NERC/Centre for Ecology and Hydrology, Oxford, Oxford OX1 3SR, UK.
J Virol Methods. 2010 Sep;168(1-2):223-7. doi: 10.1016/j.jviromet.2010.06.003. Epub 2010 Jun 15.
Small RNA sequences were obtained from leaf extracts of wild Dactylis glomerata (cocksfoot grass) using deep sequencing (454 Life Sciences, Roche Diagnostics), and were screened against virus sequences in GenBank using a local BLASTn search program (BioEdit). Putative small interfering (si)RNAs complementary in sequence to Cereal yellow dwarf virus (CYDV, genus Luteovirus) genomes were identified. Primer sequences were made against the "high scoring" siRNA sequences and RT-PCR was used to amplify a 438 bp CYDV fragment in total RNA extracts from D. glomerata leaves. Sequencing of the RT-PCR product confirmed the occurrence of a previously undescribed CYDV population with phylogenetic affinity to CYDV-RPS. In D. glomerata the CYDV infection rates were 42.3% (n=78) in 2008 and 50.0% (n=48) in 2009. Specific RT-PCR tests also showed that this D. glomerata population harboured Cocksfoot streak virus (CSV, genus Potyvirus). Dual infections by these viruses were observed in 20.5-22.9% of all plants tested in 2008-2009. Interestingly, infections of either CYDV or CSV enhanced the occurrence of the other virus in individual grasses. Opportunities are discussed for using siRNA sequencing approaches in virus survey and other ecology studies under field conditions.
采用深度测序技术（454 Life Sciences，Roche Diagnostics）从小麦根蘖草（野黑麦草）的叶片提取物中获得小 RNA 序列，并使用本地 BLASTn 搜索程序（BioEdit）在 GenBank 中筛选病毒序列。鉴定出与禾谷花叶病毒（CYDV，属 Luteovirus）基因组序列互补的推定小干扰（si）RNA。针对“高分”siRNA 序列设计引物，并在 D. glomerata 叶片总 RNA 提取物中使用 RT-PCR 扩增 438 bp 的 CYDV 片段。RT-PCR 产物的测序证实了以前未描述的 CYDV 群体的存在，其与 CYDV-RPS 的系统发育关系密切。在 D. glomerata 中，2008 年 CYDV 的感染率为 42.3%（n=78），2009 年为 50.0%（n=48）。特异性 RT-PCR 测试还表明，该 D. glomerata 群体携带黑麦草线条病毒（CSV，属 Potyvirus）。在 2008-2009 年测试的所有植物中，这两种病毒的双重感染率为 20.5-22.9%。有趣的是，CYDV 或 CSV 的感染增强了个别草中另一种病毒的发生。讨论了在田间条件下使用 siRNA 测序方法进行病毒调查和其他生态学研究的机会。
