Plant Breeding Institute, University of Sydney, PMB 4011, Narellan NSW 2567, Australia.
BMC Plant Biol. 2009 Dec 4;9:143. doi: 10.1186/1471-2229-9-143.
TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful tool for reverse genetics, combining traditional chemical mutagenesis with high-throughput PCR-based mutation detection to discover induced mutations that alter protein function. The most popular mutation detection method for TILLING is a mismatch cleavage assay using the endonuclease CelI. For this method, locus-specific PCR is essential. Most wheat genes are present as three similar sequences with high homology in exons and low homology in introns. Locus-specific primers can usually be designed in introns. However, it is sometimes difficult to design locus-specific PCR primers in a conserved region with high homology among the three homoeologous genes, or in a gene lacking introns, or if information on introns is not available. Here we describe a mutation detection method which combines High Resolution Melting (HRM) analysis of mixed PCR amplicons containing three homoeologous gene fragments and sequence analysis using Mutation Surveyor software, aimed at simultaneous detection of mutations in three homoeologous genes.
We demonstrate that High Resolution Melting (HRM) analysis can be used in mutation scans in mixed PCR amplicons containing three homoeologous gene fragments. Combining HRM scanning with sequence analysis using Mutation Surveyor is sensitive enough to detect a single nucleotide mutation in the heterozygous state in a mixed PCR amplicon containing three homoeoloci. The method was tested and validated in an EMS (ethylmethane sulfonate)-treated wheat TILLING population, screening mutations in the carboxyl terminal domain of the Starch Synthase II (SSII) gene. Selected identified mutations of interest can be further analysed by cloning to confirm the mutation and determine the genomic origin of the mutation.
Polyploidy is common in plants. Conserved regions of a gene often represent functional domains and have high sequence similarity between homoeologous loci. The method described here is a useful alternative to locus-specific based methods for screening mutations in conserved functional domains of homoeologous genes. This method can also be used for SNP (single nucleotide polymorphism) marker development and eco-TILLING in polyploid species.
TILLING（靶向诱导基因组局部突变）是一种强大的反向遗传学工具，它将传统的化学诱变与基于高通量 PCR 的突变检测相结合，以发现改变蛋白质功能的诱导突变。TILLING 最流行的突变检测方法是使用内切酶 CelI 的错配切割测定法。对于这种方法，基因座特异性 PCR 是必不可少的。大多数小麦基因以三个相似的序列存在，它们在exon 中具有高度同源性，在 intron 中具有低度同源性。通常可以在 intron 中设计基因座特异性引物。然而，在三个同源基因高度同源的保守区域、在缺乏内含子的基因中，或者在内含子信息不可用时，设计基因座特异性 PCR 引物有时会很困难。在这里，我们描述了一种突变检测方法，该方法结合了含有三个同源基因片段的混合 PCR 扩增物的高分辨率熔解（HRM）分析和使用 Mutation Surveyor 软件的序列分析，旨在同时检测三个同源基因中的突变。
我们证明，高分辨率熔解（HRM）分析可用于含有三个同源基因片段的混合 PCR 扩增物中的突变扫描。将 HRM 扫描与使用 Mutation Surveyor 的序列分析相结合，足以检测到含有三个同源基因的混合 PCR 扩增物中杂合状态下的单个核苷酸突变。该方法在 EMS（乙基甲磺酸）处理的小麦 TILLING 群体中进行了测试和验证，筛选了淀粉合酶 II（SSII）基因羧基末端结构域中的突变。选择有意义的鉴定突变可通过克隆进一步分析，以确认突变并确定突变的基因组来源。
多倍体在植物中很常见。基因的保守区域通常代表功能域，并且在同源基因座之间具有高度的序列相似性。这里描述的方法是筛选同源基因保守功能域中突变的一种有用替代方法，而不是基于基因座的方法。该方法还可用于 SNP（单核苷酸多态性）标记的开发和多倍体物种的生态 TILLING。
