PT-Flax (phenotyping and TILLinG of flax): development of a flax (Linum usitatissimumL.) mutant population and TILLinG platform for forward and reverse genetics

Abstract

BackgroundFlax (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.ResultsA 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.ConclusionsWe 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.

Ectopic Lignification in the Flax lignified bast fiber1 Mutant Stem Is Associated with Tissue-Specific Modifications in Gene Expression and Cell Wall Composition

“…In flax stems, the outer tissues containing the cellulose-rich bast fibers can be easily separated from the inner tissues containing the lignified secondary xylem cells, thereby allowing comparative studies of cell wall formation in these two tissues (Fenart et al, 2010;Huis et al, 2012). To learn more about the mechanisms regulating cell wall biosynthesis in flax, we used a combination of UV autofluorescence and phloroglucinol-HCl staining to screen a flax EMS mutant population for mutants showing altered bast fiber lignification patterns (Chantreau et al, 2013). This approach allowed us to identify 93 families showing increased lignification in bast fibers, and we then went on to characterize one of these mutants (lbf1) in detail.…”

“…To identify mutants showing increased lignification in bast fibers, we first screened 8999 plants from 3391 M2 families (Chantreau et al, 2013). Examination of transversal hand-sections of stem from individual plants by UV microscopy allowed us to identify 540 families showing increased autofluorescence in bast fibers.…”

“…For example, the groups A to E show increased lignification uniquely in fiber cells, whereas the groups F to H also show increased lignification in surrounding cells. We then classified the flax lbf mutants into different categories based on previously established visual phenotypes of the flax EMS mutant population (Supplemental Table 1; Chantreau et al, 2013). Thirty-two percent of the core collection families showed no obvious morphological phenotype, 31% were smaller to wildtype plants, and 14% showed increased stem branching ( Figure 2).…”

“…Flax (Linum usitatissimum) EMS mutants used in this study come from the PT-Flax Collection (Chantreau et al, 2013). M2 to M5 plants were grown in greenhouses or outside at the University of Lille, France.…”

“…More recently, we generated and characterized a flax ethyl methanesulfonate (EMS) mutant population that has allowed us to obtain mutants for the CAD and C3H lignin genes through a TILLinG reverse genetics approach (Chantreau et al, 2013). In this article, we report the screening of this population and the identification of 319 independent mutants showing altered lignification profiles in bast fibers.…”