Abstract：In the past decade genetic analyses have shown that surf clam Coelomactra antiquata consists of two cryptic species, a northern lineage and a southern lineage. In order to accurately distinguish the two cryptic species of surf clam and evaluate its germplasm resources by molecular makers, microsatellite (SSR) sequences in both transcriptomes of the two cryptic species were explored and analyzed based on transcriptome data. SSRs in orthologous genes were also explored, and SSRs on 4 groups of orthologous genes were randomly selected for experimental validation. The resulted showed that an average of one SSR was found per 16.6 unigenes and the appearance frequency was 6.0% in northern lineage, involving 63 repeating motifs. In southern lineage an average of one SSR was found per 10.5 unigenes and the occurrence frequency was 8.2%, involving 89 repeating motifs. Besides the mononucleotide repeats, in both transcriptomes of the two cryptic species the trinucleotide repeats appeared to be the most abundant SSRs. In the transcriptome of northern and southern C. antiquata AAC/GTT and ATC/ATG was the dominant repeating motif of trinucleotide repeats respectively, and the most common SSR length was 15 bp. In both transcriptomes of the two cryptic species the repeat copy numbers of SSR were mainly distributed from 5 to 10 times. A total of 409 groups of orthologous genes showing SSR polymorphism between the two cryptic species were screened, which could be used as potential molecular markers.Validation experiments showed that the PCR products with clear and no impurity peak were amplified by the 4 pairs of fluorescence-labeled SSR primers, and that the combination of the 2 pairs of primers could preliminarily distinguish the two cryptic species.
王雨吉, 孟学平, 易乐飞. 基于转录组西施舌微卫星标记开发及隐种鉴定[J]. 水产科学, 2022, 41(2): 289-295.
WANG Yuji, MENG Xueping, YI Lefei. Isolation of Microsatellite Markers in Transcriptome of Surf Clam Coelomactra antiquata and Their Application in Cryptic Species Identification. Fisheries Science, 2022, 41(2): 289-295.
[1] HYLLEBERG J, KNUDSEN J, VONGPANICH V. The species described by Lorentz Spengler in his “Beskrivelse over det toskallede conchylie-slaegt Mactra”. A translation into English[J]. Phuket Marine Biological Center Special Publication,1999,21(2):499-518. [2] 徐凤山,张素萍,王少青.中国海产双壳类图志[M].北京:科学出版社,2008. [3] 刘德经,朱善央.福建与江苏西施舌群体形态差异研究[J].南方水产,2010,6(2):29-34. [4] 包永波,余冬冬,尤仲杰,等.中国沿海5个自然西施舌群体等位酶的遗传变异研究[J].水生态学杂志,2010,31(3):76-79. [5] YUAN Y, KONG L F, LI Q. Mitogenome evidence for the existence of cryptic species in Coelomactra antiquata[J].Genes & Genomics,2013,35(6):693-701. [6] 孟学平,申欣,赵娜娜,等.漳州西施舌线粒体基因组全序列:腔蛤蜊属(Coelomactra)存在新种的证据[J].海洋学报(中文版),2013,35(3):204-214. [7] 孟学平,申欣,屠海淼,等.基于nad5的西施舌漳州群体遗传分化水平分析——以蛤蜊属2个物种差异水平为参照[J].生态学报,2015,35(8):2635-2642. [8] YI L F, MA K Y, QIN J, et al. Insights into cryptic diversity and adaptive evolution of the clam Coelomactra antiquata (Spengler, 1802) from comparative transcriptomics[J].Marine Biodiversity,2019,49(5):2311-2322. [9] KONG L F, LI Q, QIU Z X. Genetic and morphological differentiation in the clam Coelomactra antiquata (Bivalvia:Veneroida) along the coast of China[J].Journal of Experimental Marine Biology and Ecology,2007,343(1):110-117. [10] 何平.真核生物中的微卫星及其应用[J].遗传,1998,20(4):42-47. [11] 孙效文,张晓锋,赵莹莹,等.水产生物微卫星标记技术研究进展及其应用[J].中国水产科学,2008,15(4):689-703. [12] 狄明玉,周遵春,李云峰,等.海蜇4个自然群体遗传多样性的微卫星标记分析[J].水产科学,2018,37(6):762-768. [13] 杨计平,李新辉,李跃飞,等.四大家鱼微卫星标记的筛选及其在物种鉴定中的应用[J].广东农业科学,2011,38(12):142-145. [14] 赵哲霞,蒋珊,王滨花,等.黄颡鱼属SSR分子鉴定及其遗传多样性[J].南昌大学学报(理科版),2014,38(5):498-501. [15] 范晓,李琪,何庆国,等.紫扇贝与海湾扇贝通用SSR的检测及其在杂交子代鉴定中的应用[J].生命科学研究,2012,16(4):287-294. [16] KALIA R K, RAI M K, KALIA S, et al. Microsatellite markers:an overview of the recent progress in plants[J].Euphytica,2011,177(3):309-334. [17] 李晶晶.两种双壳贝类和光棘球海胆微卫星标记的分离及特性研究[D].青岛:中国海洋大学,2008. [18] 朱立静,孙中响,陈淑吟,等.磁珠富集法筛选西施舌微卫星序列[J].南方水产科学,2011,7(4):10-15. [19] BOLGER A M, LOHSE M, USADEL B. Trimmomatic:a flexible trimmer for Illumina sequence data[J].Bioinformatics,2014,30(15):2114-2120. [20] WOOD D E, SALZBERG S L. Kraken:ultrafast metagenomic sequence classification using exact alignments[J].Genome Biology,2014,15(3):R46. [21] GRABHERR M G, HAAS B J, YASSOUR M, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome[J].Nature Biotechnology,2011,29(7):644-652. [22] PERTEA G, HUANG X Q, LIANG F, et al. TIGR gene indices clustering tools (TGICL):a software system for fast clustering of large EST datasets[J].Bioinformatics,2003,19(5):651-652. [23] EMMS D M, KELLY S. OrthoFinder:solving fundamental biases in whole genome comparisons dramatically improves orthogroup inference accuracy[J].Genome Biology,2015,16(1):157. [24] NGUYEN L T, SCHMIDT H A, von Haeseler A, et al. IQ-TREE:a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies[J].Molecular Biology and Evolution,2015,32(1):268-274. [25] YANG Y, SMITH S A. Orthology inference in nonmodel organisms using transcriptomes and low-coverage genomes:improving accuracy and matrix occupancy for phylogenomics[J].Molecular Biology and Evolution,2014,31(11):3081-3092. [26] BEIER S, THIEL T, MÜNCH T, et al. MISA-web:a web server for microsatellite prediction[J].Bioinformatics,2017,33(16):2583-2585. [27] TEMNYKH S, DECLERCK G, LUKASHOVA A, et al. Computational and experimental analysis of microsatellites in rice (Oryza sativa L.):frequency, length variation, transposon associations, and genetic marker potential[J].Genome Research,2001,11(8):1441-1452. [28] 石耀华,洪葵,郭希明,等.马氏珠母贝EST微卫星的筛选[J].水产学报,2008,32(2):174-181. [29] 王忠良,丁燏,许尤厚,等.基于转录组数据的马氏珠母贝EST-SSR位点的信息分析及其多态性检测[J].海洋与湖沼,2015,46(3):687-693. [30] 谭杰,姚高友,刘志刚,等.扇贝“渤海红”与墨西哥湾扇贝转录组SSR信息分析及unigene功能注释[J].基因组学与应用生物学,2018,37(12):5242-5250. [31] CHEN X M, LI J K, XIAO S J, et al. De novo assembly and characterization of foot transcriptome and microsatellite marker development for Paphia textile[J].Gene,2016,576(1):537-543. [32] 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(1):132-137. [33] 李云峰,刘卫东,高祥刚,等.虾夷扇贝外套膜和肾脏组织cDNA文库构建以及EST的初步分析[J].中国水产科学,2010,17(3):578-585. [34] YUE G H, KOVACS B, ORBAN L. A new problem with cross-species amplification of microsatellites:generation of non-homologous products[J].动物学研究,2010,31(2):131-140. [35] 李小白,向林,罗洁,等.转录组测序(RNA-seq)策略及其数据在分子标记开发上的应用[J].中国细胞生物学学报,2013,35(5):720-726.
2022, Vol. 41 
