长江刀鲚选育群体转录组EST-SSR的分布特征分析
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摘要
本研究利用MISA软件挖掘长江刀鲚(Coilia ectenes)肌肉和肝脏转录组中的微卫星标记,为刀鲚选育群体的种质资源评估和分子标记辅助育种奠定基础。结果显示,从71869条Unigenes中共获得33896条重复单元长度为1～6碱基的微卫星序列;刀鲚转录组中不同类型微卫星的重复基序具有不同的分布特征,其中,单核苷酸重复、二核苷酸重复和三核苷酸重复为主要的微卫星重复类型,分别占总微卫星数目的 34.94%、49.47%和13.34%;不同微卫星重复类型的优势重复基序亦有所不同,其中,A/T为单核苷酸重复基序的优势重复基序占86.25%,AC/GT为二核苷酸重复基序的为优势重复基序占75.25%,AGG/CCT为三核苷酸重复基序的优势重复基序占28.57%;不同微卫星重复基序核苷酸的数量和重复次数亦有所不同,重复次数伴随着重复单元中核苷酸数量的增加而呈现降低的趋势;从100对四核苷酸重复的SSR引物中筛选获得了16对多态性微卫星标记,并以此为基础,对长江刀鲚选育群体(F_3)的遗传学特征进行了初步评估,结果显示,长江刀鲚选育群体F3的平均有效等位基因数(N_e)、平均观测杂合度(H_o)、平均期望杂合度(H_e)和Shannon多样性指数I分别为1.7580、0.3414、0.3977和0.6278。以上结果表明,基于刀鲚转录组数据批量开发微卫星是切实可行的,所开发的多态性微卫星标记能够应用于长江刀鲚选育群体的遗传背景评估和进一步的遗传育种研究。
The objective of this study was to identify microsatellites from the transcriptome sequences of Coilia ectenes using MISA software. These microsatellites could create very useful resources in the evaluation of germplasm resource and marker-assisted breeding of C. ectenes. The results showed that a total of 33,896 microsatellites were identified with repeating units, at lengths of 1～6 bases, from 71,869 unigenes. Different types of repeat SSRs had considerably different distribution characteristics. The majority of the microsatellite loci consisted of mono-, di-, and tri-nucleotide motifs(34.94%, 49.47%, and 13.34%, respectively). Dinucleotide microsatellite repeating units were the most abundant in the C. ectenes transcriptome, and the AC/GT repeating units were the most ascendant repeating unit(75.25%). The dominant repeating units for the mononucleotide and trinucleotide motifs were A/T(86.25%) and AGG/CCT(28.57%), respectively. Different nucleotide repeat motifs had different repetitions that had a reducing trend, with the increase in the number of nucleotides in repeat motifs. Among the 100 designed primer pairs, 16 pairs proved to be polymorphic microsatellite markers. In the present study, 16 polymorphic microsatellite loci were characterized to evaluate the genetic diversity of the selected C. ectenes(F_3). The results showed that the average number of effective alleles(N_e), the average observed heterozygosity(H_o), the average expected heterozygosity(He), and the average Shannon′s information index(I) of the selected C.ectenes(F_3) were 1.7580, 0.3414, 0.3977, and 0.6278, respectively. These results indicate that it is feasible to develop microsatellite markers based on the C. ectenes transcriptome and polymorphic microsatellite loci obtained in this study will facilitate further studies on the population genetic management and conservation of C. ectenes.
The objective of this study was to identify microsatellites from the transcriptome sequences of Coilia ectenes using MISA software. These microsatellites could create very useful resources in the evaluation of germplasm resource and marker-assisted breeding of C. ectenes. The results showed that a total of 33,896 microsatellites were identified with repeating units, at lengths of 1～6 bases, from 71,869 unigenes. Different types of repeat SSRs had considerably different distribution characteristics. The majority of the microsatellite loci consisted of mono-, di-, and tri-nucleotide motifs(34.94%, 49.47%, and 13.34%, respectively). Dinucleotide microsatellite repeating units were the most abundant in the C. ectenes transcriptome, and the AC/GT repeating units were the most ascendant repeating unit(75.25%). The dominant repeating units for the mononucleotide and trinucleotide motifs were A/T(86.25%) and AGG/CCT(28.57%), respectively. Different nucleotide repeat motifs had different repetitions that had a reducing trend, with the increase in the number of nucleotides in repeat motifs. Among the 100 designed primer pairs, 16 pairs proved to be polymorphic microsatellite markers. In the present study, 16 polymorphic microsatellite loci were characterized to evaluate the genetic diversity of the selected C. ectenes(F_3). The results showed that the average number of effective alleles(N_e), the average observed heterozygosity(H_o), the average expected heterozygosity(He), and the average Shannon′s information index(I) of the selected C.ectenes(F_3) were 1.7580, 0.3414, 0.3977, and 0.6278, respectively. These results indicate that it is feasible to develop microsatellite markers based on the C. ectenes transcriptome and polymorphic microsatellite loci obtained in this study will facilitate further studies on the population genetic management and conservation of C. ectenes.
引文
Bai CC,Liu SF,Zhuang ZM.Characteristic analysis of microsatellite DNA in the genome of Gobiidae.Progress in Fishery Sciences,2016,37(5):9-15[白翠翠,柳淑芳,庄志猛.虾虎鱼科(Gobiidae)基因组微卫星DNA的分布特征.渔业科学进展,2016,37(5):9-15]
Botstein D,White RL,Skolnick M,et al.Construction of a genetic linkage map in man using restriction fragment length polymorphisms.American Journal of Human Genetics,1980,32(3):314-331
Cai L,Yu LJ,Chen XQ,et al.A preliminary screening and characteristic analysis of microsatellite markers from transcriptome sequences in Mugilogobius chulae.Biotechnology Bulletin,2015(9):146-151[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.生物技术通报,2015(9):146-151]
Chen F,Zeng LY,Cheng QQ.Development of thirty-four novel polymorphic microsatellite markers in Coilia ectenes(Clupeiformes:Engraulidae)and cross-species amplification in two closely related taxa.Journal of Genetics,2012,91(1):1-7
Cheng QQ,Zhang QY,Ma CY,et al.Genetic structure and differentiation of four lake populations of Coilia ectenes(Clupeiformes:Engraulidae)based on mtDNA control region sequences.Biochemical Systematics and Ecology,2011,39(4-6):544-552
Deng PP,Shi YH,Zhang GY,et al.Isolation of microsatellite in C.ectenes by magnetic beads.Biotechnology Bulletin,2014(6):145-149[邓平平,施永海,张根玉,等.磁珠富集法分离刀鲚微卫星标记.生物技术通报,2014(6):145-149]
Ge GC,Cao T,Chen CJ,et al.Analysis of genetic diversity based on amplified fragment length polymorphism fingerprint of Coilia nasus from Yangtze River.Journal of Nanjing University(Natural Sciences),2008,44(3):332-338[葛家春,曹廷,陈婵娟,等.利用扩增片断长度多态性技术分析长江刀鲚的遗传多样性.南京大学学报(自然科学版),2008,44(3):332-338]
Gong SQ,Wang ZY,Xiao SJ,et al.Development and verification of SSR based on transcriptome of yellow drum,Nibea albiflora.Journal of Jimei University(Natural Science),2016,21(4):241-246[龚诗琦,王志勇,肖世俊,等.黄姑鱼转录组SSR的开发与验证.集美大学学报(自然科学版),2016,21(4):241-246]
Gu Y,Lu CY,Zhang Q,et al.Analysis of genetic diversity and growth traits in Cyprinus carpio var.haematopterus using microsatellite QTL markers.Freshwater Fisheries,2016,46(4):9-18[顾颖,鲁翠云,张芹,等.微卫星QTL标记分析豫选黄河鲤群体遗传结构及生长性状相关性.淡水渔业,2016,46(4):9-18]
Guan A,Wu YT,Chen Y,et al.Deep sequence-based transcriptome analysis of microsatellites in the cuttlefish(Sepiella japonica).Progress in Fishery Sciences,2018,39(3):144-151[管奥,毋玉婷,陈宇,等.曼氏无针乌贼转录组微卫星特征分析.渔业科学进展,2018,39(3):144-151]
Guo X,Li Q,Kong LF,et al.Microsatellite-based consensus map of the Pacific oyster(Crassostrea gigas).Journal of Fisheries of China,2013,37(6):823-829[郭香,李琪,孔令锋,等.基于微卫星标记整合长牡蛎遗传图谱.水产学报,2013,37(6):823-829]
Li DM,Yang AG,Wu B,et al.Development and application of the EST-SSR markers in Atrina pectinata.Progress in Fishery Sciences,2017,38(2):137-142[李东明,杨爱国,吴彪,等.栉江珧(Atrina pectinata)EST-SSR标记的开发与应用.渔业科学进展,2017,38(2):137-142]
Ma CY,Cheng QQ,Zhang QY.Genetic variation of Coilia ectenes(Clupeiformes:Engraulidae)revealed by the complete cytochrome b sequences of mitochondrial DNA.Journal of Experimental Marine Biology and Ecology,2010,385(1):14-19
Ma CY,Cheng QQ,Zhang QY.Development of 12 polymorphic microsatellite markers in Coilia ectenes Jordan and Seale,1905(Clupeiformes:Engraulidae)and cross-species amplification in Coilia mystus Linnaeus,1758.Environmental Biology of Fishes,2011,91(2):243-249
Ma CY,Liu M,Ma LB,et al.Genetic diversity in Coilia ectenes by RAPD analysis.Marine Fisheries Research,2004,25(5):19-24[马春艳,刘敏,马凌波,等.长江口刀鲚遗传多样性的随机扩增多态DNA(RAPD)分析.海洋水产研究,2004,25(5):19-24]
Ni SS,Yang Y,Liu SF,et al.Microsatellite analysis of Patinopecten yessoensis using next-generation sequencing method.Progress in Fishery Sciences,2018,39(1):107-113[倪守胜,杨钰,柳淑芳,等.基于高通量测序的虾夷扇贝基因组微卫星特征分析.渔业科学进展,2018,39(1):107-113]
Peakall R,Smouse PE.GENALEX 6:Genetic analysis in Excel.Population genetic software for teaching and research.Molecular Ecology Resources,2006,6(1):288-295
Rong XJ,Xu YJ,Wang QY,et al.Isolation and characterization of polymorphic microsatellite markers from Coilia ectenes.Genetics and Molecular Research,2013,12(4):6011-6017
Shi YH,Zhang GY,Zhang HM,et al.Analysis and comparison of nutritional quality of muscle from Coilia nasus FEDformulated feed and live feed.Chinese Journal of Animal Nutrition,2014,26(2):427-436[施永海,张根玉,张海明,等.配合饲料和活饵料喂养刀鲚肌肉营养品质分析与比较.动物营养学报,2014,26(2):427-436]
Shi YH,Zhang GY,Zhang HM,et al.Complete artificial propagation and embryonic development of Coilia nasus.Journal of Shanghai Ocean University,2015,24(1):36-43[施永海,张根玉,张海明,等.刀鲚的全人工繁殖及胚胎发育.上海海洋大学学报,2015,24(1):36-43]
Sun JJ,Li GF,Liu L,et al.Analysis of microsatellite DNAmarkers emphasis on correlation with some economically important traits in Mandarin fish Siniperca chuatsi.Chinese Journal of Fisheries,2017,30(1):11-18[孙际佳,李桂峰,刘丽,等.翘嘴鳜微卫星标记及其与主要经济性状的相关分析.水产学杂志,2017,30(1):11-18]
Teng SS,Fang J,Shao YQ,et al.Microsatellite analysis of genetic variation in the fast growth families of the second generation of Tegillarca granosa and correlation with growth traits.Acta Hydrobiologica Sinica,2018,42(4):681-689[滕爽爽,方军,邵艳卿,等.泥蚶G2代快速生长家系遗传结构的微卫星分析及其与生长性状的关联.水生生物学报,2018,42(4):681-689]
Wei GL,Xu GC,Gu RB,et al.Studies on the genetic diversity of farmed and wild populations of Coilia nasus by analysing mitochondrial DNA Cytb genes.Acta Agricultural Universitatis Jiangxiensis,2012,34(6):1216-1221[魏广莲,徐钢春,顾若波,等.基于mt DNA Cytb序列分析养殖与野生刀鲚群体的遗传多样性.江西农业大学学报,2012,34(6):1216-1221]
Wei GL,Xu GC,Gu RB,et al.Biology and artificial breeding progress in Coilia nasus.Journal of Yangtze University(Natural Science),2013,9(7):31-35[魏广莲,徐钢春,顾若波,等.刀鲚的生物学及人工养殖研究进展.长江大学学报(自然科学版),2013,9(7):31-35]
Yang M,Yu Y,Zhang XJ,et al.2017.Development of microsatellite markers from the transcriptome sequences of Pacific white shrimp(Litopenaus vannamei).Marine Sciences,41(2):96-102[杨铭,于洋,张晓军,等.基于转录组数据的凡纳滨对虾微卫星标记开发.海洋科学,2017,41(2):96-102]
Yuan CM.The resources of Coilia ectenes in middle and lower Yangtze River,and the status and the causation of the population formation changes.Chinese Journal of Zoology,1988,23(3):12-15[袁传宓.长江中下游刀鲚资源和种群组成变动状况及其原因.动物学杂志,1988,23(3):12-15]
Yue HM,Zhai Q,Song MY,et al.Development of microsatellite markers in Cyprinus carpio var.singuonensis using nextgeneration sequencing.Freshwater Fisheries,2016,46(1):24-28[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.淡水渔业,2016,46(1):24-28]
Zhang D,Fu JJ,Zhang LD,et al.The parentage analysis of bighead carp(Hypophthalmichthys nobilis)based on ten microsatellite markers.Genomics and Applied Biology,2018,1-11[张丹,傅建军,张利德,等.鳙基于10个微卫星标记的亲子鉴定分析.基因组学与应用生物学,2018,1-11]
Zhang YP,He G,Wang S,et al.Analysis of genetic diversity of Coilia nasus and Coilia brachygnathus based on mitochondrial DNA D-loop sequence.Jiangsu Agricultural Sciences,2017,45(20):59-64[张燕萍,贺刚,王生,等.基于线粒体DNA D-loop序列分析刀鲚与短颌鲚的遗传多样性.江苏农业科学,2017,45(20):59-64]
Botstein D,White RL,Skolnick M,et al.Construction of a genetic linkage map in man using restriction fragment length polymorphisms.American Journal of Human Genetics,1980,32(3):314-331
Cai L,Yu LJ,Chen XQ,et al.A preliminary screening and characteristic analysis of microsatellite markers from transcriptome sequences in Mugilogobius chulae.Biotechnology Bulletin,2015(9):146-151[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.生物技术通报,2015(9):146-151]
Chen F,Zeng LY,Cheng QQ.Development of thirty-four novel polymorphic microsatellite markers in Coilia ectenes(Clupeiformes:Engraulidae)and cross-species amplification in two closely related taxa.Journal of Genetics,2012,91(1):1-7
Cheng QQ,Zhang QY,Ma CY,et al.Genetic structure and differentiation of four lake populations of Coilia ectenes(Clupeiformes:Engraulidae)based on mtDNA control region sequences.Biochemical Systematics and Ecology,2011,39(4-6):544-552
Deng PP,Shi YH,Zhang GY,et al.Isolation of microsatellite in C.ectenes by magnetic beads.Biotechnology Bulletin,2014(6):145-149[邓平平,施永海,张根玉,等.磁珠富集法分离刀鲚微卫星标记.生物技术通报,2014(6):145-149]
Ge GC,Cao T,Chen CJ,et al.Analysis of genetic diversity based on amplified fragment length polymorphism fingerprint of Coilia nasus from Yangtze River.Journal of Nanjing University(Natural Sciences),2008,44(3):332-338[葛家春,曹廷,陈婵娟,等.利用扩增片断长度多态性技术分析长江刀鲚的遗传多样性.南京大学学报(自然科学版),2008,44(3):332-338]
Gong SQ,Wang ZY,Xiao SJ,et al.Development and verification of SSR based on transcriptome of yellow drum,Nibea albiflora.Journal of Jimei University(Natural Science),2016,21(4):241-246[龚诗琦,王志勇,肖世俊,等.黄姑鱼转录组SSR的开发与验证.集美大学学报(自然科学版),2016,21(4):241-246]
Gu Y,Lu CY,Zhang Q,et al.Analysis of genetic diversity and growth traits in Cyprinus carpio var.haematopterus using microsatellite QTL markers.Freshwater Fisheries,2016,46(4):9-18[顾颖,鲁翠云,张芹,等.微卫星QTL标记分析豫选黄河鲤群体遗传结构及生长性状相关性.淡水渔业,2016,46(4):9-18]
Guan A,Wu YT,Chen Y,et al.Deep sequence-based transcriptome analysis of microsatellites in the cuttlefish(Sepiella japonica).Progress in Fishery Sciences,2018,39(3):144-151[管奥,毋玉婷,陈宇,等.曼氏无针乌贼转录组微卫星特征分析.渔业科学进展,2018,39(3):144-151]
Guo X,Li Q,Kong LF,et al.Microsatellite-based consensus map of the Pacific oyster(Crassostrea gigas).Journal of Fisheries of China,2013,37(6):823-829[郭香,李琪,孔令锋,等.基于微卫星标记整合长牡蛎遗传图谱.水产学报,2013,37(6):823-829]
Li DM,Yang AG,Wu B,et al.Development and application of the EST-SSR markers in Atrina pectinata.Progress in Fishery Sciences,2017,38(2):137-142[李东明,杨爱国,吴彪,等.栉江珧(Atrina pectinata)EST-SSR标记的开发与应用.渔业科学进展,2017,38(2):137-142]
Ma CY,Cheng QQ,Zhang QY.Genetic variation of Coilia ectenes(Clupeiformes:Engraulidae)revealed by the complete cytochrome b sequences of mitochondrial DNA.Journal of Experimental Marine Biology and Ecology,2010,385(1):14-19
Ma CY,Cheng QQ,Zhang QY.Development of 12 polymorphic microsatellite markers in Coilia ectenes Jordan and Seale,1905(Clupeiformes:Engraulidae)and cross-species amplification in Coilia mystus Linnaeus,1758.Environmental Biology of Fishes,2011,91(2):243-249
Ma CY,Liu M,Ma LB,et al.Genetic diversity in Coilia ectenes by RAPD analysis.Marine Fisheries Research,2004,25(5):19-24[马春艳,刘敏,马凌波,等.长江口刀鲚遗传多样性的随机扩增多态DNA(RAPD)分析.海洋水产研究,2004,25(5):19-24]
Ni SS,Yang Y,Liu SF,et al.Microsatellite analysis of Patinopecten yessoensis using next-generation sequencing method.Progress in Fishery Sciences,2018,39(1):107-113[倪守胜,杨钰,柳淑芳,等.基于高通量测序的虾夷扇贝基因组微卫星特征分析.渔业科学进展,2018,39(1):107-113]
Peakall R,Smouse PE.GENALEX 6:Genetic analysis in Excel.Population genetic software for teaching and research.Molecular Ecology Resources,2006,6(1):288-295
Rong XJ,Xu YJ,Wang QY,et al.Isolation and characterization of polymorphic microsatellite markers from Coilia ectenes.Genetics and Molecular Research,2013,12(4):6011-6017
Shi YH,Zhang GY,Zhang HM,et al.Analysis and comparison of nutritional quality of muscle from Coilia nasus FEDformulated feed and live feed.Chinese Journal of Animal Nutrition,2014,26(2):427-436[施永海,张根玉,张海明,等.配合饲料和活饵料喂养刀鲚肌肉营养品质分析与比较.动物营养学报,2014,26(2):427-436]
Shi YH,Zhang GY,Zhang HM,et al.Complete artificial propagation and embryonic development of Coilia nasus.Journal of Shanghai Ocean University,2015,24(1):36-43[施永海,张根玉,张海明,等.刀鲚的全人工繁殖及胚胎发育.上海海洋大学学报,2015,24(1):36-43]
Sun JJ,Li GF,Liu L,et al.Analysis of microsatellite DNAmarkers emphasis on correlation with some economically important traits in Mandarin fish Siniperca chuatsi.Chinese Journal of Fisheries,2017,30(1):11-18[孙际佳,李桂峰,刘丽,等.翘嘴鳜微卫星标记及其与主要经济性状的相关分析.水产学杂志,2017,30(1):11-18]
Teng SS,Fang J,Shao YQ,et al.Microsatellite analysis of genetic variation in the fast growth families of the second generation of Tegillarca granosa and correlation with growth traits.Acta Hydrobiologica Sinica,2018,42(4):681-689[滕爽爽,方军,邵艳卿,等.泥蚶G2代快速生长家系遗传结构的微卫星分析及其与生长性状的关联.水生生物学报,2018,42(4):681-689]
Wei GL,Xu GC,Gu RB,et al.Studies on the genetic diversity of farmed and wild populations of Coilia nasus by analysing mitochondrial DNA Cytb genes.Acta Agricultural Universitatis Jiangxiensis,2012,34(6):1216-1221[魏广莲,徐钢春,顾若波,等.基于mt DNA Cytb序列分析养殖与野生刀鲚群体的遗传多样性.江西农业大学学报,2012,34(6):1216-1221]
Wei GL,Xu GC,Gu RB,et al.Biology and artificial breeding progress in Coilia nasus.Journal of Yangtze University(Natural Science),2013,9(7):31-35[魏广莲,徐钢春,顾若波,等.刀鲚的生物学及人工养殖研究进展.长江大学学报(自然科学版),2013,9(7):31-35]
Yang M,Yu Y,Zhang XJ,et al.2017.Development of microsatellite markers from the transcriptome sequences of Pacific white shrimp(Litopenaus vannamei).Marine Sciences,41(2):96-102[杨铭,于洋,张晓军,等.基于转录组数据的凡纳滨对虾微卫星标记开发.海洋科学,2017,41(2):96-102]
Yuan CM.The resources of Coilia ectenes in middle and lower Yangtze River,and the status and the causation of the population formation changes.Chinese Journal of Zoology,1988,23(3):12-15[袁传宓.长江中下游刀鲚资源和种群组成变动状况及其原因.动物学杂志,1988,23(3):12-15]
Yue HM,Zhai Q,Song MY,et al.Development of microsatellite markers in Cyprinus carpio var.singuonensis using nextgeneration sequencing.Freshwater Fisheries,2016,46(1):24-28[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.淡水渔业,2016,46(1):24-28]
Zhang D,Fu JJ,Zhang LD,et al.The parentage analysis of bighead carp(Hypophthalmichthys nobilis)based on ten microsatellite markers.Genomics and Applied Biology,2018,1-11[张丹,傅建军,张利德,等.鳙基于10个微卫星标记的亲子鉴定分析.基因组学与应用生物学,2018,1-11]
Zhang YP,He G,Wang S,et al.Analysis of genetic diversity of Coilia nasus and Coilia brachygnathus based on mitochondrial DNA D-loop sequence.Jiangsu Agricultural Sciences,2017,45(20):59-64[张燕萍,贺刚,王生,等.基于线粒体DNA D-loop序列分析刀鲚与短颌鲚的遗传多样性.江苏农业科学,2017,45(20):59-64]