紫扇贝与海湾扇贝杂交一代EST-SSR分析及分子标记开发
|
摘要
利用紫扇贝与海湾扇贝的杂交扇贝作为试验对象,通过转录组测序对紫海杂交一代扇贝EST(expressed sequence tags)序列进行挖掘、验证,分析微卫星(simple sequence repeats,SSR)在这些序列中的总体分布特征,开发紫海杂交一代扇贝EST-SSR引物。结果显示,紫海杂交扇贝的基因组中含有大量的微卫星DNA,且种类丰富。各种类型出现的频率相差很大,在1～6核苷酸重复基元中,1～2核苷酸是主要重复类型,占总SSR的92.75%。三核苷酸重复序列占比较少,为SSR总数的6.00%,四核苷酸重复序列、五核苷酸重复序列和六核苷酸重复序列的比例仅为1.25%。应用20个杂交一代个体对20对微卫星引物进行PCR扩增筛选,其中13对引物显示出多态性,共检测到36个等位基因,每个位点的等位基因变化从2个到4个不等(平均每个位点2.77),平均观测杂合度(Ho)为0.67,平均期望杂合度为(He)0.53,多态信息含量(PIC)变化范围为0.32～0.69,平均多态信息含量(PIC)为0.44。表明紫海杂交一代扇贝保持了较高的遗传多样性。试验获得的EST-SSR引物,可用于紫海杂交一代扇贝的种质资源开发、定向选育及遗传结构研究。
The F1 hybrid scallops of Argopecten purpuratus(♂)and Argopecten irradians irradians(♀)were used as experimental objects in this study.By Transcriptome sequencing to mine、verify and analyze the general distribution characteristics of microsatellite(SSR)and to develop the EST-SSR primers.The results showed that there are a large amount of microsatellite DNA and abundant in variety in its genome.The frequency of each type varies greatly,among 1～6 nucleotide repeat types,1～2 nucleotide repeats are the main types,accounting for 92.75% of all SSR.The 3 nucleotide repeats only accounted for 6.00% of total number of SSRs,while the least proportioned are 4 nucleotide repeats,5 nucleotide repeats and 6 nucleotide repeats,the proportion of the three was only 1.25%.Twenty pairs of microsatellite primers were used for PCR amplification screening in 20 individuals.A total of 36 alleles were identified from 13 polymorphic SSR loci,ranging from 2 to 4,with an average of 2.77 alleles.The average observed heterozygosity(Ho)and expected heterozygositywas(He)were 0.67 and 0.53,and the polymorphic information content(PIC)ranged from 0.32 to 0.69,and the average polymorphic information content(PIC)was 0.44.It indicated that the hybrid scallops between A.purpuratus and A.irradians maintained a high genetic diversity.The EST-SSR primers obtained in the experiment can be used for the development of germplasm resources,directed breeding and genetic structure of hybrid scallops between A.purpuratus and A.irradians.
The F1 hybrid scallops of Argopecten purpuratus(♂)and Argopecten irradians irradians(♀)were used as experimental objects in this study.By Transcriptome sequencing to mine、verify and analyze the general distribution characteristics of microsatellite(SSR)and to develop the EST-SSR primers.The results showed that there are a large amount of microsatellite DNA and abundant in variety in its genome.The frequency of each type varies greatly,among 1～6 nucleotide repeat types,1～2 nucleotide repeats are the main types,accounting for 92.75% of all SSR.The 3 nucleotide repeats only accounted for 6.00% of total number of SSRs,while the least proportioned are 4 nucleotide repeats,5 nucleotide repeats and 6 nucleotide repeats,the proportion of the three was only 1.25%.Twenty pairs of microsatellite primers were used for PCR amplification screening in 20 individuals.A total of 36 alleles were identified from 13 polymorphic SSR loci,ranging from 2 to 4,with an average of 2.77 alleles.The average observed heterozygosity(Ho)and expected heterozygositywas(He)were 0.67 and 0.53,and the polymorphic information content(PIC)ranged from 0.32 to 0.69,and the average polymorphic information content(PIC)was 0.44.It indicated that the hybrid scallops between A.purpuratus and A.irradians maintained a high genetic diversity.The EST-SSR primers obtained in the experiment can be used for the development of germplasm resources,directed breeding and genetic structure of hybrid scallops between A.purpuratus and A.irradians.
引文
[1]张福绥,何义朝,刘祥生,等.海湾扇贝(Argopecten irradians)引种、育苗及试养[J].海洋与湖沼,1986,17(5):367-374
[2]Guo X.Use and exchange of genetic resources in molluscan aquaculture[J].Reviews in Aquaculture,2010,1(3/4):251-259
[3]张福绥,何义朝,杨红生.海湾扇贝引种工程及其综合效应[J].中国工程科学,2000,2(2):30-35
[4]Huaiping Zheng,Guofan Zhang,Ximing Guo.et al.Inbreeding depression for various traits in two cultured populations of the American bay scallop,Argopecten irradians irradians Lamarck(1819)introduced into China[J].Journal of Experimental Marine Biology and Ecology,2008,364(1):42-47
[5]王春德,刘保忠,李继强,等.紫扇贝与海湾扇贝种间杂交的研究[J].海洋科学,2009,33(10):84-87+91
[6]Gonzalez M L,Perez M C,Lopez D A.Breeding cycle of the northern scallop,Argopecten purpuratus(Lamarck,1819)in southern chile[J].Aquaculture Reasearch,2002,33(11):847-852
[7]胡丽萍,姜黎明,黄晓婷,等.基于16SrDNA基因序列探讨引进物种紫扇贝(Argopecten purpuratus)在海湾扇贝属(Argopecten)中的分类地位[J].海洋与湖沼,2016,47(06),1149-1157
[8]Zhan A,Hu J,Wang X,et al.A panel of polymorphic EST-derived microsatellite loci for the bay scallop(Argopecten irradians)[J].Journal of Molluscan Studies,2006,72(4):436-438
[9]Jizeng J.Progress in Expressed Sequence Tags(EST)Project of Plant Genome[J].Progress in Biochemistry&Biophysics,2001,28(4):494-497
[10]王宣朋,张晓峰,李文升,等.利用SSR、EST-SSR、SNP标记对鲤食物转化率、体厚、体质量3种性状的分析[J].中国水产科学,2011,18(03):565-573
[11]杨成君,王军,穆立蔷,等.人参EST-SSR标记的建立[J].农业生物技术学报,2008,16(1):114-120
[12]李鸥,曹顶臣,张研,等.利用EST-SSR分子标记研究鲤的饲料转化率性状[J].水产学报,2009,33(4):624-631
[13]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(05):1003-1011
[14]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(01):132-137
[15]齐晓艳,董迎辉,姚韩韩,等.文蛤30个微卫星标记的开发及在斧文蛤和帘文蛤中的通用性检测[J].水产学报,2013,37(08):1147-1154
[16]董迎辉,吴国星,姚韩韩,等.泥蚶34个EST-SSR标记的开发及在格粗饰蚶中的通用性检测[J].水产学报,2013,37(01):70-77
[17]Hou R,Bao Z M,Wang S,et al.Transcriptome sequencing and denovo analysis for Yesso scallop(Patinopecten yessoensis)using 454GS FLX[J].PloS One,2011,6(6):e21560
[18]Wang H X,Huan P,Lu X,et al.Ming of EST-SSR markers in clean Meretrix meretrixlarvae from 454shotgun transcriptome[J].Genes and Genetic System,2011,86(3):197-205
[19]Clark M,Thorne M A,Vieira F A,et al.Insight into shell deposition in the Antarctic bivalve Laternula elliptica:gene discovery in the mantle transcriptome using 454pyrosequencing[J].BMC Genmics,2010,11:362
[20]闫国跃,杨帆,白燕远,等.苦玄参转录组EST-SSR引物开发及群体遗传多样性分析[J].中草药,2019,50(01):195-202
[21]应东山,唐浩,韩瑞玺,等.基于转录组测序的油梨EST-SSR引物开发[J].热带作物学报,2018,39(12):2446-2451
[22]牛红艳,吴小平,周春花,等.猪蛔虫全基因组微卫星分布规律[J].基因组学与应用生物学,2018(5):1933-1940
[23]李云峰,刘卫东,高祥刚,等.虾夷扇贝外套膜和肾脏组织cD-NA文库构建以及EST的初步分析[J].中国水产科学,2010,17(03):578-585
[23]李云峰,刘卫东,高祥刚,等.虾夷扇贝外套膜和肾脏组织cD-NA文库构建以及EST的初步分析[J].中国水产科学,2010,17(3):578-585
[24]李军,李白,李斌,等.基于转录组测序的槜李EST-SSR标记开发[J/OL].分子植物育种:1-9[2019-05-06].http://kns.cnki.net/kcms/detail/46.1068.S.20190220.0845.002.html
[25]Zhan AB,Bao ZM,Hu XL,et al.Characterization of 95novel microsatellite markers for Zhikong scallop Chlamys farreri using FIASCO-colony hybridization and EST database mining[J].Fisheries Science,2008,74(3):516-526
[26]张飞飞,陈璇,陈志永,等.黄鳝EST-SSR标记的开发及其通用性检测[J].江西农业大学学报,2017,39(04):792-800
[27]张振,张含国,莫迟,等.红松转录组SSR分析及EST-SSR标记开发[J].林业科学,2015,51(8):114-120
[28]李满堂,张仕林,邓鹏,等.洋葱转录组SSR信息分析及其多态性研究[J].园艺学报,2015,42(6):1103-1111
[29]杜晓华,杨雅萍,朱小佩,等.三色堇转录组SSR分析及分子标记开发[J].园艺学报,2019,46(04):797-806
[2]Guo X.Use and exchange of genetic resources in molluscan aquaculture[J].Reviews in Aquaculture,2010,1(3/4):251-259
[3]张福绥,何义朝,杨红生.海湾扇贝引种工程及其综合效应[J].中国工程科学,2000,2(2):30-35
[4]Huaiping Zheng,Guofan Zhang,Ximing Guo.et al.Inbreeding depression for various traits in two cultured populations of the American bay scallop,Argopecten irradians irradians Lamarck(1819)introduced into China[J].Journal of Experimental Marine Biology and Ecology,2008,364(1):42-47
[5]王春德,刘保忠,李继强,等.紫扇贝与海湾扇贝种间杂交的研究[J].海洋科学,2009,33(10):84-87+91
[6]Gonzalez M L,Perez M C,Lopez D A.Breeding cycle of the northern scallop,Argopecten purpuratus(Lamarck,1819)in southern chile[J].Aquaculture Reasearch,2002,33(11):847-852
[7]胡丽萍,姜黎明,黄晓婷,等.基于16SrDNA基因序列探讨引进物种紫扇贝(Argopecten purpuratus)在海湾扇贝属(Argopecten)中的分类地位[J].海洋与湖沼,2016,47(06),1149-1157
[8]Zhan A,Hu J,Wang X,et al.A panel of polymorphic EST-derived microsatellite loci for the bay scallop(Argopecten irradians)[J].Journal of Molluscan Studies,2006,72(4):436-438
[9]Jizeng J.Progress in Expressed Sequence Tags(EST)Project of Plant Genome[J].Progress in Biochemistry&Biophysics,2001,28(4):494-497
[10]王宣朋,张晓峰,李文升,等.利用SSR、EST-SSR、SNP标记对鲤食物转化率、体厚、体质量3种性状的分析[J].中国水产科学,2011,18(03):565-573
[11]杨成君,王军,穆立蔷,等.人参EST-SSR标记的建立[J].农业生物技术学报,2008,16(1):114-120
[12]李鸥,曹顶臣,张研,等.利用EST-SSR分子标记研究鲤的饲料转化率性状[J].水产学报,2009,33(4):624-631
[13]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(05):1003-1011
[14]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(01):132-137
[15]齐晓艳,董迎辉,姚韩韩,等.文蛤30个微卫星标记的开发及在斧文蛤和帘文蛤中的通用性检测[J].水产学报,2013,37(08):1147-1154
[16]董迎辉,吴国星,姚韩韩,等.泥蚶34个EST-SSR标记的开发及在格粗饰蚶中的通用性检测[J].水产学报,2013,37(01):70-77
[17]Hou R,Bao Z M,Wang S,et al.Transcriptome sequencing and denovo analysis for Yesso scallop(Patinopecten yessoensis)using 454GS FLX[J].PloS One,2011,6(6):e21560
[18]Wang H X,Huan P,Lu X,et al.Ming of EST-SSR markers in clean Meretrix meretrixlarvae from 454shotgun transcriptome[J].Genes and Genetic System,2011,86(3):197-205
[19]Clark M,Thorne M A,Vieira F A,et al.Insight into shell deposition in the Antarctic bivalve Laternula elliptica:gene discovery in the mantle transcriptome using 454pyrosequencing[J].BMC Genmics,2010,11:362
[20]闫国跃,杨帆,白燕远,等.苦玄参转录组EST-SSR引物开发及群体遗传多样性分析[J].中草药,2019,50(01):195-202
[21]应东山,唐浩,韩瑞玺,等.基于转录组测序的油梨EST-SSR引物开发[J].热带作物学报,2018,39(12):2446-2451
[22]牛红艳,吴小平,周春花,等.猪蛔虫全基因组微卫星分布规律[J].基因组学与应用生物学,2018(5):1933-1940
[23]李云峰,刘卫东,高祥刚,等.虾夷扇贝外套膜和肾脏组织cD-NA文库构建以及EST的初步分析[J].中国水产科学,2010,17(03):578-585
[23]李云峰,刘卫东,高祥刚,等.虾夷扇贝外套膜和肾脏组织cD-NA文库构建以及EST的初步分析[J].中国水产科学,2010,17(3):578-585
[24]李军,李白,李斌,等.基于转录组测序的槜李EST-SSR标记开发[J/OL].分子植物育种:1-9[2019-05-06].http://kns.cnki.net/kcms/detail/46.1068.S.20190220.0845.002.html
[25]Zhan AB,Bao ZM,Hu XL,et al.Characterization of 95novel microsatellite markers for Zhikong scallop Chlamys farreri using FIASCO-colony hybridization and EST database mining[J].Fisheries Science,2008,74(3):516-526
[26]张飞飞,陈璇,陈志永,等.黄鳝EST-SSR标记的开发及其通用性检测[J].江西农业大学学报,2017,39(04):792-800
[27]张振,张含国,莫迟,等.红松转录组SSR分析及EST-SSR标记开发[J].林业科学,2015,51(8):114-120
[28]李满堂,张仕林,邓鹏,等.洋葱转录组SSR信息分析及其多态性研究[J].园艺学报,2015,42(6):1103-1111
[29]杜晓华,杨雅萍,朱小佩,等.三色堇转录组SSR分析及分子标记开发[J].园艺学报,2019,46(04):797-806