基于专化的反转录转座子序列开发鉴定簇毛麦染色质的PCR分子标记

Development of a PCR Molecular Marker to Identify Haynaldia villosa Based on Its Specific Retrotransposon Sequence

通过克隆簇毛麦的专化序列,开发基于其序列的可用于鉴定簇毛麦染色质的PCR分子标记.用根据抗病基因保守域设计的XLS和A3简并引物进行PCR,检测到1条簇毛麦的特异PCR产物,酶切分析与测序结果表明:这条特异带由4类不同的片段组成,且都与反转录转座子具有同源性.以其中1个片段pHv29为探针进行Southern杂交,发现只有含有簇毛麦染色质的材料产生强的杂交信号,表明pHv29是一个对簇毛麦专化的反转录转座子序列.根据pHv29序列重新设计1对特异引物pHv29-F和pHv29-R,用一系列含有簇毛麦染色质的易位系或添加系的基因组DNA为模板进行PCR扩增,电泳结果表明pHv29433可以作为鉴定簇毛麦的染色质的分子标记.

To develop the PCR molecular marker detecting the chromatin of Haynaldia villosa, the sequence specific to H. villosa was isolated and then the molecular marker was developed based on its sequence. A PCR fragment specific to H. villosa was detected when PCR was conducted using the degenerate primers XLS and A3 designed according to the conserved domain of resistant genes. The result of restriction fragment length polymorphism and the sequence information showed that this specific amplicon was composed by four different fragments homologous to retrotransposon. The southern blotting was conducted using the pHv29 as probe and only the line containing the chromatin of H. villosa could produce intensive signal, so pHv29 was a specific retrotransposon of H. villosa. When the PCRs were conducted using the primers （pHv29-F and pHv29-R） designed according to the sequence of pHv29,only the templates containing the chromatin of H. villosa could amplify the specific fragment pHv29433, pHv29433 can be used as a PCR molecular marker to detect the chromatin of H. villosa.