CHO, SEUNGHEE - Chungnam National University|
|MOON, JA - Chungnam National University|
|GOTOH, TAKAFUMI - Kyushu University|
|KIM, HONG GI - Chungnam National University|
|LIM, HYOUN-SUB - Chungnam National University|
|SONG, KI HAK - Chungnam National University|
Submitted to: Journal of Faculty of Agriculture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/14/2014
Publication Date: 1/15/2015
Citation: Seo, E., Cho, S., Moon, J.S., Gotoh, T., Kim, H., Domier, L.L., Hammond, J., Lim, H., Song, K. 2015. A high throughput soybean gene identification system developed using soybean yellow common mosaic virus (SYCMV). Journal of Faculty of Agriculture. 60:127-138.
Interpretive Summary: Determination of the function of previously uncharacterized plant genes can be challenging, requiring over-expression or gene-knockout to determine altered phenotypes in order to understand gene function; reverse genetics approaches using Virus-induced gene silencing (VIGS) offer a more rapid alternative to generation of transgenic plants for gene knockdown studies, but suitable VIGS vectors are not available for all crops. Although a Bean pod mottle virus (BPMV) VIGS vector is available, BPMV is only known to occur in North America, and is not appropriate for use in Asia where BPMV is not present. An infectious clone of Soybean yellow common mosaic virus (SYCMV), a virus recently reported from Korea, was developed into a VIGS vector and modified to allow Gateway cloning for high-throughput insertion of gene fragments for reverse genetics to identify the function of previously uncharacterized genes. A method for preparing a random library of short complementary DNA (cDNA) fragments suitable for VIGS was also developed, and the cDNA library transferred to the SYCMV vector by Gateway cloning. Individual clones from this cDNA library were used to establish VIGS in soybean, and four gene fragments inducing visible phenotypes were identified for further examination and characterization. The newly developed system offers the opportunity to determine the function of previously uncharacterized soybean genes, and this system could be further adapted to other crops, for the benefit of plant geneticists and plant breeders.
Technical Abstract: Soybean yellow common mosaic virus (SYCMV) was recently reported from Korea, and a subsequent survey of soybean fields found that SYCMV, Soybean yellow mottle mosaic virus (SYMMV), and Soybean mosaic virus (SMV) infections were widespread. SYCMV has recently been developed into a Virus Inducing Gene Silencing (VIGS) vector for use as a reverse genetics tool for soybean, and here we report a modified SYCMV VIGS vector containing a new restriction enzyme site in the 3' non-coding region into which we inserted the Gateway system. Ultrasonically generated c.300 bp random fragments of Glycine max cDNA were inserted into the SYCMV VIGS vector, and individual colonies containing G. max cDNA were inoculated to cultivar Williams 82. We monitored the phenotype of inoculated soybean, and selected obvious visible phenotypes caused by SYCMV-induced gene silencing which could enable annotation of gene function of unknown gene fragments inserted into SYCMV. Here, we describe development of a high-throughput SYCMV VIGS vector for gene function identification in soybean.