基于锈斑蟳简化基因组数据的微卫星DNA标记开发

Development of Microsatellite DNA Markers Based on RAD-seq Data of 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  RADseq  microsatellite  development of primers  genetic diversity

摘要:

锈斑蟳(Charybdis feriata)是我国重要的海洋水产种质资源保护对象，具有较高的经济价值和遗传育种价值，开发微卫星DNA (Microsatellite DNA)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锈斑蟳简化基因组序列信息，开展微卫星相关信息分析，并开发微卫星DNA标记。共检测到2 419 242个微卫星位点，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Primer3 v2.3.6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(H_e)和观测杂合度(H_o)分别为0.503-0.892和0.167-1.000，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、种群遗传分析等研究。

Abstract:

Charybdis feriata is an important marine aquatic species for conservation of germplasm resources in China,with a high economic value and a high genetic breeding value.Developing microsatellite DNA markers is helpful for studying the population ecology and germplasm resources of this species.In this study,the simplified genome information of C.feriata was obtained by restriction site-associated DNA sequencing (RAD-seq),and the microsatellite-related information was analyzed to develop microsatellite DNA markers.The results showed that a total of 2 419 242 Simple Sequence Repeat (SSR) loci were identified,among which the di-nucleotide and mono-nucleotide motifs were the most,each accounting for 43.95% and 28.71% of the total loci.Hexanucleotide repeat motifs were the most diverse,amounting to 287 types;followed by pentanucleotide repeat motifs (275 types).The number of repeats varied among different types of motifs in SSR loci.A total of 21 330 pairs of microsatellite primers for C.feriata were designed by primer3 v2.3.6,and 192 pairs of primers with high polymorphism were selected to be verified in 12 samples of C.feriata.The results showed that 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 (H_e) and observed heterozygosity (H_o) were 0.503-0.892 and 0.167-1.000,respectively,and the Polymorphic Information Content (PIC) was 0.456-0.882.This study showcases that this method is feasible in screening SSR markers for C.feriata,and the obtained primers can be applied in the research of the genetic diversity and population genetics of C.feriata.