基于转录组数据的毛蚶SSR分子标记开发与评价

Development and Evaluation of SSR Markers Based on Transcriptome Sequencing in Scapharca kagoshimensis

关键词:  毛蚶  转录组  SSR  遗传多样性

Key words:  Scapharca kagoshimensis  Transcriptome  SSR  Genetic diversity

摘要:

本研究基于毛蚶(Scapharca subcrenata)的转录组数据，利用MISA软件对其中的微卫星位点进行挖掘。从35 555条unigene中共获得3987个SSR，SSR出现频率达11.21%。SSR重复类型主要以二核苷酸重复为主(58.06%)，其次为三核苷酸重复(19.04%)。共有182种重复基元，不同类型SSR的重复基元分布特征不同，其中，二核苷酸重复基元中AC/GT类型比例最高，为45.70%。毛蚶转录组中SSR重复次数主要集中在5~7次，SSR长度主要集中在12~29 bp，多态性均在中等以上。利用筛选出的14对SSR引物在山东潍坊毛蚶野生群体中进行遗传多样性分析，结果显示，平均有效等位基因数(Na)、平均观测杂合度(Ho)、平均期望杂合度(He)和多态性信息含量(PIC)分别为15.4、0.682、0.852和0.817，从PIC值来看，本研究开发的14个微卫星标记均属高多态性标记(PIC≥0.5)。此外，有7个位点显著偏离哈迪–温伯格平衡(Hardy-Weinberg equilibrium, HWE) (P<0.05)。结果表明，基于毛蚶转录组数据开发微卫星标记是切实可行的，研究结果丰富了毛蚶的分子标记数量，对毛蚶的种群遗传学分析、遗传图谱构建及分子辅助育种等研究具有重要意义。

Abstract:

Scapharca kagoshimensis is a marine shellfish of great economic value. In recent decades, populations of S. kagoshimensis have declined due to environmental destruction and overfishing. To enhance our understanding of the genetic diversity and population-level genetic structure of S. kagoshimensis, microsatellite loci were detected based on the transcriptome data of S. kagoshimensis using MISA software. A total of 3987 Single Sequence Repeats (SSRs) were identified from 35,555 unigenes and the frequency of their occurrence was 11.21%. The main types of repeats were dinucleotides and trinucleotides, which accounted for 58.06% and 19.04%, respectively. A total of 182 types of repeat motifs were classified in all SSRs, and AC/GT was the most abundant among dinucleotide repeats (45.70%). The repeat numbers of SSRs primarily ranged between five and seven, and the number of SSRs gradually decreased as repeat number increased．Motif length was predominantly between 12 and 29 bp, and the SSR polymorphism level was above moderate. Among the 60 designed primer pairs, 14 pairs proved to be polymorphic microsatellite markers and were amplified in 30 wild individuals sampled from Weifang in Shandong Province. The results showed that the average number of alleles (Na), average observed heterozygosity (Ho), average expected heterozygosity (He), and polymorphism information content (PIC) were 15.4, 0.682, 0.852, and 0.817, respectively. All 14 loci were highly polymorphic (PIC≥0.5). After Bonferroni correction, seven of the 14 loci deviated significantly from the Hardy-Weinberg equilibrium (P<0.05). These results indicate that it is feasible to develop microsatellite markers based on the S. kagoshimensis transcriptome. The polymorphic microsatellite loci obtained in this study will facilitate further studies on population genetic management, genetic mapping, and molecular assisted breeding of S. kagoshimensis.