基于SLAF-seq技术的黑棘鲷微卫星标记开发及其在鲷科鱼类中的通用性研究
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摘要
黑棘鲷(Acanthopagrusschlegelii)是广泛分布于西北太平洋的暖温性中下层经济鱼类,也是我国沿海重要的海洋捕捞鱼类和增养殖对象。然而,目前有关黑棘鲷的微卫星标记研究报道较少,难以对其种质资源状况作出精确评估。本研究采用SLAF-seq技术测序共获得22489个二至六碱基重复的黑棘鲷微卫星序列,短重复序列(二、三碱基)占总微卫星序列的90.8%,长重复序列(四至六碱基)占有9.2%。经过157对随机合成引物的多态性筛选,开发出49个高多态性的黑棘鲷微卫星标记,其中短重复序列位点有25个,长重复序列位点有24个。每个位点的等位基因数(Na)为2—20(均值为8.3),观测杂合度(Ho)和期望杂合度(He)分别为0.097—0.938和0.122—0.922(均值分别为0.663和0.701),多态信息含量(PIC)为0.118—0.897(均值为0.655)。经Bonferroni校正后,有47个位点符合哈迪-温伯格平衡(HWE),各位点间未检测到连锁不平衡现象,仅2个位点偏离HWE。结果表明,所开发的大部分微卫星标记具有高多态性,蕴含的遗传信息含量较为丰富,能够为黑棘鲷的种群遗传资源评估提供数量充足、类型多样的有效分子标记。跨物种扩增结果显示,有43个黑棘鲷微卫星标记可在9种鲷科鱼类中成功扩增,其中28个标记在太平洋棘鲷(Acanthopagruspacificus)、黄鳍棘鲷(Acanthopagruslatus)和澳洲棘鲷(Acanthopagrusaustralis)中具有较好的通用性,2个标记在平鲷(Rhabdosargussarba)、蓝点赤鲷(Pagruscaeruleostictus)、真赤鲷(Pagrusmajor)、二长棘犁齿鲷(Evynnis cardinalis)及黄牙鲷(Dentex hypselosomus)中具有通用性。这些通用性标记可为阐明鲷科属、种间的系统进化关系和棘鲷属鱼类的群体遗传学分析提供新的标记来源和研究角度。
The black porgy(Acanthopagrus schlegelii)(Perciformes: Sparidae) is a meso-demersal warm-water fish and widely distributed in the western Pacific coastal waters. It has become an important target species of mariculture in China. However, studies on the microsatellite markers of A. schlegelii are scarce. To make an accurate assessment of its current germplasm resources, based on specific-locus amplified fragment sequencing(SLAF-seq) technology, we identified22 489 di-to hexanucleotide repeats microsatellites from the genomic DNA of A. schlegelii. The shorter repeats motifs(2—3 mers) accounted for 90.8%, while the longer repeat motifs(4—6 mers) for 9.2% of the total microsatellite sequences.Finally, 49 high polymorphic microsatellite markers were developed from 157 pairs of randomly synthesized primers,among which 25 are shorter repeats motifs loci and the other 24 are longer repeat motifs loci. The allele number per locus ranges from 2 to 20(mean 8.3), and the observation and expected heterozygosity varies from 0.097 to 0.938(mean 0.663),and 0.122 to 0.922(mean 0.701), respectively. The polymorphism information content(PIC) ranges from 0.118 to 0.897(mean 0.655). Only two microsatellite loci are deviated from the Hardy-Weinberg equilibrium(HWE) after the Bonferroni correction, while the other 47 loci are consistent with HWE and no linkage disequilibrium was detected among these 47 loci. The results show that most of the microsatellite markers are highly polymorphic, and contained abundant genetic information. These polymorphic microsatellite markers provide abundant and various effective molecular markers to assess the population genetic resources. In addition, as shown in cross-species amplification, 43 microsatellite markers could be successfully amplified in nine species of family Sparidae. Among them, 28 are transferable in Acanthopagrus pacificus,Acanthopagrus latus, and Acanthopagrus australis; 2 are transferable in Rhabdosargus sarba, Pagrus caeruleostictus,Pagrus major, Evynnis cardinalis, and Dentex hypselosomus. The transferable markers provide new analytical tools for elucidating phylogenetic relationships among species and genus of the family Sparidae, and for understanding the population genetic of Acanthopagrus species in the future.
The black porgy(Acanthopagrus schlegelii)(Perciformes: Sparidae) is a meso-demersal warm-water fish and widely distributed in the western Pacific coastal waters. It has become an important target species of mariculture in China. However, studies on the microsatellite markers of A. schlegelii are scarce. To make an accurate assessment of its current germplasm resources, based on specific-locus amplified fragment sequencing(SLAF-seq) technology, we identified22 489 di-to hexanucleotide repeats microsatellites from the genomic DNA of A. schlegelii. The shorter repeats motifs(2—3 mers) accounted for 90.8%, while the longer repeat motifs(4—6 mers) for 9.2% of the total microsatellite sequences.Finally, 49 high polymorphic microsatellite markers were developed from 157 pairs of randomly synthesized primers,among which 25 are shorter repeats motifs loci and the other 24 are longer repeat motifs loci. The allele number per locus ranges from 2 to 20(mean 8.3), and the observation and expected heterozygosity varies from 0.097 to 0.938(mean 0.663),and 0.122 to 0.922(mean 0.701), respectively. The polymorphism information content(PIC) ranges from 0.118 to 0.897(mean 0.655). Only two microsatellite loci are deviated from the Hardy-Weinberg equilibrium(HWE) after the Bonferroni correction, while the other 47 loci are consistent with HWE and no linkage disequilibrium was detected among these 47 loci. The results show that most of the microsatellite markers are highly polymorphic, and contained abundant genetic information. These polymorphic microsatellite markers provide abundant and various effective molecular markers to assess the population genetic resources. In addition, as shown in cross-species amplification, 43 microsatellite markers could be successfully amplified in nine species of family Sparidae. Among them, 28 are transferable in Acanthopagrus pacificus,Acanthopagrus latus, and Acanthopagrus australis; 2 are transferable in Rhabdosargus sarba, Pagrus caeruleostictus,Pagrus major, Evynnis cardinalis, and Dentex hypselosomus. The transferable markers provide new analytical tools for elucidating phylogenetic relationships among species and genus of the family Sparidae, and for understanding the population genetic of Acanthopagrus species in the future.
引文
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许思思,2011.人为影响下渤海渔业资源的衰退机制.青岛:中国科学院研究生院(海洋研究所)博士学位论文,149
孙波,鲍毅新,赵庆洋等,2009.微卫星位点获取方法的研究进展.生态学杂志,28(10):2130-2137
李伟亚,王伟继,孔杰等,2012.中国对虾微卫星四重PCR技术的建立及其在模拟放流效果评估方面的应用.海洋学报,34(5):213-220
杨慧荣,江世贵,2006.用RAPD技术探讨5种鲷科鱼类的亲缘关系.水产学报,30(4):469-474
沈世杰,1993.台湾鱼类志.台北,中国:国立台湾大学动物学系,364
沈新强,周永东,2007.长江口、杭州湾海域渔业资源增殖放流与效果评估.渔业现代化,34(4):54-57
张浩冉,梁镇邦,吴仁协等,2019.利用SLAF-seq技术开发带鱼(Trichiurus japonicus)微卫星标记以及跨物种扩增检测.基因组学与应用生物学,38(2):574-585
张浩冉,梁镇邦,吴仁协等,2018.基于SLAF-seq技术的沙带鱼(Lepturacanthus savala)微卫星标记开发以及在近缘种中的通用性.基因组学与应用生物学,37(8):3331-3338
陈咏霞,吴仁协,梁娜等,2015.基于线粒体COI基因序列的中国鲷科鱼类系统进化关系.海洋与湖沼,46(3):611-619
罗新惠,2010.黑鲷(Acanthopagrus schlegeli)和半滑舌鳎(Cynoglossus semilaevis)微卫星标记的开发.上海:上海海洋大学硕士学位论文,9-27
赵爽,章群,乐小亮等,2010.中国近海5个黑鲷地理群体的遗传变异.海洋科学,34(2):75-79
施晓峰,苏永全,王文成等,2015.基于mtDNA控制区序列的3个黑棘鲷群体遗传结构特性研究.热带海洋学报,34(1):56-63
徐亘博,2010.半滑舌鳎等九种海水鱼微卫星分子标记的开发和应用.青岛:中国海洋大学硕士学位论文,21-36
郭宝英,谢从新,熊冬梅,2007.微卫星DNA标记技术及其在鱼类中的应用.水利渔业,27(4):5-9
曹艳,2016.基于线粒体控制区序列的中国沿海3种鲷科鱼类遗传多样性分析.广州:暨南大学硕士学位论文
梁君,王伟定,林桂装等,2010.浙江舟山人工生境水域日本黄姑鱼和黑鲷的增殖放流效果及评估.中国水产科学,17(5):1075-1084
鲁双庆,刘臻,肖调义,2003.微卫星标记在鱼类遗传育种研究中的应用.水利渔业,23(6):9-11
蒙子宁,庄志猛,金显仕等,2003.黄海和东海小黄鱼遗传多样性的RAPD分析.生物多样性,11(3):197-203
翟云,吴仁协,牛素芳等,2018.基于SLAF-seq技术开发蓝圆鲹微卫星标记及跨物种扩增检测.应用海洋学学报,37(3):426-434
Botstein D,White R L,Skolnick M et al,1980.Construction of a genetic linkage map in man using restriction fragment length polymorphisms.American Journal of Human Genetics,32(3):314-331
Carreras-Carbonell J,Macpherson E,Pascual M,2008.Utility of pairwise mtDNA genetic distances for predicting crossspecies microsatellite amplification and polymorphism success in fishes.Conservation Genetics,9(1):181-190
Castoe T A,Streicher J W,Meik J M et al,2012.Thousands of microsatellite loci from the venomous coralsnake Micrurus fulvius and variability of select loci across populations and related species.Molecular Ecology Resources,12(6):1105-1113
Chapuis M P,Estoup A,2007.Microsatellite null alleles and estimation of population differentiation.Molecular Biology and Evolution,24(3):621-631
Chen Y X,Liu J,Wu R X,2015.A new record of blue-spotted seabream Pagrus caeruleostictus from Chinese coastal waters documented from morphology and DNA barcoding.Chinese Journal of Oceanology and Limnology,33(2):500-505
DeWoody J A,Avise J C,2000.Microsatellite variation in marine,freshwater and anadromous fishes compared with other animals.Journal of Fish Biology,56(3):461-473
Gonzalez E B,Murakami T,Teshima Y et al,2009.Paternity testing of wild black rockfish Sebastes inermis(brownish type)from the Seto Inland Sea of Japan.Ichthyological Research,56(1):87-91
Goudet J,2001.FSTAT:a program to estimate and test gene diversities and fi xation indices(Version 2.9.3.2).https://www2.unil.ch/popgen/softwares/fstat.htm.Accessed on 2018-05-14
Grover A,Sharma P C,2016.Development and use of molecular markers:past and present.Critical Reviews in Biotechnology,36(2):290-302
Iwatsuki Y,Carpenter K,2006.Acanthopagrus taiwanensis,a new sparid fish(Perciformes),with comparisons to Acanthopagrus berda(Forssk?l,1775)and other nominal species of Acanthopagrus.Zootaxa,1202:1-19
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