试栽羊肚菌分子系统分析

Molecular system analysis of experimental cultivation morels

[目的]为了弄清陕西汉中试栽羊肚菌的种类归属。[方法]收集到汉中地区试栽羊肚菌子实体19个样品,采用CTAB法提取其基因组DNA,使用通用引物PCR扩增5个基因序列(ITS、LSU、EF1-α、RPB1和RPB2)保守区片段,用1. 0%的琼脂糖凝胶电泳对PCR产物进行检测,并对PCR产物送样测序。依据测序结果构建系统发育树,结合系统发育树进行物种归属鉴定。[结果]获取了所有样品的5个基因序列保守区片段,通过测序比对19个样品5个基因序列保守区片段并进行序列联合,对总长度3 025 bp的保守区序列分析并构建系统发育树,依据系统发育树鉴定出汉中试栽羊肚菌分别为六妹羊肚菌(Morchella sextelata)、梯棱羊肚菌(M. importuna)、隐形羊肚菌(M. cryptica)和北方羊肚菌(M. septentrionalis)。[结论]采用多基因联合分析法弄清楚汉中试栽羊肚菌的种类归属。

[Objective]The classification of planting morel species in Hanzhong of Shaanxi province was identified.[Method]A total of 19 samples of morels fruiting bodies were collected from the Hanzhong area and the genomic DNA was extracted by CTAB method. Polymerase chain reaction( PCR) was used to amplify nuclear ribosomal internal transcribed spacer( ITS),domains D1 and D2 of the Ribosomal 28 S large subunit( LSU),translation elongation factor 1-alpha( EF1-α),RNA polymeraseⅡ largest( RPB1) and second largest subunit( RPB2) using universal primers. Five conserved region gene fragments were detected by 1. 0% agarose gel electrophoresis,and the PCR products were sent for sequencing.[Result]Phylogenetic tree was constructed based on the sequencing results,and species identification was performed in combination with phylogenetic tree. As a result,ITS,LSU,EF1-α,RPB1 and RPB2 sequences of all species were obtained. Through sequencing and comparison,every samples had 3 025 bp conserved region. Phylogenetic tree was constructed by the joint analysis of 3 025 bp sequences. The morels were identified as Morchella sextelata,M. importuna,M. cryptica,and M. septentrionalis by the phylogenetic tree.[Conclusion] The species of test cultivation morels were preliminarily ascertained in Hanzhong area of Shaanxi province.