Dipartimento di Scienze delle Produzioni Agrarie e Alimentari, University of Catania, Catania, Italy.
PLoS One. 2012;7(8):e44202. doi: 10.1371/journal.pone.0044202. Epub 2012 Aug 30.
High resolution melting curve analysis (HRM) has been used as an efficient, accurate and cost-effective tool to detect single nucleotide polymorphisms (SNPs) or insertions or deletions (INDELs). However, its efficiency, accuracy and applicability to discriminate microsatellite polymorphism have not been extensively assessed. The traditional protocols used for SSR genotyping include PCR amplification of the DNA fragment and the separation of the fragments on electrophoresis-based platform. However, post-PCR handling processes are laborious and costly. Furthermore, SNPs present in the sequences flanking repeat motif cannot be detected by polyacrylamide-gel-electrophoresis based methods. In the present study, we compared the discriminating power of HRM with the traditional electrophoresis-based methods and provided a panel of primers for HRM genotyping in Citrus. The results showed that sixteen SSR markers produced distinct polymorphic melting curves among the Citrus spp investigated through HRM analysis. Among those, 10 showed more genotypes by HRM analysis than capillary electrophoresis owing to the presence of SNPs in the amplicons. For the SSR markers without SNPs present in the flanking region, HRM also gave distinct melting curves which detected same genotypes as were shown in capillary electrophoresis (CE) analysis. Moreover, HRM analysis allowed the discrimination of most of the 15 citrus genotypes and the resulting genetic distance analysis clustered them into three main branches. In conclusion, it has been approved that HRM is not only an efficient and cost-effective alternative of electrophoresis-based method for SSR markers, but also a method to uncover more polymorphisms contributed by SNPs present in SSRs. It was therefore suggested that the panel of SSR markers could be used in a variety of applications in the citrus biodiversity and breeding programs using HRM analysis. Furthermore, we speculate that the HRM analysis can be employed to analyse SSR markers in a wide range of applications in all other species.
高分辨率熔解曲线分析（HRM）已被用作一种高效、准确且具有成本效益的工具，用于检测单核苷酸多态性（SNP）或插入/缺失（INDEL）。然而，其效率、准确性以及用于区分微卫星多态性的适用性尚未得到广泛评估。传统的 SSR 基因分型协议包括 DNA 片段的 PCR 扩增和电泳平台上的片段分离。然而，PCR 后处理过程繁琐且昂贵。此外，基于聚丙烯酰胺凝胶电泳的方法无法检测重复基序侧翼序列中的 SNP。在本研究中，我们比较了 HRM 与传统电泳方法的区分能力，并为柑橘 HRM 基因分型提供了一组引物。结果表明，通过 HRM 分析，16 个 SSR 标记在调查的柑橘属中产生了不同的多态性熔解曲线。其中，10 个 SSR 标记通过 HRM 分析显示出比毛细管电泳更多的基因型，这是由于扩增子中存在 SNP。对于侧翼区域不存在 SNP 的 SSR 标记，HRM 也给出了不同的熔解曲线，检测到与毛细管电泳（CE）分析相同的基因型。此外，HRM 分析允许区分大多数 15 个柑橘基因型，所得的遗传距离分析将它们聚类为三个主要分支。总之，已经证实 HRM 不仅是 SSR 标记基于电泳方法的高效且具有成本效益的替代方法，也是一种揭示由 SSR 中存在的 SNP 贡献的更多多态性的方法。因此，建议使用 HRM 分析使用 SSR 标记面板进行柑橘生物多样性和育种计划的各种应用。此外，我们推测 HRM 分析可用于分析其他物种中 SSR 标记的广泛应用。
