青海小海绵羊肚菌M12-10转录组SSR信息分析及其分子标记开发

Analysis on SSR information in transcriptome and development of molecular markers of Morchella spongiola M12-10 in Qinghai

为明确羊肚菌转录组SSR总体特征、开发出适用于分子育种的SSR引物,对分离自青海大通的小海绵羊肚菌M12-10菌丝体高通量转录组测序,采用MISA分析小海绵羊肚菌M12-10转录组SSR位点信息,利用Primer 3.0设计引物,并选择12对引物将19株不同种、不同采集地的野生羊肚菌进行聚类分析。结果表明:高通量测序组装得到153326个转录本,100672个SSR位点分布在20043条Unigenes基因上;SSR发生频率为19.9%,单核苷酸(9268,46.0%)和三核苷酸(5183,25.0%)是主要的重复类型,优势重复基元为A/T(33.0%)、GA/AG(12.0%)和TGC/CAG(40.0%);基序长度集中在10~20bp的比例为73.9%,具有高多态性。基于SSR的20043条Unigene成功设计了14894对引物,随机筛选的12对SSR引物中4对引物表现稳定可重复的多态性;遗传相似系数为0.55时,19株羊肚菌菌株分为两类;相似系数为0.84时,19株羊肚菌全部分开。故基于小海绵羊肚菌M12-10转录组数据SSR标记开发是可行有效的,筛选的4对高频率、高多态性的SSR引物将有助于开展种质资源评价和遗传多样性分析。

In order to characterize SSRs derived from transcriptome of Morchella and to develop SSRmarkers which is suitable for molecular breeding,the study performed Illamin High-through transcriptome sequencing on the mycelium of M.spongiola M12-10.MISA software was employed to analyze the distribution frequency and the basic characteristics of repeat motifs of SSR loci from the transcriptome of mycelium M.spongiola M12-10.SSR primers were designed using Primer 3.Twelve pairs of primers were polymorphism and cluster analysis detection in 19 different species different collection sites of wild morel.The results showed that a total of 153326 Unigenes were obtained by co-assembly,and 100672 SSR loci were distributed on 20043 Unigenes genes.The SSR frequency was 19.9%,and single nucleotide(9268,46.0%)and trinucleotide(5183,25.0%)were the main repeat types.The dominant repeat motifs were A/T(33.0%),GA/AG(12.0%)and TGC/CAG(40.0%);and the ratio of motifs length to 10-20bp was 73.9%,with high polymorphism.Based on the aequences of 20043 Unigenes,14894 pairs of SSR primers were successfully designed.Four pairs of primers in 12 pairs of SSR primers had stable and repeatable polymorphisms.When the genetic similarity coefficient was 0.55,19 Morchella strains were divided into two types.When the similarity coefficient was 0.84,they were all separated,respectively.The above results indicated that the development of SSR markers based on the transcriptome sequence of M.spongiola M12-10 is feasible and effective.The screening of 4 pairs of high frequency and high polymorphism SSR primers will contribute to facilitate the evaluation of germplasm resources and genetic diversity analysis.