印尼虎鱼RAD-seq数据中微卫星标记的初步筛选及特征分析

采用RAD-seq(Restriction-site associated DNA sequencing)对印尼虎鱼(Datnioides pulcher)进行简化基因组测序,获得13308 806个高质量干净读长(HQ clean reads),利用SSR搜索软件在所得序列中共检测到26359个SSR位点,由496种重复基序组成,主要分布在三、四、五碱基重复类型中,单碱基重复序列中最多的类型为A/T,二碱基重复序列中以AC/GT和AG/GT重复单元为主,三碱基重复序列中以AGG/CCT、AGC/CTG、AGG/CCT为优势类型;在数量上,二碱基重复类型SSR位点最多(19492个),占总SSR位点的73.95%;除了单核苷酸类型外,SSR基序的重复次数主要集中在4-9之间,多态性SSR数目为2 783个。根据SSR位点两翼序列,利用Primer5.0成功设计出20 066对SSR引物,引物设计成功率为76.13%,依据测序数据中的重复类型和重复数,随机选择100个二至五碱基重复类型SSR在6个虎鱼样本中验证其可用性和多态性,有73对引物通过扩增可获得有效目的片段,其中20个SSR验证为具有多态性。开发所得SSR序列可用于印尼虎鱼及其近缘物种遗传分析、遗传图谱构建以及分子标记辅助育种等工作,同时也证实了利用简化基因组测序技术开发SSR标记和发现样品间具有多态性的SSR标记的可能性。

Preliminary screening and characterization of microsatellite markers in RAD-seq data of Datnioides pulcher

RAD-seq ( Restriction-site associated DNA sequencing)was used to simplify genome sequencing of Datnioides Pulcher A total of13308 806 high-quality clean reads ( HQ clean reads)lengths were obtained and26359 simple sequence repeats ( SSR)loci were detected in the obtained sequence by SSR search software,which consisted of496 repeat motifs.The most common type of repeat was A/T in single base repeat sequence.AC/GT and A G/GT repeat units in double base repeat sequence.AGG/CCT,AGC/CTG and AGG/CCT were the dominant types in the three base repeat sequence.In quantity,the two base repeat type SSR loci were the largest (19492),accounting for 73.95% of the total SSR sites.Except for single nucleotide type,the number of repeats of SSR loci was mainly ranged from4 to 9,and the number of polymorphic SSR loci was2 783.About20 066 pairs of SSR primers were successfully designed with Primer5.0.The success rate of primer design was 76.13%.According to the repetition type and repetition number in the sequencing data,100 two to five base repetition type SSRs were randomly selected and validated by PCR and polyacrylamide gel in 6 Datnioides Pulcher samples.A total of 73 pairs of primers were amplified by electrophoresis,and20 of them were polymorphic.The developed SSR sequences can be used for genetic analysis,genetic map construction and molecular marker assisted breeding of Datnioides Pulcher and its related species.