| 摘要: |
| 【目的】挖掘美洲鲥脑组织转录组数据中的EST-SSR，规模化开发多态性较好的EST-SSR，为美洲鲥种质资源评估提供分子标记。【方法】采集2龄美洲鲥雌雄个体（各3尾）的脑组织，提取其RNA，构建cDNA文库，进行高通量转录组测序。用MISA软件进行脑组织转录组EST-SSR挖掘和特征分析，利用SSRMMD软件规模化开发多态性EST-SSR。从获得的多态性位点中随机选取20对4核苷酸重复的EST-SSR设计引物，对24尾1龄美洲鲥进行遗传多样性评估，验证这些多态性EST-SSR的应用效果。【结果】从雌性美洲鲥脑组织转录组189 428个非冗余基因中鉴定到117 751个EST-SSR，从雄性美洲鲥脑组织转录组185 419个非冗余基因中鉴定到114 809个EST-SSR。美洲鲥雌雄个体脑组织转录组中不同类型微卫星的重复基序均具有不同的数量分布特征，其中2核苷酸重复基序数量最多，分别占EST-SSR总数的72.78%和73.60%，而单核苷酸、3核苷酸、4核苷酸、5核苷酸和6核苷酸重复的EST-SSR数量随着重复碱基数量的增加而呈逐级减少趋势；不同EST SSR重复类型的优势重复基序亦有所不同，单核苷酸重复、2核苷酸重复和3核苷酸重复基序的优势基序分别为A/T、AC/GT和AGG/CCT。从7 671个雌雄美洲鲥脑组织转录组共有EST-SSR中成功鉴定到1 726个多态性EST-SSR，其中鉴定自单核苷酸重复、2核苷酸重复、3核苷酸重复的多态性EST-SSR分别有705，827和116个。20个4核苷酸重复的EST-SSR中，有17个位点多态性较好，其检测1龄美洲鲥的观测杂合度（Ho）、期望杂合度（He）和多态信息含量（PIC）均值分别为0.498，0.620和0.561，表明本单位科研基地的美洲鲥养殖群体历经数年的人工养殖，仍然具有相对较高的遗传多样性。【结论】基于美洲鲥脑组织转录组数据规模化开发了多态性EST-SSR，这些位点可用于美洲鲥养殖群体的遗传多样性评估及分子标记辅助遗传育种。 |
| 关键词: 美洲鲥 高通量测序 脑组织转录组 微卫星标记（SSR） 表达序列标签SSR(EST-SSR) |
| DOI： |
| 分类号: |
| 基金项目:上海市科技兴农重点攻关项目（2021-02-08-00-12-F00788） |
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| Development and application of polymorphic EST-SSR markers in cultured American shad (Alosa sapidissima) based on brain transcriptome datasets |
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YU Aiqing,SHI Yonghai,XU Jiabo,et al
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| Abstract: |
| 【Objective】EST-SSR in the brain transcriptome data of American shad (Alosa sapidissima) were mined and those with good polymorphism were developed on a large scale to provide molecular markers for germplasm resource evaluation.【MethodThe expressed sequence tag-simple sequence repeats (EST-SSR) were screened in the brain transcriptome database of female and male A.sapidissima by high-throughput Illumina sequencing using the MISA and SSRMMD software.Specific PCR amplification primers were also designed based on twenty tetra-nucleotide EST-SSR markers that were randomly selected from these polymorphic microsatellite loci.The genetic diversity of twenty-four one-year-old American shad was evaluated and the application of developed polymorphic EST-SSR markers was validated.【Result】A total of 117 751 EST-SSR were identified from 189 428 unigenes of female A.sapidissima transcriptome dataset and 114 809 were identified from 185 419 unigenes of male dataset.Different types of repeat SSRs had considerably different distribution characteristics,in which the number of dinucleotide microsatellite repeat motif was the largest,accounting for 72.78% and 73.60% of total numbers in female and male brain transcriptome of A.sapidissima,respectively.The numbers of mono-nucleotide,tri-nucleotide,tetra-nucleotide,penta-nucleotide and hexa-nucleotide microsatellite repeat motifs decreased with the increase of nucleotides number in repeat motifs.Different EST-SSR repeat motifs had different dominant repeat units in the brain transcriptome of A.sapidissima,and A/T,AC/GT and AGG/CCT were the dominant repeating units of mono nucleotide motif,di-nucleotide motif and tri-nucleotide motif,respectively.A total of 1 726 polymorphic EST-SSR were screened from 7 671 EST-SSR sequences in common between female and male EST-SSR.The numbers of polymorphic EST-SSR markers identified from mono-nucleotide repeat,di-nucleotide repeat and tri-nucleotide repeat SSR were 705,827 and 116,respectively.Polymorphism analysis of randomly selected 17 polymorphic EST-SSR in the cultured population of A.sapidissima showed that the average values of observed heterozygosity,expected heterozygosity and polymorphism information content were 0.498,0.620 and 0.561,respectively.【Conclusion】Based on the brain transcriptome dataset of A.sapidissima,polymorphic EST-SSR markers were developed on a large scale,which would facilitate further studies on population genetic analyses and molecular markers assisted breeding of A.sapidissima. |
| Key words: Alosa sapidissima high-throughput sequencing brain transcriptome simple sequence repeats (SSR) expressed sequence tag-simple sequence repeats (EST-SSR) |
