基于转录组数据的铜藻微卫星标记开发与验证
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摘要
根据铜藻转录组测序数据来进行微卫星标记的发掘,共获得2 800个微卫星位点。二碱基重复出现的次数最多,占32.82%,其次是三碱基重复,占25.21%;在所有重复单元类型中,AC/GT出现频率最高,占9.18%;重复序列的长度则主要集中在12～20 bp,占72.79%;重复次数主要集中在5～10次,占74.14%。随机选取74对引物进行验证,最终获得18个具有多态性的微卫星标记。对31株铜藻个体进行检测和评价,共扩增得到43个等位基因,每个位点平均等位基因数为2.39,平均有效等位基因数为1.91,平均观测杂合度(H_o)为0.318,平均期望杂合度(H_e)为0.449,平均多态信息含量(PIC)为0.365,在18个位点中有11个显著偏离Hardy-Weinberg平衡(P<0.05)。开发的微卫星标记为后续铜藻多样性、起源和迁移等遗传学问题研究提供了有力支撑。
SSRs were developed and characterized by using transcriptome data. A total of 2800 SSR loci were identified and dinucleotides were the most frequent(32.82%), followed by trinucleotides(25.21%). AC/GT was the most abundant unit(9.18%). The length of repetitive sequences was mainly concentrated between 12-20 bp(72.79%), and 5-10 repeats being the most abundant number(74.14%). 74 pairs of primers were randomly selected for detection, and 18 polymorphic SSRs were obtained finally. The characteristics of these microsatellites were determined in 31 individuals of S. horneri. A total of 43 alleles were detected, the allele number per locus ranged from 2 to 4(average 2.39), and mean effective allele number was 1.91, and mean observed and expected heterozygosity were 0.318 and 0.449, respectively. The average of polymorphism information content( PIC) was 0.365, and 11 loci deviated significantly from Hardy-Weinberg equilibrium(HWE)(P<0.05). These obtained SSR markers will be helpful for the genetic studies in S. horneri.
SSRs were developed and characterized by using transcriptome data. A total of 2800 SSR loci were identified and dinucleotides were the most frequent(32.82%), followed by trinucleotides(25.21%). AC/GT was the most abundant unit(9.18%). The length of repetitive sequences was mainly concentrated between 12-20 bp(72.79%), and 5-10 repeats being the most abundant number(74.14%). 74 pairs of primers were randomly selected for detection, and 18 polymorphic SSRs were obtained finally. The characteristics of these microsatellites were determined in 31 individuals of S. horneri. A total of 43 alleles were detected, the allele number per locus ranged from 2 to 4(average 2.39), and mean effective allele number was 1.91, and mean observed and expected heterozygosity were 0.318 and 0.449, respectively. The average of polymorphism information content( PIC) was 0.365, and 11 loci deviated significantly from Hardy-Weinberg equilibrium(HWE)(P<0.05). These obtained SSR markers will be helpful for the genetic studies in S. horneri.
引文
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[21] ZANE L,BARGELLONI L,PATARNELLO T.Strategies for microsatellite isolation:a review[J].Molecular Ecology,2002,11(1):1-16.
[22] XIE C T,LI B,XU Y,et al.Characterization of the global transcriptome for Pyropia haitanensis (Bangiales,Rhodophyta) and development of cSSR markers[J].BMC Genomics,2013,14:107.
[23] 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[J].海洋科学,2017,41(2):96-102.YANG M,YU Y,ZHANG X J,et al.Development of microsatellite markers from the transcriptome sequences of Pacific white shrimp (Litopenaus vannamei)[J].Marine Sciences,2017,41(2):96-102.
[24] ZHANG J,LIU T,RUI F P.Development of EST-SSR markers derived from transcriptome of Saccharina japonica and their application in genetic diversity analysis[J].Journal of Applied Phycology,2018,30(3):2101-2109.
[25] 杨旭,毕燕会,周志刚.瓦氏马尾藻微卫星分子标记的筛选[J].海洋渔业,2014,36(5):431-436.YANG X,BI Y H,ZHOU Z G.Isolation of microsatellite markers in Sargassum vachellianum[J].Marine Fisheries,2014,36(5):431-436.
[26] 刘必谦,曾庆国,骆其君,等.条斑紫菜(Porphyra yezoensis)dbEST中筛选微卫星位点及引物种间转移扩增[J].海洋与湖沼,2005,36(3):248-254.LIU B Q,ZENG Q G,LUO Q J,et al.Isolation of microsatellite loci from dbEST of algae Porphyra yezoensis and primer amplification of interspecies transfer[J].Oceanologiaet Limnologia Sinica,2005,36(3):248-254.
[27] SHAN T F,PANG S J,WANG X M,et al.Assessment of the genetic connectivity between farmed and wild populations of Undaria pinnatifida(Phaeophyceae) in a representative traditional farming region of China by using newly developed microsatellite markers[J].Journal of Applied Phycology,2018,30(4):2707-2714.
[28] TóTH G,GáSPáRI Z,JURKA J.Microsatellites in different eukaryotic genomes:survey and analysis[J].Genome Research,2000,10(7):967-981.
[29] VARSHNEY R K,GRANER A,SORRELLS M E.Genic microsatellite markers in plants:features and applications[J].Trends in Biotechnology,2005,23(1):48-55.
[30] YANG H,MAO Y X,KONG F N,et al.Profiling of the transcriptome of Porphyra yezoensis with Solexa sequencing technology[J].Chinese Science Bulletin,2011,56(20):2119-2130.
[31] HU Y Y,SUI Z H,ZHOU W,et al.Development of genomic simple sequence repeat markers and genetic diversity analysis of Gracilariopsis lemaneiformis (Rhodophyta)[J].Journal of Applied Phycology,2018,30(1):707-716.
[32] SIA E A,KOKOSKA R J,DOMINSKA M,et al.Microsatellite instability in yeast:dependence on repeat unit size and DNA mismatch repair genes[J].Molecular and Cellular Biology,1997,17(5):2851-2858.
[33] SENANAN W,KAPUSCINSKI A R,NA-NAKORN U,et al.Genetic impacts of hybrid catfish farming (Clarias macrocephalus×C.gariepinus) on native catfish populations in central Thailand[J].Aquaculture,2004,235(1/4):167-184.
[34] BI Y H,YANG X,SUN Z,et al.Development and characterization of 12 polymorphic microsatellite markers in Sargassum vachellianum[J].Conservation Genetics Resources,2015,7(1):203-205.
[35] 李青.重要海水养殖鱼类星斑川鲽(Platichthys stellatus)微卫星开发及遗传多样性研究[D].济南:山东大学,2016.LI Q.Study on the development and genetic diversity of microsatellite in the important maricultural fish,Platichthys stellatus[D].Jinan:Shandong University,2016.
[2] 陈亮然,章守宇,陈彦,等.枸杞岛马尾藻场铜藻的生命史与形态特征[J].水产学报,2015,39(8):1218-1229.CHEN L R.ZHANG S Y,CHEN Y,et al.Life history and morphology of Sargassum horneri from the Sargassum seaweed bed of GouqiIsland[J].Journal of Fisheries of China,2015,39(8):1218-1229.
[3] 张鹏,王铁杆,钟晨辉,等.铜藻苗种繁育研究[J].水生生物学报,2018,42(2):428-438.ZHANG P,WANG T G,ZHONG C H,et al.study on seed breeding of Sargassum horneri[J].Acta Hydrobiologica Sinica,2018,42(2):428-438.
[4] REN J R,YANG R,HE Y Y,et al.Genetic variation of Sargassum horneri populations detected by inter-simple sequence repeats[J].Genetics and Molecular Research:GMR,2015,14(1):619-625.
[5] HU Z M,UWAI S,YU S H,et al.Phylogeographic heterogeneity of the brown macroalga Sargassum horneri(Fucaceae) in the northwestern Pacific in relation to late Pleistocene glaciation and tectonic configurations[J].Molecular Ecology,2011,20(18):3894-3909.
[6] UWAI S,KOGAME K,YOSHIDA G,et al.Geographical genetic structure and phylogeography of the Sargassum horneri/filicinum complex in Japan,based on the mitochondrial cox3 haplotype[J].Marine Biology,2009,156(5):901-911.
[7] YU S H,CHON G Z,ZHAO F J,et al.Population genetics of Sargassum horneri (Fucales,Phaeophyta) in China revealed by ISSR and SRAP markers[J].Chinese Journal of Oceanology and Limnology,2013,31(3):609-616.
[8] KUBO N,DOUKE A,NISHIGAKI T,et al.Development and characterization of simple sequence repeat markers for genetic analyses of Sargassum horneri (Sargassaceae,Phaeophyta) populations in Kyoto,Japan[J].Journal of Applied Phycology,2017,29(3):1729-1733.
[9] 刘占江.水产基因组学技术[M].北京:化学工业出版社,2011.LIU Z J.Aquaculture genome technologies[M].Beijing:Chemical Industry Press,2011.
[10] GUICHOUX E,LAGACHE L,WAGNER S,et al.Current trends in microsatellite genotyping[J].Molecular Ecology Resources,2011,11(4):591-611.
[11] SHAN T F,PANG S J,LI J,et al.Isolation and characterization of eight novel microsatellite loci from the brown alga Sargassum horneri[J].Journal of Applied Phycology,2015,27(6):2419-2421.
[12] TRIWITAYAKORN K,CHATKULKAWIN P,KANJANAWATT-ANAWONG S,et al.Transcriptome sequencing of Heveabrasiliensis for development of microsatellite markers and construction of a genetic linkage map[J].DNA Research,2011,18(6):471-482.
[13] Yü Z M,LI H M,LIU L Q,et al.Rapid development of microsatellite markers from the large yellow croaker (Pseudosciaena crocea) using next generation DNA sequencing technology[J].Biochemical Systematics and Ecology,2013,51:314-319.
[14] LI Q Y,ZHANG J,YAO J T,et al.Development of Saccharina japonica genomic SSR markers using next-generation sequencing[J].Journal of Applied Phycology,2016,28(2):1387-1390.
[15] NEI M.Estimation of average heterozygosity and genetic distance from a small number of individuals[J].Genetics,1978,89(3):583-590.
[16] BOTSTEIN D,WHITE R L,SKOLNICK M,et al.Construction of a genetic linkage map in man using restriction fragment length polymorphisms[J].American Journal of Human Genetics,1980,32(3):314-331.
[17] YANG X H,XU Y B,SHAH T,et al.Comparison of SSRs and SNPs in assessment of genetic relatedness in maize[J].Genetica,2011,139(8):1045-1054.
[18] OSTRANDER E A,JONG P M,RINE J,et al.Construction of small-insert genomic DNA libraries highly enriched for microsatellite repeat sequences[J].Proceedings of the National Academy of Sciences of the United States of America,1992,89(8):3419-3423.
[19] LYALL J E W,BROWN G M,FURLONG R A,et al.A method for creating chromosome-specific plasmid libraries enriched in clones containing microsatellite repeat sequences directly from flow-sorted chromosomes[J].Nucleic Acids Research,1993,21(19):4641-4642.
[20] KIJAS J M H,FOWLER J C S,GARBETT C A,et al.Enrichment of microsatellites from the citrus genome using biotinylated oligonucleotide sequences bound to streptavidin-coated magnetic particles[J].Biotechniques,1994,16(4):656-660,662.
[21] ZANE L,BARGELLONI L,PATARNELLO T.Strategies for microsatellite isolation:a review[J].Molecular Ecology,2002,11(1):1-16.
[22] XIE C T,LI B,XU Y,et al.Characterization of the global transcriptome for Pyropia haitanensis (Bangiales,Rhodophyta) and development of cSSR markers[J].BMC Genomics,2013,14:107.
[23] 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[J].海洋科学,2017,41(2):96-102.YANG M,YU Y,ZHANG X J,et al.Development of microsatellite markers from the transcriptome sequences of Pacific white shrimp (Litopenaus vannamei)[J].Marine Sciences,2017,41(2):96-102.
[24] ZHANG J,LIU T,RUI F P.Development of EST-SSR markers derived from transcriptome of Saccharina japonica and their application in genetic diversity analysis[J].Journal of Applied Phycology,2018,30(3):2101-2109.
[25] 杨旭,毕燕会,周志刚.瓦氏马尾藻微卫星分子标记的筛选[J].海洋渔业,2014,36(5):431-436.YANG X,BI Y H,ZHOU Z G.Isolation of microsatellite markers in Sargassum vachellianum[J].Marine Fisheries,2014,36(5):431-436.
[26] 刘必谦,曾庆国,骆其君,等.条斑紫菜(Porphyra yezoensis)dbEST中筛选微卫星位点及引物种间转移扩增[J].海洋与湖沼,2005,36(3):248-254.LIU B Q,ZENG Q G,LUO Q J,et al.Isolation of microsatellite loci from dbEST of algae Porphyra yezoensis and primer amplification of interspecies transfer[J].Oceanologiaet Limnologia Sinica,2005,36(3):248-254.
[27] SHAN T F,PANG S J,WANG X M,et al.Assessment of the genetic connectivity between farmed and wild populations of Undaria pinnatifida(Phaeophyceae) in a representative traditional farming region of China by using newly developed microsatellite markers[J].Journal of Applied Phycology,2018,30(4):2707-2714.
[28] TóTH G,GáSPáRI Z,JURKA J.Microsatellites in different eukaryotic genomes:survey and analysis[J].Genome Research,2000,10(7):967-981.
[29] VARSHNEY R K,GRANER A,SORRELLS M E.Genic microsatellite markers in plants:features and applications[J].Trends in Biotechnology,2005,23(1):48-55.
[30] YANG H,MAO Y X,KONG F N,et al.Profiling of the transcriptome of Porphyra yezoensis with Solexa sequencing technology[J].Chinese Science Bulletin,2011,56(20):2119-2130.
[31] HU Y Y,SUI Z H,ZHOU W,et al.Development of genomic simple sequence repeat markers and genetic diversity analysis of Gracilariopsis lemaneiformis (Rhodophyta)[J].Journal of Applied Phycology,2018,30(1):707-716.
[32] SIA E A,KOKOSKA R J,DOMINSKA M,et al.Microsatellite instability in yeast:dependence on repeat unit size and DNA mismatch repair genes[J].Molecular and Cellular Biology,1997,17(5):2851-2858.
[33] SENANAN W,KAPUSCINSKI A R,NA-NAKORN U,et al.Genetic impacts of hybrid catfish farming (Clarias macrocephalus×C.gariepinus) on native catfish populations in central Thailand[J].Aquaculture,2004,235(1/4):167-184.
[34] BI Y H,YANG X,SUN Z,et al.Development and characterization of 12 polymorphic microsatellite markers in Sargassum vachellianum[J].Conservation Genetics Resources,2015,7(1):203-205.
[35] 李青.重要海水养殖鱼类星斑川鲽(Platichthys stellatus)微卫星开发及遗传多样性研究[D].济南:山东大学,2016.LI Q.Study on the development and genetic diversity of microsatellite in the important maricultural fish,Platichthys stellatus[D].Jinan:Shandong University,2016.