基于刺参转录组测序的SSR标记和防御机制相关SNP标记开发
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摘要
海参是棘皮动物门（Echinodermata）海参纲（Holothuroidea）动物的统称，主要分布在中国北方沿海，日本，韩国，俄罗斯等地。由于其极高的经济价值和药用价值，已成为我国重要的海水养殖种类。但是由于过度捕捞和环境恶化，刺参野生群体在急剧下降。所以为了保护野生群体及刺参养殖业的健康发展，开发和利用分子标记对刺参养殖群体和野生群体进行遗传分析是必要的。这对于其优良性状的保持、经济效益的提高等方面具有重要的意义。
（1）利用本实验室采用Illumina HiSeq2000测序技术得到12000多条刺参Unigene序列，应用SSRHunter软件搜索到355个微卫星位点，设计出110对引物，最终45个位点扩增出符合预期大小、清晰、具有多态性的条带。进一步的结果分析显示这些位点的等位基因数目在2-20之间，平均每个位点5.53个等位基因；观测杂合度和期望杂合度为0.1333-0.8668和0.1266-0.9463；没有观察到连锁不平衡位点（LD）；17个位点显著偏离哈迪温伯格平衡（HWE）。
（2）本实验利用高分辨率溶解曲线（HRM）技术对刺参防御机制功能相关性Unigene进行SNP位点筛选，得到34个单核苷酸突变位点。碱基置换类型为转换和颠换的百分比分别为70.59%和23.53%， A/G(C/T)、A/C(G/T)、A/T、C/G的位点数目分别为20个、4个、1个、7个，分别占总数的58.82%、11.76%、2.94%、20.59%；1个碱基置换类型模糊位点以及1个三态位点有碱基缺失的位点。34个SNP位点中有26个位点能利用小扩增子法进行基因分型。分型结果显示其中有11个位点将50个刺参个体分为2个基因型，15个位点分为3个基因型。进一步数据分析显示26个位点均具有2个等位基因，最小等位基因频率为0.038-0.500；观测杂合度和期望杂合度分别为0.054-0.792和0.064-0.547，没有观察到连锁不平衡位点（LD）；2个位点显著偏离哈迪温伯格平衡（PHWE<0.01）。
本实验所开发的基于刺参转录组测序的SSR标记及刺参防御机制相关SNP标记期望对刺参遗传连锁图谱的构建以及比较基因组研究有所帮助。
Sea cucumber (Apostichopus japonicus) belongs to the Holothurioider of Echinodermata,mainly distributed in the northern coast of China, Japan, Korea, and Far Eastern Russia. Due toits high nutritional and medicinal properties, sea cucumber has long been exploited as aneconomically important aquaculture species in China. However, the number of wild seacucumber has severely declined because of over-exploitation and environment deterioration.Thus, it is necessary to implement research on sea cucumber genetics to protect sea cucumberresource. It's useful to keep the good trait and improve economic returns.
（1）From more than12000unigene sequences were obtained from a transcriptome ofApostichopus japonicus using the Illumina HiSeq2000Sequencing Technology, total355microsatellite loci were identified,110primer pairs synthesized and45amplified expectedproducts and showed polymorphism using50field specimens tested. The number of allelesranged from2to20with an average of5.53alleles per locus, and the observed and expectedheterozygosities varied from0.1333to0.8668and from0.1266to0.9463with an average of0.4486and0.6579, respectively. No significant linkage disequilibrium (LD) between pairs ofloci was found and17loci significantly deviated from the Hardy-Weinberg equilibrium (HWE).
（2）In this study,34Single nucleotide polymorphism (SNPs) sites related to defensemechanism of the sea cucumber were found by the high resolution melting (HRM) assays.Among them, the percentage of transition is70.56%while the transversion is23.53%, thenumber of A/G(C/T), A/C(G/T), A/T and C/G mutation type is20,4,1and7, accounting for58.82%,11.76%,2.94%and20.59%of the total, respectively;1site is tri-base deletion and onesite is fuzzy base substitution type. In these34SNP sites,26sites could be used to geneotypingby small amplicon method. The results of genotyping revealed that11sites were genopypeed tothree genetypes while the other15sites were were genopypeed to two genetypes; all loci hadtwo alleles and the minor allele frequency ranged from0.038to0.500; The observed andexpected heterozygosity ranged from0.054to0.792and0.064to0.547, respectively; Two locisignificantly depart from Hardy Weinberg equilibrium and no significant linkage disequilibrium(LD) was detected.
These SSR markers and SNP markers should be useful for the construction of geneticlinkage map and comparative genome analysis in A. japonicus.
（1）利用本实验室采用Illumina HiSeq2000测序技术得到12000多条刺参Unigene序列，应用SSRHunter软件搜索到355个微卫星位点，设计出110对引物，最终45个位点扩增出符合预期大小、清晰、具有多态性的条带。进一步的结果分析显示这些位点的等位基因数目在2-20之间，平均每个位点5.53个等位基因；观测杂合度和期望杂合度为0.1333-0.8668和0.1266-0.9463；没有观察到连锁不平衡位点（LD）；17个位点显著偏离哈迪温伯格平衡（HWE）。
（2）本实验利用高分辨率溶解曲线（HRM）技术对刺参防御机制功能相关性Unigene进行SNP位点筛选，得到34个单核苷酸突变位点。碱基置换类型为转换和颠换的百分比分别为70.59%和23.53%， A/G(C/T)、A/C(G/T)、A/T、C/G的位点数目分别为20个、4个、1个、7个，分别占总数的58.82%、11.76%、2.94%、20.59%；1个碱基置换类型模糊位点以及1个三态位点有碱基缺失的位点。34个SNP位点中有26个位点能利用小扩增子法进行基因分型。分型结果显示其中有11个位点将50个刺参个体分为2个基因型，15个位点分为3个基因型。进一步数据分析显示26个位点均具有2个等位基因，最小等位基因频率为0.038-0.500；观测杂合度和期望杂合度分别为0.054-0.792和0.064-0.547，没有观察到连锁不平衡位点（LD）；2个位点显著偏离哈迪温伯格平衡（PHWE<0.01）。
本实验所开发的基于刺参转录组测序的SSR标记及刺参防御机制相关SNP标记期望对刺参遗传连锁图谱的构建以及比较基因组研究有所帮助。
Sea cucumber (Apostichopus japonicus) belongs to the Holothurioider of Echinodermata,mainly distributed in the northern coast of China, Japan, Korea, and Far Eastern Russia. Due toits high nutritional and medicinal properties, sea cucumber has long been exploited as aneconomically important aquaculture species in China. However, the number of wild seacucumber has severely declined because of over-exploitation and environment deterioration.Thus, it is necessary to implement research on sea cucumber genetics to protect sea cucumberresource. It's useful to keep the good trait and improve economic returns.
（1）From more than12000unigene sequences were obtained from a transcriptome ofApostichopus japonicus using the Illumina HiSeq2000Sequencing Technology, total355microsatellite loci were identified,110primer pairs synthesized and45amplified expectedproducts and showed polymorphism using50field specimens tested. The number of allelesranged from2to20with an average of5.53alleles per locus, and the observed and expectedheterozygosities varied from0.1333to0.8668and from0.1266to0.9463with an average of0.4486and0.6579, respectively. No significant linkage disequilibrium (LD) between pairs ofloci was found and17loci significantly deviated from the Hardy-Weinberg equilibrium (HWE).
（2）In this study,34Single nucleotide polymorphism (SNPs) sites related to defensemechanism of the sea cucumber were found by the high resolution melting (HRM) assays.Among them, the percentage of transition is70.56%while the transversion is23.53%, thenumber of A/G(C/T), A/C(G/T), A/T and C/G mutation type is20,4,1and7, accounting for58.82%,11.76%,2.94%and20.59%of the total, respectively;1site is tri-base deletion and onesite is fuzzy base substitution type. In these34SNP sites,26sites could be used to geneotypingby small amplicon method. The results of genotyping revealed that11sites were genopypeed tothree genetypes while the other15sites were were genopypeed to two genetypes; all loci hadtwo alleles and the minor allele frequency ranged from0.038to0.500; The observed andexpected heterozygosity ranged from0.054to0.792and0.064to0.547, respectively; Two locisignificantly depart from Hardy Weinberg equilibrium and no significant linkage disequilibrium(LD) was detected.
These SSR markers and SNP markers should be useful for the construction of geneticlinkage map and comparative genome analysis in A. japonicus.
引文
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