山东农业科学2016，Vol.48Issue(7)：118-124,132,8.DOI:10.14083/j.issn.1001-4942.2016.07.027
基于 SYBR Green 检测小麦黄花叶病毒的两步法 qPCR
SYBR Green -Based Two -Step Real Time Quantitative PCR (qPCR)for Detection of Wheat yellow mosaic virus
摘要
Abstract
Wheat yellow mosaic virus disease,a common disease in wheat growing regions,can cause severe wheat yield loss.In order to establish proper real time quantitative PCR (qPCR)method for the detec-tion of Wheat yellow mosaic virus (WYMV)copy number,the sequences of different WYMV isolates were a-ligned,as a result,total 11 pairs of primers were designed based on the conserved region sequence,and their annealing temperatures were optimized.Then,the standard curves of these 11 primer pairs were established, respectively.The results showed that the PCR efficiencies of 11 primer pairs ranged from 50.60% to 116.04%,and only two primer pairs could be used for further research with the PCR efficiencies of 97.21%(WY -05 and WY -06)and 91.85% (WY -F2 and WY -R2).In order to testify their utility,the qPCR using WY -F2 and WY -R2 as primer pair was performed on WYMV resistant wheat varieties,susceptible wheat varieties and F2 segregating population consisting of 266 individual plants which derived from wheat re-sistant/susceptible combination.The results indicated that the WYMV copy numbers of resistant and suscepti-ble wheat varieties were highly significantly different.The sensitivity of the qPCR method was more higher than that of enzyme -linked immunosorbent assay,so it was more suitable for phenotype identification of the segre-gating population,and could be used for genetic mapping and map -based cloning of WYMV resistant gene.关键词
小麦黄花叶病毒/两步法 qPCR/酶联免疫吸附剂测定法Key words
Wheat yellow mosaic virus/Two -step real time quantitative PCR/Enzyme -linked immu-nosorbent assay分类
植物保护学引用本文复制引用
刘成,刘建军,小松田隆夫,铃木孝子,佐久间俊,宋健民,李豪圣,刘爱峰,曹新有,韩冉,宫文萍..基于 SYBR Green 检测小麦黄花叶病毒的两步法 qPCR[J].山东农业科学,2016,48(7):118-124,132,8.基金项目
日本农林水产省和学术振兴会项目（TRS1003、P11515）；国家现代农业产业技术体系专项（CARS －03－1－8）；山东省农业科学院青年拔尖人才科研启动费（1－18－24）；泰山学者种业计划项目；山东省自主创新重大关键技术项目 （）
