云南省6种鹅膏菌DNA条形码序列分析

DNA barcode sequences analysis of 6 kinds of Amanita species from Yunnan province

目的对云南省6种鹤膏菌的核糖体大亚基(nuclear large-subunit ribosomal DNA,nLSU rDNA)及内转录间隔区(internal transcribed spacer,1TS)DNA序列进行比对分析,探究其亲缘关系以找到合适的鉴定鹅膏菌D N A条形码序列的方法。方法采用试剂盒离心柱法提取6种鹅膏菌DNA,以ITS4/ITS5作为丨T S序列引物和LR5/LROR作为nLSU序列的引物进行PC R扩增,后送至华大基因科技有限公司进行测序。所返回的序列进行遗传距离分析及邻接树构建。结果球基鹅膏、红托鹅膏与小豹斑鹅膏3种鹅膏菌亲缘关系较近。以丨T S序列分析,三者遗传距离在0.0202〜0.0801之间。以nL SU序列分析,三者遗传距离在0.0031~0.0303之间。黄毒蝇鹅膏与赭盖鹅膏菌H 2在IT S序列中与黄毒蝇鹅膏20、21、H 7、D l、D16、D 24亲缘关系较近,遗传距离在0.0462~0.0479。赭盖鹅膏菌7与黄毒蝇鹅膏D 14两株菌遗传距离仅有0.1528。从邻接树来看ITS序列存在种内多,聚类分析结果不稳定,明显不同的2个种聚为一支。而nLSU序列可将各种分离开,结论建议鹅膏菌快速鉴定选用nLSU序列作为主要鉴定序列,IT S序列作为辅助序列。

Objective To compare and analyze the nuclear large-subunit ribosomal DNA (nLSU rDNA) and theinternal transcribed spacer (ITS) DNA sequence of 6 kinds of Am anita species from Yunnan province, and exploretheir kinship to find a suitable DNA barcode sequence for identifying Am anita. M ethods DNA of 6 species ofagaricola bacteria was extracted by centrifugation column method, ITS4/ITS5 was used as ITS sequence primer andLR5/LROR was used as nLSU sequence primer for PCR amplification, which was then sent to Bgi for sequencing.The returned sequence was used for genetic distance analysis and adjacency tree construction. R esults Threespecies of A m anita were closely related to ball-based amanita, red agate cream and small leopard spotted amanita. ByITS sequence analysis, the genetic distance of the 3 was between 0.0202 and 0.0801. The genetic distance of nLSUwas between 0.0031 and 0.0303. In ITS sequence, goose ointment and ochratella ochratae H2 were closely related togoose ointment 20, 21, H7, D l, D16 and D24, with a genetic distance of 0.0462-0.0479. The genetic distancebetween ochratoxin 7 and gossamer fly D14 was only 0.1528. From the adjacency tree, ITS sequence had multiple intra-species copies, and the result of clustering analysis was not stable. The nLSU sequence could separate them.Conclusion It is suggested that nLSU sequence should be used as the main identification sequence and ITSsequence as the auxiliary sequence.