基于RAD测序技术的黄绿卷毛菇(Floccularia luteovirens)微卫星引物的开发

Development of Microsatellite Primers Floccularia luteovirens based on RAD-seq

黄绿卷毛菇(Floccularia luteovirens)是广泛分布于青藏高原高寒草甸的一种重要的可食用的菌根真菌。先前的研究利用EST-SSR引物已经对该物种的遗传多样性及群落结构进行了研究,但研究表明EST-SSR位点多态性较低,因此想要进一步探讨形成现有遗传分布格局的机制(该物种小尺度空间范围内基株的密度及大小)需要重新开发具有更高多态性的基于核基因的微卫星位点。本研究利用RAD测序技术使用直接测序分析的方法重新开发了12对具有多态性的引物。测序结果去接头拼装后共获得46 036条重叠群。在这些序列当中共扫描到342条含有重复单元的序列,在这当中70.47%为三碱基重复(241),8.19%为二碱基重复(28)。随机选取48对检测,12对具有多态性且测序结果良好,基于3个居群63个个体的遗传多样性研究显示,所有样品中共获得60个单倍型,每个位点的单倍型数量介于2 (GSSR26L)至9 (GSSR7L,GSSR11L)之间。Gst值介于-0.03(GSSR46L)至0.28(GSSR6L)之间、Fst值介于-0.03(GSSR33L)至0.42(GSSR6L)之间。在引物GSSR47L扩增测序中发现,其扩增出的片段不仅具有重复单元数量的变化,重复单元本身也存在突变的现象,这是之前使用聚丙烯凝胶电泳所检测不到的。本研究获得的微卫星引物将为接下来小尺度空间下研究黄绿卷毛菇基株的密度、大小及动态变化提供有力的支持。

Floccularia luteovirens(Physalacriaceae)is an important edible mycorrhizal fungus mushroom,which is widely distributed on alpine meadows of the Qinghai-Tibet Plateau.Expressed sequence tag-simple sequence repeat(EST-SSR)markers have been used to investigate its genetic diversity and community structure in the previous studies,but the results showed that the polymorphism of EST-SSR locus is low.Therefore,in order to further explore the mechanism that led to its current genotype distribution pattern(the densityand size of the genets in the small scale space ofthe species),we need to redevelop the nuclear gene based microsatellite sites with higher polymorphism.In this study,12 pairs of primers with polymorphism were re-developed by direct sequencing analysis using RAD sequencing technology.After Illumina pair-end sequencing and assembled to yield A total of 46 036 contigs were obtained after reassembling the sequences.In these sequences,a total of342 SSRs were detected,ofwhich 70.47% were tri-nucleotide repeats(241)and 8.19% were bi-nucleotide repeats(28).Ofthe 48 pairs randomlyselected,,twelve were polymorphic and sequenced well.Genetic diversity studies based on 63 individuals from 3 populations showed that 60 haplotypes were obtained in all samples,with number of haplotypes ranging from 2(GSSR26L)to 9(GSSR7L,GSSR11L)per locus.Gst value ranged from-0.03(GSSR46L)to 0.28(GSSR6L)and Fst value ranged from-0.03(GSSR33L)to 0.42(GSSR6L).In the amplified sequences of primer GSSR47L,it was found that the amplified fragment not only had the change of the number ofrepeat units,but also the repeat units themselves had mutations,which could not be detected bypolyacrylam-idegel electrophoresis before.The microsatellite markers obtained in this study would provide a powerful support for the studyofthe density,size and dynamic changes ofthe genets of Floccularia luteovirens in small scale space.