茅山地区南苍术的特异性分子标记研究

摘要

以中国野生南苍术〔Atractylodes lancea(Thunb.)DC.〕全部分布区9个居群(包括江苏句容的茅山居群、射乌山居群和小九华山居群,安徽金寨的燕子河居群和天堂寨居群,河南嵩县的五马寺居群和纸坊居群,湖北的保康后坪居群和红安七里坪居群)为研究对象,进行rbcL、matK、psbA-trnH、ITS和nadF片段序列比对及SRAP指纹图谱比较来筛选江苏句容茅山居群南苍术的特异性分子标记.结果表明:通过5个DNA片段的序列比对,江苏句容茅山居群南苍术在nadF片段的第581位点处碱基为T,其余8个居群为A,可以将江苏句容茅山居群与其余8个居群区分开.利用SRAP随机引物筛选出1条江苏句容茅山居群南苍术特有的长度为323 bp的条带,根据该条带序列设计1对SCAR引物,正向引物序列为5′-ATCTTTGCTATTAACTATTAAGCTATTCTTCGCTATTCA-3′,反向引物序列为5′-CGATGTTGAAATTGCTGTCTCTGCTTTCTATTC-3′,该引物可在江苏句容茅山居群所有南苍术单株中扩增得到长度为268 bp的特异性条带,而在其他居群中均不能扩增出该条带.本研究筛选出的2种特异性标记可以为鉴定南苍术的道地性提供可靠的技术支持.%The study focuses on nine populations from all distribution areas of wild Atractylodes lancea ( Thunb.) DC. in China, including Maoshan population, Shewushan population and Xiaojiuhuashan population in Jurong of Jiangsu, Yanzihe population and Tiantangzhai population in Jinzhai of Anhui, Wumasi population and Zhifang population in Songxian of He' nan, and Houping population in Baokang and Qiliping population in Hongan of Hubei. Taking these nine populations as objects, specific molecular marker of A. lancea from Maoshan population in Jurong of Jiangsu is screened by using sequence alignment of fragments of rbcL, matK, psbA-trnH, ITS and nadF and comparison on SRAP fingerprints. The results show that through sequence alignment of five DNA fragments, the base at the 581th locus in nadF fragment of A. lancea from Maoshan population in Jurong of Jiangsu is A, and that from other eight populations is T, which can distinguish Maoshan population in Jurong of Jiangsu from other eight populations. A specific band with length of 323 bp in A. lancea from Maoshan population in Jurong of Jiangsu is screened by SRAP random primers, and a pair of SCAR primers is designed according to the sequence of this band. The forward primer sequence is 5′-ATCTTTGCTATTAACTATTAAGCTATTCTT CGCTATTCA-3′, and the reverse primer sequence is 5′-CGATGTTGAAATTGCTGTCTCTGCTTTCTA TTC-3′, which can amplify specific band with length of 268 bp from all individuals of A. lancea from Maoshan population in Jurong of Jiangsu, and cannot amplify any band from other populations. The two specific markers screened in this study can provide reliable technical support for identifying the genuineness of A. lancea.