title: High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes.
abstract: BACKGROUND
  Targeted Induced Loci Lesions IN Genomes (TILLING) is increasingly being used to generate and identify mutations in target genes of crop genomes. TILLING populations of several thousand lines have been generated in a number of crop species including Brassica rapa. Genetic analysis of mutants identified by TILLING requires an efficient, high-throughput and cost effective genotyping method to track the mutations through numerous generations. High resolution melt (HRM) analysis has been used in a number of systems to identify single nucleotide polymorphisms (SNPs) and insertion/deletions (IN/DELs) enabling the genotyping of different types of samples. HRM is ideally suited to high-throughput genotyping of multiple TILLING mutants in complex crop genomes. To date it has been used to identify mutants and genotype single mutations. The aim of this study was to determine if HRM can facilitate downstream analysis of multiple mutant lines identified by TILLING in order to characterise allelic series of EMS induced mutations in target genes across a number of generations in complex crop genomes.
  
  
  RESULTS
  We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner™ system we were able to detect mutations in heterozygous and homozygous states for both genes.
  
  
  CONCLUSIONS
  Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations) from receiving M3 seed of Brassica rapa from the RevGenUK TILLING service.
authors.affiliation: School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK.
authors.forename: Seosamh Ó
authors.initials: SÓ
authors.lastname: Lochlainn
authors.affiliation: Rothamsted Research, Harpenden AL5 2JQ, UK.
authors.forename: Stephen
authors.initials: S
authors.lastname: Amoah
authors.affiliation: School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK.
authors.forename: Neil S
authors.initials: NS
authors.lastname: Graham
authors.affiliation: School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK.
authors.forename: Khalid
authors.initials: K
authors.lastname: Alamer
authors.affiliation: School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK.
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Rios
authors.affiliation: Rothamsted Research, Harpenden AL5 2JQ, UK.
authors.forename: Smita
authors.initials: S
authors.lastname: Kurup
authors.affiliation: Rothamsted Research, Harpenden AL5 2JQ, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Stoute
authors.affiliation: School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK.
authors.forename: John P
authors.initials: JP
authors.lastname: Hammond
authors.affiliation: Department of Crop Genetics, John Innes Centre, Norwich, NR4 7UH, UK.
authors.forename: Lars
authors.initials: L
authors.lastname: Østergaard
authors.affiliation: Southern Cross Plant Science, Southern Cross University, PO Box 157Lismore NSW 2480, Australia.
authors.forename: Graham J
authors.initials: GJ
authors.lastname: King
authors.affiliation: The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK.
authors.forename: Phillip J
authors.initials: PJ
authors.lastname: White
authors.affiliation: School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK.
authors.forename: Martin R
authors.initials: MR
authors.lastname: Broadley