title: DHPLC technology for high-throughput detection of mutations in a durum wheat TILLING population.
abstract: BACKGROUND
  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. 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.
  
  
  RESULTS
  In 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.
  
  
  CONCLUSIONS
  We 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.
authors.affiliation: Department of Soil, Plant and Food Sciences, section of Genetic and Plant Breeding, University of Bari "Aldo Moro", Via G. Amendola 165/A, 70126, Bari, Italy. pattybiotec@yahoo.it.
authors.forename: Pasqualina
authors.initials: P
authors.lastname: Colasuonno
authors.affiliation: Department of Soil, Plant and Food Sciences, section of Genetic and Plant Breeding, University of Bari "Aldo Moro", Via G. Amendola 165/A, 70126, Bari, Italy. ornellaincerti87@libero.it.
authors.forename: Ornella
authors.initials: O
authors.lastname: Incerti
authors.affiliation: Department of Soil, Plant and Food Sciences, section of Genetic and Plant Breeding, University of Bari "Aldo Moro", Via G. Amendola 165/A, 70126, Bari, Italy. marialuisalozito@gmail.com.
authors.forename: Maria Luisa
authors.initials: ML
authors.lastname: Lozito
authors.affiliation: Department of Soil, Plant and Food Sciences, section of Genetic and Plant Breeding, University of Bari "Aldo Moro", Via G. Amendola 165/A, 70126, Bari, Italy. rosanna.simeone@uniba.it.
authors.forename: Rosanna
authors.initials: R
authors.lastname: Simeone
authors.affiliation: Department of Soil, Plant and Food Sciences, section of Genetic and Plant Breeding, University of Bari "Aldo Moro", Via G. Amendola 165/A, 70126, Bari, Italy. agata.gadaleta@uniba.it.
authors.forename: Agata
authors.initials: A
authors.lastname: Gadaleta
authors.affiliation: Department of Soil, Plant and Food Sciences, section of Genetic and Plant Breeding, University of Bari "Aldo Moro", Via G. Amendola 165/A, 70126, Bari, Italy. antonio.blanco@uniba.it.
authors.forename: Antonio
authors.initials: A
authors.lastname: Blanco
mesh_terms: D000483:Alleles; D002338:Carotenoids / Q000378:metabolism; D018744:DNA, Plant / Q000235:genetics; D005819:Genetic Markers; D018745:Genome, Plant; D023281:Genomics / Q000379:methods; D060005:Genotyping Techniques; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D019753:Intramolecular Lyases / Q000235:genetics; D009154:Mutation; D010940:Plant Proteins / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA; D014908:Triticum / Q000235:genetics*
chemical_list: D018744:DNA, Plant; D005819:Genetic Markers; D010940:Plant Proteins; D002338:Carotenoids; D019753:Intramolecular Lyases; C070925:lycopene cyclase-isomerase