PT-Flax (phenotyping and TILLinG of flax) : development of a flax (Linum usitatissimum L.) mutant population and TILLinG platform for forward and reverse genetics
2013
Chantreau , Maxime (INRA , Peronne (France). UMR 1281 Stress Abiotiques et Différenciation des Végétaux Cultivés) | Grec , Sébastien (INRA , Peronne (France). UMR 1281 Stress Abiotiques et Différenciation des Végétaux Cultivés) | Gutierrez , Laurent (Université de Picardie Jules Verne, Amiens(France). Centre de Ressources Régionales en Biologie Moléculaire (CRRBM) ) | Dalmais , Marion (INRA , Evry (France). UMR 1165 Génomique Végétale) | Pineau , Christophe (Linea, Grandvilliers(France).) | Demailly , Hervé (Université de Picardie Jules Verne, Amiens(France). Centre de Ressources Régionales en Biologie Moléculaire (CRRBM)) | Paysant-Leroux , Christine ( Terre de Lin Société Coopérative Agricole, Saint-Pierre-Le-Viger(France).) | Tavernier , Reynald (Linea, Grandvilliers(France).) | Trouvé , Jean-Paul (Terre de Lin Société Coopérative Agricole, Saint-Pierre-Le-Viger(France).) | Chatterjee , Manash (Bench Bio Private LimitedNational University of Ireland, GujaratGalway(Inde).) | Guillot , Xavier (Laboulet Semences S.A., Airaines(France).) | Brunaud , Veronique (INRA , Evry (France). UMR 1165 Génomique Végétale) | Chabbert , Brigitte(auteur de correspondance) (INRA , Reims (France). UMR 0614 Fractionnement des AgroRessources et Environnement) | van Wuytswinkel , Olivier (Université de Picardie Jules Verne, Amiens(France). UFR des Sciences - EA 3900-BioPI) | Bendahmane , Abdelhafid (INRA , Evry (France). UMR 1165 Génomique Végétale) | Thomasset , Brigitte (Centre National de la Recheche Scientifique(France). FRE 3580, GEC, ) | Hawkins , Simon (INRA , Peronne (France). UMR 1281 Stress Abiotiques et Différenciation des Végétaux Cultivés)
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.
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