DHPLC technology for high-throughput detection of mutations in a durum wheat TILLING population

Abstract

BackgroundDurum wheat (Triticum turgidum L.) is a cereal crop widely grown in the Mediterranean regions; the amber grain is mainly used for the production of pasta, couscous and typical breads. Single nucleotide polymorphism (SNP) detection technologies and high-throughput mutation induction represent a new challenge in wheat breeding to identify allelic variation in large populations. The TILLING strategy makes use of traditional chemical mutagenesis followed by screening for single base mismatches to identify novel mutant loci. Although TILLING has been combined to several sensitive pre-screening methods for SNP analysis, most rely on expensive equipment. Recently, a new low cost and time saving DHPLC protocol has been used in molecular human diagnostic to detect unknown mutations.ResultsIn this work, we developed a new durum wheat TILLING population (cv. Marco Aurelio) using 0.70-0.85 % ethyl methane sulfonate (EMS). To investigate the efficiency of the mutagenic treatments, a pilot screening was carried out on 1,140 mutant lines focusing on two target genes (Lycopene epsilon-cyclase, ε-LCY, and Lycopene beta-cyclase, β-LCY) involved in carotenoid metabolism in wheat grains. We simplify the heteroduplex detection by two low cost methods: the enzymatic cleavage (CelI)/agarose gel technique and the denaturing high-performance liquid chromatography (DHPLC). The CelI/agarose gel approach allowed us to identify 31 mutations, whereas the DHPLC procedure detected a total of 46 mutations for both genes. All detected mutations were confirmed by direct sequencing. The estimated overall mutation frequency for the pilot assay by the DHPLC methodology resulted to be of 1/77 kb, representing a high probability to detect interesting mutations in the target genes.ConclusionWe demonstrated the applicability and efficiency of a new strategy for the detection of induced variability. We produced and characterized a new durum wheat TILLING population useful for a better understanding of key gene functions. The availability of this tool together with TILLING technique will expand the polymorphisms in candidate genes of agronomically important traits in wheat.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-016-0350-0) contains supplementary material, which is available to authorized users.

A roadmap for gene functional characterisation in wheat

“…Lastly, if the gene of interest is missing or is already a null allele in Kronos or Cadenza, which can be determined using the full genome sequences of the two cultivars, mutant populations of other genotypes are available (e.g. Dong et al (2009); Chen et al (2012); Bovina et al (2014); Sestili et al (2015); Colasuonno et al (2016)), although these would need to be screened using conventional PCR-based approaches. Additional practical information about selecting mutant lines and downstream analyses can be found at www.wheat-training.com/tilling-mutant-resources and in Uauy et al (2017).…”

Recent Biotechnological Advances in the Improvement of Cassava

“…TILLING involves induction of mutations in the plant genome using classical mutagenesis approaches followed by traditional or high throughput deep sequencing to identify the mutations in the gene of interest [77][78][79]. This technique has been used in allele discovery in different plant species [80][81][82][83]. EcoTILLING, which is an adaptation of the TILLING, is used in detecting rare single nucleotide polymorphism (SNPs) or small INDELs in target genes in natural populations [84].…”

DNA Fingerprinting of Vegetable Soybean Cultivar ‘Zhexian No.9’ Using 101 New Developed HRM-Based SNP Markers

“…Several methods were reported in the detection of SNP markers, including SSCP (Kozlowski and Krzyzosiak, 2001), DGGE (Guldberg et al 1993), CAPS (Li et al 2012), DNA-chip (Singh et al 2015), DHPLC (Wolford et al 2000;Colasuonno et al 2016), mass spectrometric detection (Park et al 2017), and HRM (high-resolution melting) (Villano et al 2016;Kim et al 2016). The recently developed HRM analysis method provided us a novel, quick, and close-tube PCR approach to analyze SNPs (Nguyen et al 2012).…”