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梨品种S基因型鉴定对梨栽培中授粉品种选择和遗传育种都具有重要意义。本研究利用梨S-RNase基因荧光标记的特异引物PCR扩增获得梨品种荧光标记的cDNA特异产物;进一步完善梨S-RNase基因cDNA芯片,以被检测梨品种cDNA特异序列与梨S-RNase基因cDNA芯片杂交检测不同梨品种S基因型,并发现新的S-RNase基因。结果表明:利用梨S-RNase基因cDNA芯片鉴定了泸定王皮梨、兴山24号、弥渡百合等35个未知S基因型梨品种,确定了各品种的S基因型。结合PCRRFLP及DNA克隆和测序等技术,发现了7个新的S-RNase基因资源,获得了新S-RNase基因序列。序列分析表明各新S-RNase基因均具有S-RNase基因特异区域序列的典型特征;进化分析显示7个新S-RNase基因主要属于蔷薇科苹果亚科S-RNase类群,且存在种间和属间比种内和属内进化关系更近的现象。7个新的S基因分别命名为:PpS_(53)(Pyrus pyrifolia S53)、PpS_(54)、PpS_(55)、PpS_(56)、PpS_(57)、PpS_(58)和PpS_(59),GenBank登录号分别为:KX581753、KX581754、KX581755、KX581756、KX581757、KX581751和KX581752。
Identification of S genotypes of pear cultivars is of great importance to selection of pollination varieties and genetic breeding in pear culture. In this study, pear-RNase gene fluorescent-labeled specific primers were used to amplify the fluorescently-labeled cDNA-specific products of pear cultivars. The cDNA sequence of pear S-RNase gene was further optimized. cDNA microarray hybridization to detect S pear genotypes and found a new S-RNase gene. The results showed that S - RNase gene cDNA microarray identified 35 unknown S genotype pear cultivars including Luding Wangpi pear, Xingshan 24 and Midu lily, and identified the S genotypes of each species. Combined with PCRRFLP and DNA cloning and sequencing techniques, seven new S-RNase gene resources were discovered and the new S-RNase gene sequence was obtained. Sequence analysis showed that all the novel S-RNase genes had the typical features of S-RNase gene specific region sequences. Phylogenetic analysis showed that the seven new S-RNase genes belonged to the S-RNase family of Rosaceae apple subfamily, Between species and within the evolutionary relationship closer phenomenon. Seven new S genes were named as PsS_ (53) (Pyrus pyrifolia S53), PpS_ (54), PpS_ (55), PpS_ (56), PpS_ (57), PpS_ (58) and PpS_ (59) GenBank accession numbers are: KX581753, KX581754, KX581755, KX581756, KX581757, KX581751 and KX581752, respectively.