title: PT-Flax (phenotyping and TILLinG of flax): development of a flax (Linum usitatissimum L.) mutant population and TILLinG platform for forward and reverse genetics.
abstract: BACKGROUND
  Flax (Linum usitatissimum L.) is an economically important fiber and oil crop that has been grown for thousands of years. The genome has been recently sequenced and transcriptomics are providing information on candidate genes potentially related to agronomically-important traits. In order to accelerate functional characterization of these genes we have generated a flax EMS mutant population that can be used as a TILLinG (Targeting Induced Local Lesions in Genomes) platform for forward and reverse genetics.
  
  
  RESULTS
  A population of 4,894 M2 mutant seed families was generated using 3 different EMS concentrations (0.3%, 0.6% and 0.75%) and used to produce M2 plants for subsequent phenotyping and DNA extraction. 10,839 viable M2 plants (4,033 families) were obtained and 1,552 families (38.5%) showed a visual developmental phenotype (stem size and diameter, plant architecture, flower-related). The majority of these families showed more than one phenotype. Mutant phenotype data are organised in a database and can be accessed and searched at UTILLdb (http://urgv.evry.inra.fr/UTILLdb). Preliminary screens were also performed for atypical fiber and seed phenotypes. Genomic DNA was extracted from 3,515 M2 families and eight-fold pooled for subsequent mutant detection by ENDO1 nuclease mis-match cleavage. In order to validate the collection for reverse genetics, DNA pools were screened for two genes coding enzymes of the lignin biosynthesis pathway: Coumarate-3-Hydroxylase (C3H) and Cinnamyl Alcohol Dehydrogenase (CAD). We identified 79 and 76 mutations in the C3H and CAD genes, respectively. The average mutation rate was calculated as 1/41 Kb giving rise to approximately 9,000 mutations per genome. Thirty-five out of the 52 flax cad mutant families containing missense or codon stop mutations showed the typical orange-brown xylem phenotype observed in CAD down-regulated/mutant plants in other species.
  
  
  CONCLUSIONS
  We have developed a flax mutant population that can be used as an efficient forward and reverse genetics tool. The collection has an extremely high mutation rate that enables the detection of large numbers of independant mutant families by screening a comparatively low number of M2 families. The population will prove to be a valuable resource for both fundamental research and the identification of agronomically-important genes for crop improvement in flax.
authors.forename: Maxime
authors.initials: M
authors.lastname: Chantreau
authors.forename: Sébastien
authors.initials: S
authors.lastname: Grec
authors.forename: Laurent
authors.initials: L
authors.lastname: Gutierrez
authors.forename: Marion
authors.initials: M
authors.lastname: Dalmais
authors.forename: Christophe
authors.initials: C
authors.lastname: Pineau
authors.forename: Hervé
authors.initials: H
authors.lastname: Demailly
authors.forename: Christine
authors.initials: C
authors.lastname: Paysant-Leroux
authors.forename: Reynald
authors.initials: R
authors.lastname: Tavernier
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Trouvé
authors.forename: Manash
authors.initials: M
authors.lastname: Chatterjee
authors.forename: Xavier
authors.initials: X
authors.lastname: Guillot
authors.forename: Véronique
authors.initials: V
authors.lastname: Brunaud
authors.forename: Brigitte
authors.initials: B
authors.lastname: Chabbert
authors.forename: Olivier
authors.initials: O
authors.lastname: van Wuytswinkel
authors.forename: Abdelhafid
authors.initials: A
authors.lastname: Bendahmane
authors.forename: Brigitte
authors.initials: B
authors.lastname: Thomasset
authors.affiliation: Université Lille Nord de France, Lille 1 UMR 1281, Villeneuve d'Ascq cedex F-59650, France. simon.hawkins@univ-lille1.fr.
authors.forename: Simon
authors.initials: S
authors.lastname: Hawkins
mesh_terms: D020029:Base Pairing / Q000235:genetics; D005020:Ethyl Methanesulfonate; D019597:Flax / Q000235:genetics*; D035264:Flowers / Q000235:genetics; D017343:Genes, Plant / Q000235:genetics; D018745:Genome, Plant / Q000235:genetics*; D005838:Genotype; D008031:Lignin / Q000235:genetics; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D059645:Mutation Rate; D059372:Nucleotide Motifs / Q000235:genetics; D010641:Phenotype; D010802:Phylogeny; D059386:Reverse Genetics / Q000379:methods*; D012639:Seeds / Q000235:genetics
chemical_list: D008031:Lignin; D005020:Ethyl Methanesulfonate