基于锈斑蟳简化基因组数据的微卫星标记开发

Development of microsatellite markers based on RAD-seq data of striped swimming crab Charybdis feriata

关键词:  锈斑蟳  简化基因组  微卫星  引物开发  遗传多样性

Key words:  Charybdis feriata, RAD-seq, SSR, Development of primers, Genetic diversity

摘要:

锈斑蟳(Charybdis feriata)是我国重要海洋水产种质资源保护对象,具有较高经济价值和遗传育种价值,开发微卫星标记有助于锈斑蟳种群生态、种质资源等方面研究。本研究基于简化基因组测序技术获得了锈斑蟳简化基因组序列信息,开展了微卫星相关信息分析,并开发了微卫星分子标记。结果显示,通过MISA软件共检测到2 419 242个SSR位点,其中二核苷酸和单核苷酸重复基序数量最多,各占总位点的43.95%和28.71%。六核苷酸重复基序的类型最多,达287种；其次是五核苷酸的重复基序的类型,为275种。不同类型SSR位点基序的重复次数也不同。使用Primer premier v6.0软件共设计出21 330对锈斑蟳微卫星引物,选取多态性高的192对引物在12个锈斑蟳样本中进行验证,有14对引物具有单一条带且多态性较好,每个引物的等位基因数为4-14,期望杂合度(He)和观测杂合度(Ho)分别为0.503-0.892和0.167-1,多态信息含量(PIC)为0.456-0.882。研究表明,本方法在筛选锈斑蟳SSR标记中是可行的,所得引物可以应用于锈斑蟳遗传多样性、种群遗传分析等问题的研究。

Abstract:

Charybdis feriata is an important marine aquatic germplasm resource conservation object in China, with high economic value and genetic breeding value, and the development of microsatellite markers is helpful for the study of its population ecology and germplasm resources. In this study, we obtained the simplified genome sequence information of the striped swimming crab based on RAD- sequencing technology, carried out the analysis of microsatellite related information, and developed microsatellite markers. The results showed that a total of 2,419,242 SSR loci were identified by MISA software, among which the di-nucleotide and single-nucleotide repetitive motifs were the most in number, each accounting for 43.95% and 28.71% of the total loci. The types of hexanucleotide repeat motifs were the most numerous, amounting to 287 types; followed by the types of pentanucleotide repeat motifs, at 275 types. The number of repeats of motifs for different types of SSR loci also varied. A total of 21,330 pairs of microsatellite primers for the striped swimming crab were designed using Primer premier v6.0 software, and 192 pairs of primers with high polymorphism were selected to be verified in 12 samples of the striped swimming crab, 14 pairs of primers had a single band and good polymorphism, and the number of alleles of each primer ranged from 4 to 14, The expected heterozygosity (He) and observed heterozygosity (Ho) were 0.503-0.892 and 0.167-1, respectively, and the polymorphic information content (PIC) was 0.456-0.882. The study showed that this method was feasible in screening SSR markers for the striped swimming crab, and the obtained primers could be applied in the study of the striped swimming crab "s genetic diversity and the genetic analysis of the populations.