基于AOX1和MCM5基因水芹地方品种的遗传关系分析及‘玉祁红芹’ISSR-SCAR标记建立

Analysis on genetic relationship of local cultivars of Oenanthe javanica based on AOX1 and MCM5 genes and establishment of ISSR-SCAR marker for‘Yuqihongqin’

基于低拷贝核基因AOX1和MCM5的扩增结果,对采自南京的水芹〔Oenanthe javanica(Bl.)DC.〕野生种和太湖流域6个地方品种进行了遗传多样性分析,并基于ISSR标记开发了两步法鉴定‘玉祁红芹’(‘Yuqihongqin’)的SCAR标记。结果显示:水芹6个地方品种的AOX1基因中检测到22个SNP位点和7个InDel位点;MCM5基因中检测到33个SNP位点,其中3个位于编码区且均为同义突变,还有1个位于非编码区的InDel位点。与MCM5基因相比,AOX1基因提供的信息位点更多,基于AOX1基因构建的水芹野生种和6个地方品种的ML遗传关系树的支持率相对较高;其中‘玉祁红芹’、‘苏州圆叶芹’(‘Suzhouyuanyeqin’)和‘梅南水芹’(‘Meinanshuiqin’)的遗传关系较近,而‘常熟白芹’(‘Changshubaiqin’)、‘溧阳白芹’(‘Liyangbaiqin’)和‘江阴青芹’(‘Jiangyinqingqin’)的遗传关系较近。基于ISSR-880引物扩增得到‘溧阳白芹’的特异性片段,设计的第1对SCAR引物鉴定出‘玉祁红芹’和‘溧阳白芹’2个地方品种,再利用ISSR-836引物扩增得到‘玉祁红芹’特异性片段,设计的第2对SCAR引物在‘玉祁红芹’所有单株中扩增出特异性条带,而在‘溧阳白芹’所有单株中均未出现此条带。本研究开发的两步法鉴别‘玉祁红芹’的ISSR-SCAR标记可为水芹其他地方品种的鉴定、保存和利用提供参考。

The genetic diversity of wild species of Oenanthe javanica(Bl.)DC.collected from Nanjing and 6 local cultivars in Taihu Lake basin was analyzed based on the amplification results of low copy nuclear gene AOX1 and MCM5,and SCAR markers of a two-step identification method for‘Yuqihongqin’were developed based on ISSR markers.The results show that 22 SNP loci and 7 InDel loci are detected in AOX1 gene in 6 local cultivars of O.javanica;33 SNP loci are detected in MCM5 gene,in which,3 SNP loci are synonymic mutations and located in coding region,and 1 InDel locus is located in non-coding region.Compared with MCM5 gene,AOX1 gene provides more information sites,and the support rate of ML genetic relationship tree of wild species and 6 local cultivars of O.javanica based on AOX1 gene is relatively high;in which,the genetic relationship of‘Yuqihongqin’,‘Suzhouyuanyeqin’,and‘Meinanshuiqin’is relatively close,and that of‘Changshubaiqin’,‘Liyangbaiqin’,and‘Jiangyinqingqin’is relatively close.The specific fragment of‘Liyangbaiqin’is amplified with ISSR-880 primer,and 2 local cultivars of‘Yuqihongqin’and‘Liyangbaiqin’are identified with the first designed pair of SCAR primers.Afterwards,the specific fragment of‘Yuqihongqin’is amplified by using ISSR-836 primer,and specific bands can only be amplified in all individuals of‘Yuqihongqin’with the second designed pair of SCAR primers,while these bands are not appearing in all individuals of‘Liyangbaiqin’.The ISSR-SCAR markers of the two-step identification method for‘Yuqihongqin’developed in this study can provide references for the identification,preservation,and utilization of other local cultivars of O.javanica.