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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 is its common disease, often resulting in serious cut. In order to establish a qPCR method that can be used to quantitatively detect the copy number of wheat yellow mosaic virus (WYMV), we compared the WYMV races of different origins with 11 pairs of primers in the conserved regions of the sequence and optimized their annealing temperature , And then establish the q PCR standard curve of each primer. The results showed that the amplification efficiency of 11 pairs of primers ranged from 50.60% to 116.04%, only 2 pairs of primers could be used for subsequent studies. The amplification efficiency was 97.21% (WY-05 and WY-06) and 91.85% (WY- F2 and WY-R2). WY-F2 and WY-R2 were used to analyze the wheat cultivars with high resistance and high susceptibility to WYMV. The results showed that the WYMV copy number of resistant wheat was significantly different. The results showed that the q PCR method was more sensitive than enzyme-linked immunosorbent assay and more suitable for the phenotypic identification of resistant segregant populations, and therefore could be used for the inheritance of resistance to WYMV gene Locate and map cloning work.