Simultaneous mutation detection of three homoeologous genes in wheat by High Resolution Melting analysis and Mutation Surveyor®

Abstract

BackgroundTILLING (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.ResultsWe 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.ConclusionPolyploidy 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.

iTILLING: A Personalized Approach to the Identification of Induced Mutations in Arabidopsis

“…In contrast, iTILLING is a gel-free system that uses high-resolution melt-curve analysis of PCR products to reveal mutations of interest. High-resolution melting has recently been shown to be an effective alternative to enzymatic cleavage for mutation detection in TILLING populations (Dong et al, 2009;Gady et al, 2009). Gady et al (2009) used a LightScanner System (Idaho Technology) high-resolution melting machine for their TILLING screen, whereas we utilized the CFX96 PCR Detection System from Bio-Rad Laboratories.…”

“…More recently, high-resolution melting analysis of PCR products has been used to identify heteroduplexes when performing TILLING (Dong et al, 2009;Gady et al, 2009). High-resolution melting analysis was originally developed for use in clinical settings to identify known single-nucleotide polymorphisms and small insertions/deletions potentially linked to genetic diseases (Erali et al, 2008).…”

“…In order to find individual plants carrying point mutations of interest, a process called TILLING (for Targeting Induced Local Lesions IN Genomes) was developed whereby genes are screened for mutations using a PCR-based assay (McCallum et al, 2000). Although originally developed for use with Arabidopsis, the TILLING process has been subsequently applied to a wide range of plants, including barley (Hordeum vulgare; Caldwell et al, 2004), Brassica napus , Brassica oleracea (Himelblau et al, 2009), Brassica rapa (Stephenson et al, 2010), Lotus japonicus (Perry et al, 2009), maize (Zea mays; Till et al, 2004), Medicago truncatula (Le Signor et al, 2009), oat (Avena sativa; Chawade et al, 2010), pea (Pisum sativum; Triques et al, 2007), potato (Solanum tuberosum; Elias et al, 2009), rice (Oryza sativa; Till et al, 2007), sorghum (Sorghum bicolor;Xin et al, 2008), soybean (Glycine max; Cooper et al, 2008), tomato (Solanum lycopersicum ; Gady et al, 2009), and wheat (Triticum aestivum; Dong et al, 2009). TILLING has also been used in Drosophila (Winkler et al, 2005), zebrafish (Wienholds et al, 2003), and Caenorhabditis elegans (Gilchrist et al, 2006).…”

Application of real-time PCR-based SNP detection for mapping of <i>Net2</i>, a causal D-genome gene for hybrid necrosis in interspecific crosses between tetraploid wheat and <i>Aegilops tauschii</i>

“…This technique has been successfully applied to EMS-induced mutant screening by targeting induced local lesions in genomes in common wheat (Dong et al, 2009;Botticella et al, 2011). Therefore, we considered that HRM analysis might help efficiently detect SNPs and construct high-density maps of the wheat genome.…”

Nuevas herramientas en la lucha contra las virosis del pimiento

“…Recientemente se han publicado nuevos enfoques basados en estas técnicas (Dong et al, 2009;Gady et al, 2009;Rigola et al, 2009), y es muy probable que se desarrollen otras estrategias basadas en la NGS. En este escenario, nuestras plataformas de Capsicum (ADNc y ADN) facilitarán la identificación de nuevas variaciones naturales en otros genes, relacionados con respuestas a factores bióticos o abióticos, o con caracteres útiles para la mejora de las especies domesticadas de pimiento.…”