柱状田头菇和杨柳田头菇B交配型位点特征分析及其应用

Characterization of B Mating Loci Identification and Application in Cyclocybe aegerita and C.salicacola

柱状田头菇(茶树菇)Cyclocybe aegerita在我国广泛种植,其菌株资源分为多个类群且遗传背景多样。多种命名和复杂的交配特征给柱状田头菇及近缘种的育种研究带来障碍。以两个物种柱状田头菇和杨柳田头菇C.salicacola菌株群体为研究材料,通过基因组测序及序列分析,设计特异引物扩增获取菌株群体的交配区域片段,用于菌株鉴定及系统构建。克隆获得了两个柱状田头菇菌株的B交配位点,其中约8 kb的片段序列非常保守,包括信息素受体rcb1、rcb2和部分非编码区。基于该区域含有变异区的片段设计特异引物,经对35个菌株扩增及克隆测序后,每个实验菌株中均获得1-2类型约3.2 kb片段(cbmr)。使用该片段与线粒体小亚基构建的聚类树契合度较高,但前者在类群的划分上更为精细。此外,该片段的变异区也为类群和交配型鉴定提供了靶位点。依据B交配型部分位点设计的引物在所有的实验菌株中均获得相应的片段,可以作为分子标记用于柱状田头菇和杨柳田头菇菌株及B交配型分子鉴定。

The mushroom Cyclocybe aegerita(Agrocybe aegerita)is cultivated widely in China,with rich natural resources and varied genetic background.The confused nomenclature and complex mating behavior hamper the cognition of genetic breeding for C.aegerita.Strain groups of two species,C.aegerita and C.salicacola were employed,and special primer was designed for amplifying to acquire the mating locus fragment of strain group via genome sequencing and sequence analysis for the strain identification and phylogenetic construction.As results,two B mating loci were cloned from two stains of C.aegerita,and a fragment about 8 kb including rcb1,rcb2 and partial noncoding region were conserved.The special primers were designed based the fragments containing variable regions,and then 1-2 types of about 3.2 kb fragments(cbmr)from each tested strain were obtained after the amplification and clone sequencing of 35 strains in the conserved B mating region.The classification of strains based on the cbmr showed high fitness in phylogenetic tree with that of mitochondrial small-subunit(mtSSU),but the former revealed the genetic background of strains more finely.Additionally,the variable regions provided efficient targeting sites for the group and mating type identification.In conclusion,based on the sequence characterization of B mating locus,the corresponding products can be obtained from all tested strains,which can be used as molecular markers for strain and B mating type identification of C.aegerita and C.salicacola.