11份蜜环菌种质rDNA-ITS序列分析

Sequence Analysis of rDNA-ITS of 11 Armillaria mellea Strains

本研究以收集自贵州省(贵州大学农学院M-01~M-07、都匀市M-08、凯里市M-09和福泉市M-11)和湖北省宜昌市(M-10)的11份蜜环菌种质为研究对象,提取其基因组DNA,采用通用引物ITS4/ITS5对核糖体转录间隔区(rDNA-ITS)进行扩增,利用DNAMAN软件对11份蜜环菌种质进行构建同源树并分析其序列特征,采用MEGA软件最近邻距法进行系统发育分析及遗传距离的计算。结果表明,11份蜜环菌种质可分为两个大类4个亚类,第Ⅰ-Ⅰ类包括M-01、M-02、M-04、M-05和M-08-M-10,第Ⅰ-Ⅱ类为M-03,第Ⅱ-Ⅰ类包括M-06和M-07,第Ⅱ-Ⅱ类为M-11,具有较强的同源性(93%);两个大类样品的特征序列均位于ITS1和ITS2区表现为碱基的转换或颠换;共有序列的相似度为88.71%在ITS区存在丰富的变异表现为碱基的转换、颠换和缺失,第Ⅰ类的GC含量(44.2%)高于第Ⅱ类(42.6%);种内遗传距离均为0,第Ⅰ-Ⅱ类和第Ⅱ-Ⅱ类的种间遗传距离最小(0.002),第Ⅱ-Ⅰ类和第Ⅱ-Ⅱ类的种间遗传距离最大(0.035);经NCBI数据库BLAST对比及系统发育分析这4个亚类的样品均不能与下载的蜜环菌归为一支。因此,11份密环菌种质可能是一个较为庞大的复合群,采用通用引物ITS4/ITS5扩增的核糖体基因可以对此类蜜环菌进行分类但不适合作为鉴定的持家基因。

In this study,the 11 Armillaria mellea strains were collected from Guizhou Province(M-01~M-07 of Agricultural College of Guizhou University,M-08 of Duyun City,M-09 of Kaili City and M-11 of Fuquan City)and Yichang city of Hubei Province(M-10)were used as the object of study.Their genome DNA was extracted as templates of PCR amplification and gene segments of ribosomal DNA ITS were amplified using PCR amplifying technology.DNAMAN was used to construct homologous trees and analyze the sequence characteristics of 11 Armillaria mellea strains,MEGA was used to analyze the phylogeny and calculate the genetic distance.The results showed that the 11 Armillaria mellea strains could be divided into two groups and four subclass,the classⅠ-Ⅰincludes M-01,M-02,M-04,M-05 and M-08-M-10,the classⅠ-Ⅱwas M-03,the classⅡ-Ⅰincludes M-06 and M-07,the classⅡ-Ⅱwas M-11,which had strong homology(93%);The characteristic sequences of the two types of samples were located in ITS1 and ITS2 regions,showing the conversion or transformation of bases;The similarity of the common sequences was 88.71%,and there were abundant variations in ITS region,showing the conversion,transformation and absence of bases,and the GC content of the classⅠ(44.2%)higher than that of the classⅡ(42.6%);The intraspecific genetic distance were 0,the genetic distance between species of the classⅠ-ⅡandⅡ-Ⅱwas minimal(0.002),and the classⅡ-ⅠandⅡ-Ⅱwas the largest(0.035);According to NCBI database BLAST comparison and analyze the phylogeny,These four subclass of samples could not be classified with the downloaded Armillaria mellea.So the 11 Armillaria mellea strains be a relatively large composite group,the ribosomal genes amplified by universal primers ITS4/ITS5 could be used to classify Armillaria mellea but were not suitable for identification as housekeeping genes.