基于转录组的曼氏无针乌贼SSR与SNP位点信息分析
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摘要
利用IlluminaHiseq平台对曼氏无针乌贼进行了转录组测序,采用生物信息学方法分析了转录组序列中的SSR、SNP位点信息并设计了SSR引物。利用MISA工具筛选了转录组测序获得的127 575条Unigene序列,共得到分布于50 626条序列中的SSR位点108 685个,SSR发生率39.68%,出现频率为85.19%。平均每949 bp含有1个SSR位点。在50626条含有SSR位点的Unigene序列中,有25 548条Unigene包含SSR位点数目在2个及以上。其中单碱基重复是EST微卫星序列的主要形式(42.84%),其次是二碱基重复(28.73%),三碱基重复(14.93%)和四碱基重复(12.79%)。在所有微卫星序列所包含的重复单元中,优势重复基元类型为A/T(占总SSRs的42.23%),其次为AT/AT (13.33%),AC/GT (9.32%),AAAG/CTTT(10.00%)。运用Primer premier 5软件共设计了46 520对SSR引物。在12 323条Unigene中发掘出SNP位点64 732个,每条Unigene平均含5.25个SNP位点。其中转换(Transition) 45 975个(71.02%),颠换(Transversion) 18 757个(28.98%)。本研究通过第二代高通量测序技术,结合生物信息学方法对曼氏无针乌贼进行了SSR位点与SNP位点的开发,为其遗传多样性分析、分子辅助育种和增殖放流效果评估等提供了有力研究工具。
Transcriptome sequencing was performed on Sepiella japonica using the Illumina Hiseq platform. SSR and SNP locus information in transcriptome sequences was analyzed using bioinformatics methods,and SSR primers were designed. By using MISA, 127 575 Unigene sequences obtained from transcriptome sequencing were screened. A total of 108 685 SSRs were found in 50 626 sequences. The incidence of SSR was39.68% and the frequency of occurrence was 85.19%. On average, every 949 bp contains 1 SSR site. 25 548 Unigene contain two or more SSR sites. Mono-nucleotide SSR is the main type of microsatellites(42.84%), foll owed by di-nucleotide(28.73%), tri-nucleotide(14.93%) and quad-nucleotide(12.79%). A/T(42.23%), AT/AT(13.33%), AC/GT(9.32%), AAAG/CCCT(10.00%) were the main types of SSRs. A total of 46 520 SSR primers were designed using Primer premier 5 software. In 12 323 Unigene, 64 432 SNP sites were discovered,and each Unigene contained an average of 5.25 SNP sites. And the Transition sites were 45 975(71.02%), the Transversion were 18 757(28.98%). In this study, SSRs and SNPs were developed using the high-throughput sequencing technology combined with bioinformatics methods for the analysis of genetic diversity, molecularassisted breeding, and proliferation and release of S. japonica.
Transcriptome sequencing was performed on Sepiella japonica using the Illumina Hiseq platform. SSR and SNP locus information in transcriptome sequences was analyzed using bioinformatics methods,and SSR primers were designed. By using MISA, 127 575 Unigene sequences obtained from transcriptome sequencing were screened. A total of 108 685 SSRs were found in 50 626 sequences. The incidence of SSR was39.68% and the frequency of occurrence was 85.19%. On average, every 949 bp contains 1 SSR site. 25 548 Unigene contain two or more SSR sites. Mono-nucleotide SSR is the main type of microsatellites(42.84%), foll owed by di-nucleotide(28.73%), tri-nucleotide(14.93%) and quad-nucleotide(12.79%). A/T(42.23%), AT/AT(13.33%), AC/GT(9.32%), AAAG/CCCT(10.00%) were the main types of SSRs. A total of 46 520 SSR primers were designed using Primer premier 5 software. In 12 323 Unigene, 64 432 SNP sites were discovered,and each Unigene contained an average of 5.25 SNP sites. And the Transition sites were 45 975(71.02%), the Transversion were 18 757(28.98%). In this study, SSRs and SNPs were developed using the high-throughput sequencing technology combined with bioinformatics methods for the analysis of genetic diversity, molecularassisted breeding, and proliferation and release of S. japonica.
引文
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[13] JIANG Dong, YE Qingliang, WANG Fusheng, et al. The Mining of Citrus Est-snp and Its Application in Cultivar Discrimination[J]. Agricultural Sciences in China, 2010, 9(2):179-190.
[14]李齐发.中国牦牛分子系统发育的初步研究[D].南京:南京农业大学, 2004.
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[16]王艳红,胡超群,张吕平,等.凡纳滨对虾EST微卫星标记初步筛选[J].水产学报, 2011, 35(7):969-976.
[17]韩晓飞.白梭吻鲈微卫星分子标记开发及GHR基因的克隆表达分析[D].苏州:苏州大学, 2016.
[18]高峰涛,邵长伟,崔忠凯,等.基于高通量测序的青石斑鱼基因组微卫星开发及评价[J].中国海洋大学学报(自然科学版),2017, 47(4):52-57.
[19] LANDER E S. The New Genomics:Global Views of Biology[J]. Science, 1996, 274(5 287):536-539.
[20] SYV魧NEN A C. Accessing Genetic Variation:Genotyping Single Nucleotide Polymorphisms[J]. Nature Reviews Genetics, 2001,2(12):930-942.
[21]王盼盼.大黄鱼基因组和转录组SNP的挖掘与应用[D].厦门:集美大学, 2016.
[22]李伟亚.利用分子标记评估中国对虾放流效果研究[D].上海:上海海洋大学, 2012.
[23]王朝溪.青海湖裸鲤AFLP指纹图谱的构建以及与生长相关SNP位点的筛选[D].西宁:青海大学, 2015.
[24]全迎春,马冬梅,白俊杰,等.大口黑鲈转录组SNPs筛选及其与生长的关联分析[J].水生生物学报, 2016, 40(6):1 128-1134.
[25]张春兰,秦孜娟,王桂芝,等.转录组与RNA-Seq技术[J].生物技术通报, 2012(12):51-56.
[26] KARIM S, PRUDENCIO A S, GHEBINEJAD A, et al. In Silico Search, Characterization and Validation of New Est-ssr Markers in the Genus Prunus[J]. Bmc Research Notes, 2016, 9(1):336.
[27] PERTEA M, KIM D, LEEK J T, et al. Transcript-level Expression Analysis of Rna-seq Experiments with Hisat, Stringtie and Ballgown[J]. Nature Protocols, 2016, 11(9):1 650-1 667.
[28] AARON M, MATTHEW H, ERIC B, et al. The Genome Analysis Toolkit:a Mapreduce Framework for Analyzing Next-generation Dna Sequencing Data[J]. Genome Research, 2010, 20(9):1 297-1 303.
[29]吴建霞,李亚玲,张智俊,等.芦笋EST资源的SSR信息分析[J].生物信息学, 2012, 10(2):96-100.
[30]吕振明,侯龙,龚理,等.基于de novo高通量测序的曼氏无针乌贼(Sepiella japonica)ESTs中微卫星位点筛选与特征分析[J].海洋与湖沼, 2017, 48(4):877-883.
[31] KELKAR Y D, SVITLANA T, FRANCESCA C, et al. The Genome-wide Determinants of Human and Chimpanzee Microsatellite Evolution[J]. Genome Research, 2008, 18(1):30-38.
[32]李珊珊,孙春玉,蒋世翠,等. SSR分子标记及其在植物遗传育种中的应用[J].吉林蔬菜, 2014(5):33-38.
[33] YE Yingying, WU Changwen, GUO Baoying, et al. Isolation and Characterization of Ten Polymorphic Microsatellite Markers for the Blue Mussel(Mytilus edulis)[J]. Biochemical Systematics and Ecology, 2014, 54:5-7.
[34] XIA Lingmin, GUO Baoying, YE Yingying, et al. Determination of Genetic Diversity of the Cuttlefish Sepiella japonica Inhabiting Chinese Coastal Waters Using the Mitochondrial D-loop Region:the Valuable Inspiration to Artificial Releasing Project[J]. Biochemical Systematics and Ecology, 2016, 69:274-282.
[35]吴欣.基于转录组测序的石磺科贝类SSR和SNP两种分子标记的开发[D].上海:上海海洋大学, 2016.
[36]许家磊,王宇,后猛,等. SNP检测方法的研究进展[J].分子植物育种, 2015, 13(2):475-482.
[2]吴常文,赵淑江,徐蝶娜.舟山渔场针乌贼(Sepia andreana)的生物学特性及其渔场分布变迁[J].海洋与湖沼, 2006, 37(3):231-237.
[3]符方尧,蒋霞敏,柳敏海.曼氏无针乌贼的药用价值及其人工养殖[J].中国水产, 2005(12):26-27.
[4]李继姬.浙江近海曼氏无针乌贼资源演变、EGFP放流标志技术与增殖放流效果评估[D].舟山:浙江海洋学院, 2012.
[5]朱挺兵,颜文斌,杨德国.可见植入荧光标记和微金属线码标记标志拉萨裂腹鱼的研究[J].安徽农业科学, 2016, 44(13):4-5.
[6]付自东,李静,岳碧松,等.用荧光物质浸泡标记胭脂鱼仔、稚鱼耳石[J].动物学杂志, 2005, 40(4):60-65.
[7]许凌雪.杂交种(哲罗鲑♀×细鳞鲑♂)的鉴定和呼玛河哲罗鲑增殖放流效果初步评估[D].上海:上海海洋大学, 2012.
[8]季晓芬,段辛斌,刘绍平,等.基于微卫星评估草鱼放流亲本对野生群体遗传多样性的影响[J].水产学报, 2018, 42(1):10-17.
[9]刘海映,吕海波,崔帆,等.放流三疣梭子蟹遗传多样性和贡献率初步研究[J].水产科学, 2016, 35(6):613-619.
[10]王丽娟.许氏平鲉增殖放流对其荣成俚岛湾群体遗传多样性及鱼类组成的影响[D].青岛:中国科学院研究生院(海洋研究所), 2016.
[11]孙楠.魁蚶增殖放流效果评估与群体遗传多样性分析[D].青岛:中国海洋大学, 2015.
[12]成为为,汪登强,危起伟,等.基于微卫星标记对长江中上游胭脂鱼增殖放流效果的评估[J].中国水产科学, 2014, 21(3):574-580.
[13] JIANG Dong, YE Qingliang, WANG Fusheng, et al. The Mining of Citrus Est-snp and Its Application in Cultivar Discrimination[J]. Agricultural Sciences in China, 2010, 9(2):179-190.
[14]李齐发.中国牦牛分子系统发育的初步研究[D].南京:南京农业大学, 2004.
[15]李薇,李久煊,荆慧芳,等.基于高通量测序的达氏鲟微卫星标记筛选[J].动物学杂志, 2017, 52(3):449-457.
[16]王艳红,胡超群,张吕平,等.凡纳滨对虾EST微卫星标记初步筛选[J].水产学报, 2011, 35(7):969-976.
[17]韩晓飞.白梭吻鲈微卫星分子标记开发及GHR基因的克隆表达分析[D].苏州:苏州大学, 2016.
[18]高峰涛,邵长伟,崔忠凯,等.基于高通量测序的青石斑鱼基因组微卫星开发及评价[J].中国海洋大学学报(自然科学版),2017, 47(4):52-57.
[19] LANDER E S. The New Genomics:Global Views of Biology[J]. Science, 1996, 274(5 287):536-539.
[20] SYV魧NEN A C. Accessing Genetic Variation:Genotyping Single Nucleotide Polymorphisms[J]. Nature Reviews Genetics, 2001,2(12):930-942.
[21]王盼盼.大黄鱼基因组和转录组SNP的挖掘与应用[D].厦门:集美大学, 2016.
[22]李伟亚.利用分子标记评估中国对虾放流效果研究[D].上海:上海海洋大学, 2012.
[23]王朝溪.青海湖裸鲤AFLP指纹图谱的构建以及与生长相关SNP位点的筛选[D].西宁:青海大学, 2015.
[24]全迎春,马冬梅,白俊杰,等.大口黑鲈转录组SNPs筛选及其与生长的关联分析[J].水生生物学报, 2016, 40(6):1 128-1134.
[25]张春兰,秦孜娟,王桂芝,等.转录组与RNA-Seq技术[J].生物技术通报, 2012(12):51-56.
[26] KARIM S, PRUDENCIO A S, GHEBINEJAD A, et al. In Silico Search, Characterization and Validation of New Est-ssr Markers in the Genus Prunus[J]. Bmc Research Notes, 2016, 9(1):336.
[27] PERTEA M, KIM D, LEEK J T, et al. Transcript-level Expression Analysis of Rna-seq Experiments with Hisat, Stringtie and Ballgown[J]. Nature Protocols, 2016, 11(9):1 650-1 667.
[28] AARON M, MATTHEW H, ERIC B, et al. The Genome Analysis Toolkit:a Mapreduce Framework for Analyzing Next-generation Dna Sequencing Data[J]. Genome Research, 2010, 20(9):1 297-1 303.
[29]吴建霞,李亚玲,张智俊,等.芦笋EST资源的SSR信息分析[J].生物信息学, 2012, 10(2):96-100.
[30]吕振明,侯龙,龚理,等.基于de novo高通量测序的曼氏无针乌贼(Sepiella japonica)ESTs中微卫星位点筛选与特征分析[J].海洋与湖沼, 2017, 48(4):877-883.
[31] KELKAR Y D, SVITLANA T, FRANCESCA C, et al. The Genome-wide Determinants of Human and Chimpanzee Microsatellite Evolution[J]. Genome Research, 2008, 18(1):30-38.
[32]李珊珊,孙春玉,蒋世翠,等. SSR分子标记及其在植物遗传育种中的应用[J].吉林蔬菜, 2014(5):33-38.
[33] YE Yingying, WU Changwen, GUO Baoying, et al. Isolation and Characterization of Ten Polymorphic Microsatellite Markers for the Blue Mussel(Mytilus edulis)[J]. Biochemical Systematics and Ecology, 2014, 54:5-7.
[34] XIA Lingmin, GUO Baoying, YE Yingying, et al. Determination of Genetic Diversity of the Cuttlefish Sepiella japonica Inhabiting Chinese Coastal Waters Using the Mitochondrial D-loop Region:the Valuable Inspiration to Artificial Releasing Project[J]. Biochemical Systematics and Ecology, 2016, 69:274-282.
[35]吴欣.基于转录组测序的石磺科贝类SSR和SNP两种分子标记的开发[D].上海:上海海洋大学, 2016.
[36]许家磊,王宇,后猛,等. SNP检测方法的研究进展[J].分子植物育种, 2015, 13(2):475-482.
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