蜜环菌DNA分子序列杂合特性及其本质  

Characteristics and essence of heterozygosity of the DNA sequences amplified from Armillaria species

作　　者：李寿建 刘幽言 许欣蕾 刘柳 李兵[1] 郭顺星[1] LI Shoujian;LIU Youyan;XU Xinlei;LIU Liu;LI Bing;GUO Shunxing(The Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing 100193,China)

机构地区：[1]中国医学科学院北京协和医学院药用植物研究所,北京100193

出　　处：《菌物学报》2024年第10期114-129,共16页Mycosystema

基　　金：中国医学科学院医学与健康科技创新工程(2021-I2M-1-031);北京协和医学院中央高校基本科研业务费(3332023052)。

摘　　要：蜜环菌是蜜环菌属真菌的统称,由于其既是严重的林木病害病原真菌,又具有重要的食药用价值,因此一直受到广泛重视。蜜环菌属物种形态分类困难,20世纪70年代开始,蜜环菌一直采用基于交配验证的生物种鉴定方法。但随着分子序列在物种鉴定中广泛应用且优势逐渐显现,开始有学者进行蜜环菌的分子鉴定,并初步发现蜜环菌分子序列存在杂合,而DNA分子序列杂合会影响蜜环菌物种鉴定的准确性。为揭示蜜环菌DNA分子序列杂合现状及其本质,本研究随机选取了猪苓和天麻共生蜜环菌各20株,以及野外子实体分离获得的蜜环菌40株进行了ITS、IGS、tef-1α、β-tubulin和LSU 5个DNA片段的序列扩增、测序和分析,揭示了蜜环菌的高比例序列杂合特性,在测序的400条序列中有高达74.25%的序列存在杂合,包括超过50%的存在杂合位点的序列和近20%的移码双峰序列。同时对不同分子片段和不同类型的蜜环菌中的序列杂合情况进行了统计,发现不同分子序列、不同类型蜜环菌的序列杂合占比均超过了65%,甚至有些序列杂合占比高达80%–90%。通过对不同菌株的共37株单倍体菌株分子序列测序分析,揭示了蜜环菌分子序列的杂合特性主要因其两套单倍体基因组之间的序列差异造成,同时存在少量菌株IGS序列移码双峰是由于IGS序列多拷贝之间的序列差异造成。本研究系统揭示了蜜环菌DNA分子序列存在高度杂合,并揭示了其造成序列杂合的本质,为深入开展蜜环菌的分子鉴定、分子系统学研究和遗传育种提供了指导。Fungi of the genus Armillaria have been widely valued because they are not only serious pathogens of forest diseases,but also have important edible and medicinal values.Due to the difficulty of morphological identification,biological species identification methods based on mating verification are used for recognition of Armillaria speices since the 1970’s.With the extensive application and advantages of molecular sequence in species identification,some scholars began to study the molecular identification of Armillaria.Heterozygosity of the molecular sequence of Armillaria was preliminarily discovered,affecting the accuracy of Armillaria species identification.In this study,20 strains of Armillaria symbiotic with Polyporus umbellatus and Gastrodia elata,and 40 strains of wild Armillaria fruiting bodies were randomly selected,and the proportion of sequence heterozygosity of the strains was revealed through sequencing and analysis of ITS,IGS,tef-1α,β-tubulin,and LSU 5 DNA fragments.It was found that 74.25%of the 400 sequences determined were heterozygous,including more than 50%of the sequences with heterozygous loci and nearly 20%of the sequences with frame shifted bimodal.Statistical analysis of the sequence heterozygosity of different molecular fragments and different types of Armillaria strains found that the heterozygosity ratios exceeded 65%,and even as high as 80%–90%for some sequences.Additional analysis of the molecular sequence of 37 haploid strains revealed that the heterozygosity of the molecular sequence was mainly caused by the sequence difference between the two sets of haploid genomes,and the frame shifted bimodal of IGS sequence of a few strains was caused by the sequence difference between the multiple copies of IGS sequence.This study systematically revealed the DNA molecular sequence heterozygosity of Armillaria and the essence of the sequence heterozygosity,providing guidance for further molecular identification,phylogenetic studies,and genetic breeding of Armillaria species.

关 键 词：单倍体菌株 猪苓 天麻 共生 

分 类 号：S567.3[农业科学—中草药栽培]