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.
Highlights
Durum 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
Targeting Induced Local Lesions IN Genomes (TILLING) can be carried out using different procedures, such as the heteroduplex mismatch cleavage assay through single-strand-specific endonuclease CelI [10], Colasuonno et al BMC Genetics (2016) 17:43 the DNA melting temperature analysis [11] and the deep sequencing of pooled DNA [12]
Amplicon size varied on the bases of the Single nucleotide polymorphism (SNP) detection method: in the CelI assay based on agarose gel, we considered polymerase chain reaction (PCR) products up 1,000 bp, while in the denaturing high-performance liquid chromatography (DHPLC) method, we set the amplicon length with a maximum of 800 bp
Summary
Durum 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. The CelI assay represents the most popular, accessible and low-cost method to detect mutations in large populations [13] It has been combined with agarose gel detection approaches [14,15,16], with a non-denaturing polyacrylamide method [9, 17] or a gel based system (LI-COR DNA analyzer) requiring labeled PCR primers [10, 18]. Other pre-screening methods for SNP detection based on DNA melting temperature, such as Denaturing Gradient Gel Electrophoresis (DGGE) [19], Single-Strand Conformational Polymorphism (SSCP) [20] and Mass Spectrometry (MS) [21], were found to have limitations in terms of sensitivity, costs and suitability for high-throughput screening
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