印尼虎鱼RAD-seq数据中微卫星标记的初步筛选及特征分析
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摘要
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13 308 806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26 359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(19 492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Primer 5.0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。
RAD-seq(Restriction-site associated DNA sequencing)was used to simplify genome sequencing of Datnioides Pulcher A total of 13 308 806 high-quality clean reads(HQ clean reads)lengths were obtained and 26 359 simple sequence repeats(SSR)loci were detected in the obtained sequence by SSR search software,which consisted of 496 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(19 492),accounting for 73.95% of the total SSR sites.Except for single nucleotide type,the number of repeats of SSR loci was mainly ranged from 4 to 9,and the number of polymorphic SSR loci was 2 783.About 20 066 pairs of SSR primers were successfully designed with Primer 5.0.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,and 20 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.
RAD-seq(Restriction-site associated DNA sequencing)was used to simplify genome sequencing of Datnioides Pulcher A total of 13 308 806 high-quality clean reads(HQ clean reads)lengths were obtained and 26 359 simple sequence repeats(SSR)loci were detected in the obtained sequence by SSR search software,which consisted of 496 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(19 492),accounting for 73.95% of the total SSR sites.Except for single nucleotide type,the number of repeats of SSR loci was mainly ranged from 4 to 9,and the number of polymorphic SSR loci was 2 783.About 20 066 pairs of SSR primers were successfully designed with Primer 5.0.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,and 20 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.
引文
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[17]房祖业,陈晓东,吴咏诗,等.大刺鳅(Mastacembelus armatus)二、三、四碱基重复微卫星标记的筛选和特征分析[J].海洋与湖沼,2018,49(1):174-182.
[18]王久利,朱明星,徐明行,等.基于RAD-seq技术的异型花SSR信息分析[J].植物研究,2017,37(3):447-452.
[19]Wang B,Xie X,Liu S,et al.Development and characterization of novel microsatellite markers for the Common Pheasant(Phasianus colchicus)using RAD-seq [J].Avian Research,2017,8(1):4.
[20]岳华梅,翟晴,宋明月,等.基于转录组测序的兴国红鲤微卫星标记筛选[J].淡水渔业,2016,46(1):24-28.
[21]陈松波,龚丽,刘海金.牙鲆EST资源的SSR信息分析[J].东北农业大学学报,2010,41(10):82-86.
[22]刘博,邵艳卿,滕爽爽,等.缢蛏(Sinonovacula constricta)EST-SSR分布特征及引物开发利用[J].海洋与湖沼,2012,43(1):132-137.
[23]Metzgar D,Bytof J,Wills C.Selection against frameshift mutations limits microsatellite expansion in coding DNA [J].Genome Res,2000,10(1):72-80.
[24]鲁翠云,孙效文,梁利群.鳙鱼微卫星分子标记的筛选[J].中国水产科学,2005,12(2):192-196.
[25]蔡磊,余露军,陈小曲,等.诸氏鲻虾虎鱼转录组序列中微卫星标记的初步筛选及特征分析[J].生物技术通报,2015,31(9):146-151.
[2]牟希东,李小慧,胡隐昌,等.布氏罗非鱼微卫星位点筛选和群体遗传多样性分析[J].生物技术通报,2009,(S1):236-241.
[3]郑国栋,陈杰,蒋霞云,等.长江草鱼不同群体EST-SSR多态性标记的筛选及其遗传结构分析[J].水生生物学报,2015,39(5):1003-1011.
[4]杨曦.鲫鱼(Carassius auratus)表达序列标签资源的SSR构成与分布分析[J].生物技术通报,2012,(11):139-143.
[5]孔杰,蒋晓红,周洲,等.磁珠富集法开发长臀鮠微卫星分子标记[J].贵州农业科学,2016,44(7):14-17.
[6]曹柱,鲁翠云,郑先虎,等.磁珠富集法筛选方正银鲫的三、四核苷酸重复微卫星DNA[J].淡水渔业,2011,41(1):22-28.
[7]赵良杰,彭新亮,郭旭升.基于转录组数据的银鲴微卫星位点筛选[J].淡水渔业,2018,48(4):23-29.
[8]Norrell A E,Crawley D,Jones K L,et al.Development and characterization of eighty-four microsatellite markers for the red snapper(Lutjanus campechanus)using Illumina paired-end sequencing [J].Aquaculture,2014,430:128-132.
[9]孙典巧,孙悦娜,王日昕,等.鮸鱼EST序列中微卫星标记的初步筛选及特征分析[J].水生生物学报,2011,35(5):753-760.
[10]谢楠,刘凯,冯晓宇,等.乌鳢和斑鳢EST序列微卫星信息分析[J].淡水渔业,2016,46(3):16-22.
[11]谢芳靖,张子平,邹志华,等.大黄鱼EST微卫星标记初步筛选[J].福建水产,2011,33(5):9-14.
[12]石耀华,洪葵,郭希明,等.马氏珠母贝EST微卫星的筛选[J].水产学报,2008,32(2):174-181.
[13]李响,杨楠,赵凯歌,等.蜡梅转录组EST-SSR标记开发与引物筛选[J].北京林业大学学报,2013,35(S1):25-32.
[14]胡景杰,任红艳.RAD测序技术及其在水生生物研究中的应用[J].水产科学,2018,37(1):125-132.
[15]王洋坤,胡艳,张天真.RAD-seq技术在基因组研究中的现状及展望[J].遗传,2014,36(1):41-49.
[16]李海洋,李荣华,夏岩石,等.基于RAD-seq数据开发烟草多态性SSR标记[J].中国烟草科学,2018,39(1):1-9.
[17]房祖业,陈晓东,吴咏诗,等.大刺鳅(Mastacembelus armatus)二、三、四碱基重复微卫星标记的筛选和特征分析[J].海洋与湖沼,2018,49(1):174-182.
[18]王久利,朱明星,徐明行,等.基于RAD-seq技术的异型花SSR信息分析[J].植物研究,2017,37(3):447-452.
[19]Wang B,Xie X,Liu S,et al.Development and characterization of novel microsatellite markers for the Common Pheasant(Phasianus colchicus)using RAD-seq [J].Avian Research,2017,8(1):4.
[20]岳华梅,翟晴,宋明月,等.基于转录组测序的兴国红鲤微卫星标记筛选[J].淡水渔业,2016,46(1):24-28.
[21]陈松波,龚丽,刘海金.牙鲆EST资源的SSR信息分析[J].东北农业大学学报,2010,41(10):82-86.
[22]刘博,邵艳卿,滕爽爽,等.缢蛏(Sinonovacula constricta)EST-SSR分布特征及引物开发利用[J].海洋与湖沼,2012,43(1):132-137.
[23]Metzgar D,Bytof J,Wills C.Selection against frameshift mutations limits microsatellite expansion in coding DNA [J].Genome Res,2000,10(1):72-80.
[24]鲁翠云,孙效文,梁利群.鳙鱼微卫星分子标记的筛选[J].中国水产科学,2005,12(2):192-196.
[25]蔡磊,余露军,陈小曲,等.诸氏鲻虾虎鱼转录组序列中微卫星标记的初步筛选及特征分析[J].生物技术通报,2015,31(9):146-151.
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