Mining expressed sequences for single nucleotide polymorphisms in Pacific abalone Haliotis discus hannai.
Mining expressed sequences for single nucleotide polymorphisms in Pacific abalone Haliotis discus hannai.
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DOI：
10.1111/j.1365-2109.2009.02269.x
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发表时间：
2009-09
影响因子：
2
通讯作者：
Haigang Qi;Xiao Liu;Guofan Zhang;Fucun Wu
中科院分区：
文献类型：
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作者：
Haigang Qi;Xiao Liu;Guofan Zhang;Fucun Wu
单核苷酸多态性（single nucleotide polymorphisms，SNPs）是群体遗传学、系谱分析和基因组作图的重要资源，但在太平洋鲍中的研究尚未见报道。本研究采用生物信息学方法，在皱纹盘鲍（Haliotis discus hannai）表达序列（ESTs）中发现SNPs，并采用聚合酶链反应直接测序（PCR-DS）和等位基因特异性PCR（AS-PCR）分别进行SNPs检测和基因型评分。共组装了5893个EST，鉴定出302个SNP。EST中SNPs的平均密度为1%。根据EST侧翼的SNPs设计了52对测序引物，其中13对引物能扩增出预期大小的产物。聚合酶链反应直接测序的扩增产物从合并的DNA样品显示40个多态性SNP位点。采用改良的四引物AS-PCR方法，对37只野生鲍的7个线粒体和6个核SNPs进行了分型和特征分析。因此，从有限数量的EST中发现SNPs是可行的，AS-PCR作为一种简单、可靠的检测方法，可作为鲍鱼及其他非模式生物中小规模SNPs检测的主要方法。
Although single nucleotide polymorphisms (SNPs) are important resources for population genetics, pedigree analysis and genomic mapping, such loci have not been reported in Pacific abalone so far. In this study, a bioinformatics strategy was adopted to discover SNPs within the expressed sequences (ESTs) of Pacific abalone, Haliotis discus hannai, and furthermore, polymerase chain reaction direct sequencing (PCR-DS) and allele-specific PCR (AS-PCR) were used for SNPs detection and genotype scoring respectively. A total of 5893 ESTs were assembled and 302 putative SNPs were identified. The average density of SNPs in ESTs was 1%. Fifty-two sets of sequencing primers were designed from SNPs flanking ESTs to amplify the genomic DNA, and 13 could generate products of expected size. Polymerase chain reaction direct sequencing of the amplification products from pooled DNA samples revealed 40 polymorphic SNP loci. Using a modified tetra-primer AS-PCR, seven mitochondrial and six nuclear SNPs were typed and characterized among 37 wild abalones. In conclusion, it is feasible to discover SNPs from number limited ESTs and the AS-PCR as a simple, robust and reliable assay could be a primary method for small- and medium-scale SNPs detection in abalones as well as other non-model organisms.