High resolution melting analysis of almond SNPs derived from ESTs
High resolution melting analysis of almond SNPs derived from ESTs
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DOI：
10.1007/s00122-008-0870-8
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发表时间：
2008-12-01
影响因子：
5.4
通讯作者：
Sedgley, Margaret
中科院分区：
文献类型：
--
作者：
Wu, Shu-Biao;Wirthensohn, Michelle G.;Sedgley, Margaret
高分辨率熔解曲线（HRM）是近年来SNP检测的一个新进展。该技术测量温度诱导的短PCR扩增子的链分离，并且能够检测样品之间小至一个碱基差异的变化。它已被应用于分析和扫描导致人类疾病的基因突变。在植物物种中，这种方法的使用是有限的。我们应用HRM分析杏仁SNP发现和基因分型的基础上预测的SNP信息来自杏仁和桃EST数据库。通过对25个扁桃品种的HRM分析，从扁桃和桃EST重叠群中筛选出推定的SNPs。对所有4类SNPs、INDEL和微卫星进行了区分，并建立了17个扩增子的HRM图谱。含有单、双和多个SNPs的PCR扩增子产生不同的HRM谱。此外，不同基因型的INDEL和微卫星变异也通过HRM分析进行了表征。通过对PCR产物进行测序，在HRM扩增子及其侧翼区域中验证/揭示了100个SNP。结果表明，该基因区SNPs的平均频率为1：114 bp，转换与颠换比为1.16：1。稀有等位基因频率在0.02 ~ 0.5之间，多态信息含量在0.04 ~ 0.53之间，平均为0.31。HRM已被证明是一种快速、低成本和有效的SNP发现和基因分型方法，特别是对于没有太多基因组信息的物种，如杏仁。
High resolution melting curve (HRM) is a recent advance for the detection of SNPs. The technique measures temperature induced strand separation of short PCR amplicons, and is able to detect variation as small as one base difference between samples. It has been applied to the analysis and scan of mutations in the genes causing human diseases. In plant species, the use of this approach is limited. We applied HRM analysis to almond SNP discovery and genotyping based on the predicted SNP information derived from the almond and peach EST database. Putative SNPs were screened from almond and peach EST contigs by HRM analysis against 25 almond cultivars. All 4 classes of SNPs, INDELs and microsatellites were discriminated, and the HRM profiles of 17 amplicons were established. The PCR amplicons containing single, double and multiple SNPs produced distinctive HRM profiles. Additionally, different genotypes of INDEL and microsatellite variations were also characterised by HRM analysis. By sequencing the PCR products, 100 SNPs were validated/revealed in the HRM amplicons and their flanking regions. The results showed that the average frequency of SNPs was 1:114 bp in the genic regions, and transition to transversion ratio was 1.16:1. Rare allele frequencies of the SNPs varied from 0.02 to 0.5, and the polymorphic information contents of the SNPs were from 0.04 to 0.53 at an average of 0.31. HRM has been demonstrated to be a fast, low cost, and efficient approach for SNP discovery and genotyping, in particular, for species without much genomic information such as almond.