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

小麦黄花叶病毒病是其常发病,常造成严重减产。为建立可用于定量检测小麦黄花叶病毒（WYMV）拷贝数的q PCR方法,本研究对不同来源的WYMV小种序列进行比对分析,在序列保守区域设计引物11对,对其退火温度进行优化,进而建立各引物的q PCR标准曲线。结果发现,11对引物的扩增效率在50.60%~116.04%之间,仅2对引物可用于后续研究,扩增效率分别为97.21%（WY-05和WY-06）和91.85%（WY-F2和WY-R2）。利用其中的WY-F2和WY-R2对高抗和高感WYMV的小麦品种进行分析,结果显示,抗感小麦品种WYMV拷贝数差异极显著。对抗感分离群体266个单株进行分析,发现该q PCR方法灵敏度显著高于酶联免疫吸附剂测定法,更适用于抗感杂交分离群体的表型鉴定,因而可以用于抗WYMV基因的遗传定位与图位克隆工作。

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（ q PCR） method for the detection of Wheat yellow mosaic virus（ WYMV） copy number,the sequences of different WYMV isolates were aligned,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% to116. 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 q PCR 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 resistant/susceptible combination. The results indicated that the WYMV copy numbers of resistant and susceptible wheat varieties were highly significantly different. The sensitivity of the q PCR method was more higher than that of enzyme- linked immunosorbent assay,so it was more suitable for phenotype identification of the segregating population,and could be used for genetic mapping and map- based cloning of WYMV resistant gene.