【摘要】 【目的】基于第三代高通量测序技术——单分子实时(SMRT)测序开发凡纳滨对虾微卫星分子标记,为凡纳滨对虾的遗传育种研究提供基础资料。【方法】采用SMRT测序对凡纳滨对虾转录组进行测序,经Illumina测序纠错后利用MISA对获得的转录组数据进行分析;以Primer 3.0设计微卫星引物,随机挑选40对微卫星引物进行PCR扩增验证,并选用扩增成功且具有多态性的微卫星引物用于不同养殖家系凡纳滨对虾遗传多态性分析。【结果】凡纳滨对虾转录组SMRT测序共获得51367条非冗余全长转录本序列,鉴定出39674条微卫星序列;微卫星的分布密度为0.232SSR/kb;以二核苷酸重复微卫星序列分布最多,共有22223条(占56.01%)。随机选取的40对微卫星引物中有26对微卫星引物能成功扩增出特异性条带;微卫星荧光分型结果显示,有16个微卫星位点具有多态性,共获得67个等位基因,平均等位基因数(Na)为4.2个,计算获得的平均观测杂合度(Ho)为0.511,平均期望杂合度(He)为0.451,平均多态性信息含量(PIC)为0.489。在16个微卫星位点中,有2个微卫星位点为低度多态性(PIC<0.25),6个微卫星位点为中度多态性(0.25<PIC<0.50),其余8个微卫星位点为高度多态性(PIC>0.50);有4个微卫星位点偏移Hardy-Weinberg平衡(P<0.05),其余12个微卫星位点均未偏离Hardy-Weinberg平衡(P>0.05)。【结论】采用SMRT测序开发凡纳滨对虾微卫星分子标记是一种简单而高效的途径,有助于开展凡纳滨对虾的遗传育种研究工作。更多还原

【Abstract】 【Objective】Microsatellite markers of Litopenaeus vannamei based on the third-generation high-throughput sequencing technology,single-molecule real-time(SMRT)sequencing were developed in order to provide basic data for genetic and breeding research of L. vannamei.【Method】The transcriptome of L. vannamei was sequenced using SMRT.The obtained transcriptome data were corrected by Illumina sequencing,then analyzed by MISA software. The microsatellite primers were designed with Primer 3.0 software,and 40 pairs of microsatellite primers were randomly selected for PCR amplification. Microsatellite primers with successful amplification and polymorphism were selected for genetic polymorphism analysis of L. vannamei in different breeding families.【Result】In total,51367 non-redundant full length transcript sequences and 39674 SSR loci were obtained by SMRT sequencing of the L. vannamei transcriptome. The distribution density of microsatellites was 0.232 SSR/kb;the dinucleotide repeat microsatellite sequences were the most distributed,with a total of 22223(56.01%). Of the 40 randomly selected microsatellite primers,26 pairs of microsatellite primers successfully amplified specific bands. The microsatellite fluorescence phenotyping showed that 16 microsatellite loci were polymorphic. A total of 67 alleles were obtained,and the average number of alleles(Na)was 4.2. The calculated average observed heterozygosity(Ho)was 0.511,the average expected heterozygosity(He)was 0.451,and the average polymorphism information content(PIC)was 0.489. Among the 16 microsatellite loci,2 loci were low polymorphisms(PIC<0.25),6 loci were moderate polymorphisms(0.25<PIC<0.50),and the remaining 8 loci were highly polymorphic(PIC>0.50);4 microsatellite loci deviated from Hardy-Weinberg equilibrium(P<0.05),and the remaining 12 loci did not deviate from Hardy-Weinberg equilibrium(P>0.05).【Conclusion】Using SMRT sequencing to develop microsatellite markers of L. vannamei is a simple and efficient way,which is helpful for the genetic and breeding research of L. vannamei.更多还原