PT-Flax (phenotyping and TILLinG of flax): development of a flax (Linum usitatissimumL.) mutant population and TILLinG platform for forward and reverse genetics
- Maxime Chantreau1, 2,
- Sébastien Grec1, 2,
- Laurent Gutierrez3,
- Marion Dalmais4,
- Christophe Pineau5,
- Hervé Demailly3,
- Christine Paysant-Leroux6,
- Reynald Tavernier5,
- Jean-Paul Trouvé6,
- Manash Chatterjee7, 8,
- Xavier Guillot9,
- Véronique Brunaud4,
- Brigitte Chabbert10, 11,
- Olivier van Wuytswinkel12,
- Abdelhafid Bendahmane4,
- Brigitte Thomasset13 and
- Simon Hawkins1, 2Email author
© Chantreau et al.; licensee BioMed Central Ltd. 2013
Received: 5 July 2013
Accepted: 9 October 2013
Published: 15 October 2013
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.
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.
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.
KeywordsFlax TILLinG Mutants Fiber Lignin Lignan Oil Fatty acids
Flax (Linum usitatissimum L.) is an economically important oil and fiber crop that has been domesticated and grown by mankind for thousands of years. Oil extracted from flax seeds (linseed) is a considerable source of the omega-3 fatty acid, α-linolenic acid (ALA) and seeds also contain biologically active lignans with beneficial effects on human health . Flax phloem fibers have cell walls rich in cellulose and are used for textiles (linen) and for reinforcing composite polymers as an environmentally-friendly substitute for glass fibers . Flax is also used as a biological model to study the molecular mechanisms involved in the formation of hypolignified secondary cell walls characteristic of different fiber species (e.g. flax, hemp, jute, kenaf etc.) [3–6].
Recently a number of different flax resources and approaches including high-density microarray platforms, physical and genetic maps, molecular markers, metabolomics and proteomics [6–12] have been developed. The recent sequencing of the genome  has also opened the way for flax genomics leading to rapid advances in the structural identification of genes and gene families [14, 15]. However, while all of these approaches allow the identification of large numbers of genes potentially involved in a wide variety of different biological processes, the confirmation of their biological role(s) requires functional characterization. Flax can be genetically engineered and a limited number of genes have been up-/down-regulated in this species thereby providing important functional information on the role of these genes [5, 16, 17]. In order to accelerate functional characterization of genes potentially associated with different agronomical traits for crop improvement we have developed a chemically mutagenized (EMS) flax population and TILLinG (Targeting Induced Local Lesions IN Genomes) platform.
TILLinG (Targeting Induced Local Lesions IN Genomes) is a high-throughput reverse genetic method used to obtain an allelic series of a targeted mutated gene in a mutagenized population [18, 19]. Chemical mutagenesis is complementary to other approaches such as T-DNA insertion or radiation and has been applied to a wide range of different plant species [20–24]. Currently, the most usual detection method depends on the use of the specific mis-match endonuclease ENDO1 to detect chemically induced SNPs. Nevertheless, high throughput sequencing by NGS technologies coupled with variant detection algorithms is also starting to be used to detect such mutations [25, 26]. The development of such an approach in flax is timely since although this species can be transformed by Agrobacterium, the process is time consuming and relatively inefficient . Ethane Methyl-Sulphonate (EMS) has been previously used as a chemical mutagen to introduce genetic variability into flax, but has not yet been used for reverse genetics .
In this paper, we present the development and characterization of a flax EMS mutagenized population. Visual phenotyping and the successful identification of a large number of cad and c3h lignin gene mutants validated the use of our population as a valuable forward and reverse genetics tool. The use of this population in subsequent studies will greatly facilitate the functional characterization of different targeted genes in this economically important species.
Production and phenotyping of the EMS Flax mutant population
Preliminary tests and kill curve analyses (Additional file 1) were performed on flax (Linum usitatissimum L. cv Diane) seeds to determine appropriate EMS concentrations as previously described [29–32]. Based on these results 3 different EMS percentages (0.3%, 0.6% and 0.75%) were used to mutagenize 10,000 flax seeds. These mutagenized seeds were sown and gave rise to 5,000 M1 plants. M2 seeds were collected from M1 plants and all the seeds produced by an individual M1 plant were pooled to constitute the corresponding M2 family. After seed collection from M1 plants, 4,894 M2 different seed families were obtained and used as the basis for the flax TILLinG resource.
Constitution of the M2 mutant population and percentage sterility for each EMS concentration
Number of M2 families
Criteria of Phenotyping categories used to describe the Flax M2 mutant population
Data on flax mutant phenotypes were introduced into the UTILLdb database and can be consulted at http://urgv.evry.inra.fr/UTILLdb.
Flax lignin gene TILLinG
For reverse genetics, leaves of individual M2 plants were collected and pooled by families for DNA extraction. M3 seeds were collected for long-term storage and production of further material. To estimate mutation density and validate the flax population for future reverse genetics in this species, two genes were TILLed. Since flax is cultivated for both its seeds and its bast fibers and cell wall lignin content is an important factor in bast fiber quality, we decided to TILL two lignin genes (coumarate-3-hydroxylase, C3H and cinnamyl alcohol dehydrogenase, CAD). C3H acts early in the monolignol biosynthetic pathway and plays a key role in the production of G and S lignin monomers whereas CAD catalyses the final step in the production of the lignin monomers (monolignols) .
The CODDLE program (Codons Optimized to Discover Deleterious Lesions) combined with the PRIMER 3 tool were used to define the region within our two genes, with the most probability of a deleterious G/C to A/T transition and to design PCR primers compatible with the ENDO1 approach. A region of 1,077 Nt (1170–2247) was identified in the C3H [Phytozome : Lus10033524] gene. For the CAD gene [Phytozome : Lus10027864], CODDLE targeted a region between position 38 and 1336 in the genomic sequence, corresponding to the dehydrogenase alcohol domain. However, given sequence redundancy between different flax CAD genes in this region we chose to target the region between positions 828 and 1791. This region is composed of a nucleotide binding domain involved in cofactor binding and a catalytic domain involved in substrate binding .
A total of 150 CAD and C3H mutants was obtained by screening for mutations with the mismatch-specific endonuclease ENDO1 as previously described . Sixty-three, 49 and 39 mutations were detected in the 0.3%, 0.6% and 0.75% EMS populations, respectively. One cad family (0,6%) contained 2 mutations. Mutation densities varied between 1/49 kb and 1/30 kb depending on the EMS dose giving rise to an average value of 1 mutation per 41 kb.
Characteristics of mutations in CAD and C3H amplicons
% of GC in exons
Identified mutations in exon
Type of mutation (exon)
% Mutation saturation
EMS action on G/C base pairs is influenced by the local environment
Frequency of triplet motif centred on all Gs and on mutated Gs. Ratios were obtained by comparing the frequency of a particular triplet centred on the mutated G to the frequency of the same triplet centred on any available G in both amplicons
Frequencies of triplet centered on all Gs
Frequencies of triplet centered on mutated Gs
From the genotype to the phenotype
Having identified a large number of flax CAD and C3H mutants we then wished to know whether the mutations were associated with phenotypic modifications commonly observed in down-regulated plants or natural mutants in other species. Such data would also largely contribute to validating the interest of our mutant population for functional genomics in flax. For this we decided to focus on the CAD mutants since both natural CAD mutants and down-regulated CAD plants in other species show a characteristic and easily observable red-brown coloration of the wood known as the 'brown midrib’ phenotype. This coloration is mainly due to the accumulation of higher amounts of cinnamaldehydes into the lignin polymer rather than the more usual cinnamyl alcohols [42–48]. In contrast C3H down-regulation in other species is associated with modifications in lignin structure but no visible phenotype . Observation of stem cross-sections obtained from the 52 M2 mutant CAD families (108 plants) with a missense or stop codon mutation allowed us to identify 62 plants (belonging to 35 families) with a brown midrib phenotype (phenotype frequency = 0.57). A comparable screening of a randomized subset of 33 non-CAD mutant families (94 plants) identified 6 plants with a weak brown midrib phenotype (phenotype frequency = 0.06). These data strongly support the idea that the observed brown midrib phenotype results from a mutation within the targeted flax CAD gene.
PSSM/Sift scores for CAD mutant lines showing category 1 brown midrib phenotype
No. Indiv 1/total*
Mutation frequencies and ploidy levels in different published EMS plant populations
EMS concentration (%)
Frequency of mutation
Linum usitatissimum L.
-0.3, 0.6, 0.75%
Triticum aestivum L.
-0.5, 0.6, 0.7%
Brassica napus L.
-0.5, 0.8, 1, 1.2%
-from 1/12 to 1/22 kb
-from 1/27 to 1/60 kb
-1/1 710 kb
Cucumis melo L.
Heliantus annus L.
-Between 0.25 and 0.5%
-Between 0.25 and 0.56 %
Glycine max L. Merr.
Hordeum vulgare L.
-Between 0.25 and 0.75 %
-Between 0.1 and 0.3%
-1/2 000 Kb
-1/1 000 Kb
Hordeum vulgare L.
-1/1 000 Kb
With a genome size of approximately 370 Mb  we can estimate that there will be an average of approximately 9,000 mutations per genome. Despite this high value, the majority (81.6 – 93.7%) of the M2 population were viable and produced seeds suggesting that flax plants can support a high mutation level . Although the high number of mutations per genome might be considered as a disadvantage since more back-crosses will be necessary to reduce total mutation number and identify genes potentially associated with a particular phenotype via a positional cloning approach, it also presents a number of advantages. Firstly, it considerably reduces the number of families that need to be screened to identify a mutant. For example, calculations show that only 56 families have to be screened to identify a missense mutation in a 1 Kb exon target and only 650 families need to be screened to identify a codon stop mutation, thereby reducing the overall time and costs spent on mutant identification. Secondly, the high mutation rate allows the identification of a large number of independent mutant families for a given gene in a reverse genetics strategy. For example, screening of our total population allowed us to identify 67 cad mutants (exons only) and 74 c3h mutants (exons only). Subsequent characterization and identification of similar phenotypic modifications in the different lines provides strong evidence for a link between gene mutation and the observed phenotype. This was clearly demonstrated in the observation of our cad mutants where 35 (71%) of the 52 families showing a missense or codon stop mutation in the CAD gene showed the characteristic orange-brown coloration of xylem tissue previously observed in other CAD down-regulated or mutant plants [42–48]. Although a previous report of CAD down-regulation in flax RNAi plants was associated with reduced lignin, somewhat surprisingly the authors did not report the presence/absence of a brown midrib phenotype . This might be related to the relatively high (60–80%) residual activity in these plants.
Preliminary analyses of our M3 cad mutants (data not shown) indicate that the mutation is heritable, segregates and can be correlated with brown/orange coloured xylem further establishing the link between mutation and phenotype in the flax population. Wet chemistry and spectroscopy will allow confirmation of structural modifications in cell wall lignin in these mutants. Similar techniques will also be used to investigate potential changes to the lignin polymer in flax c3h mutants. Down-regulation of this gene in other species is associated with an increase in lignin condensation and reduction in lignin G and S units [49, 78] and it will be particularly interesting to assess the effects in flax lignin that is already highly condensed and contains low amounts of S lignin .
In conclusion, the generated flax EMS population represents an important biological resource for both forward and reverse genetics in this species. A large number of mutants showing biologically-interesting phenotypes has been identified and genes have been successfully TILLed using ENDO1. Further targets can be identified from the literature, as well as on the basis of recent transcriptomic studies in flax that have identified different genes potentially involved in various biological processes [6, 7, 9]. The use of the flax EMS population to identify mutants will greatly accelerate functional characterization of agronomically-interesting genes in this crop species. In order to accelerate mutant identification in our flax EMS population, we are currently developing an approach based on high throughput sequencing using NGS.
Mutagenesis and plant growth conditions
Ten batches of one hundred seeds (Linum usitatissimum L. cv Diane) were treated with 8 different EMS concentrations (0.25, 0.3, 0.5, 0.6, 0.75, 0.8, 1.0 and 2.0%). Two exposure times (5 and 8 h) were tested for two batches of different EMS concentration (5 h: 0.3, 0.6, 0.8, 1.0, 2.0% EMS; 8 h: 0.25, 0.5, 0.75, 1.0, 2.0% EMS). Untreated seeds were used as reference. All seeds were washed with tap water (3 × 5 mins, 1 × 30 mins) before transfer to wet Whatman paper in Petri dishes and incubation in a growth chamber at 20°C and 8 h photoperiod. Based on percentage germination, three treatments (0.3% EMS/5 h, 0.6% EMS/5 h and 0.75% EMS/8 h) were selected representing different balances between germination and mutation rate. Mutagenized seeds (M1) were sown in the field for M2 seed production. Collected M2 seeds were sown under greenhouse conditions together with WT seeds for phenotyping and DNA extraction. M2 and M3 seeds are stored at 4°C under low humidity conditions and constitute the EMS flax mutant collection.
Forward genetic screen
Two months after germination, M2 families were scored for phenotypes distinct from the wild type. For each family, the most affected individual was phenotyped in detail and photographed. For identification of the brown midrib phenotype, freehand sections of individual stems were made from cad mutant family plants and compared to control sections under a stereo microscope. The presence of a brown-orange coloration in xylem tissue was considered indicative of the brown midrib phenotype and was noted from 1 to 3 depending upon the intensity of the coloration with 1 being the most intense. The category indicated for a family is that of the most intense phenotype observed in any single individual belonging to that family.
Genomic DNA extraction and pooling
Leaf material was collected from individual M2 plants and pooled by family before being dried overnight at 65°C in a ventilated oven. Total DNA was extracted by using either the Dneasy Plant 96 Qiagen Kit (Qiagen, Hilden, Germany) or according to the protocol described by Carrier et al. . DNA quality for each extraction was monitored by electrophoresis on 0.8% agarose gels. The DNA of 3,515 M2 family was quantified with Picogreen (InVitrogen) using a M1000 microplate reader (TECAN-Switzerland), normalized to 1 ng.μL-1 and arrayed in a total of five 96 wells plates by an 8-fold pooling strategy (Additional file 4) using a GENESYS 150 workstation (TECAN-Switzerland).
PCR amplification and mutation detection
Primers used in TILLinG experiments
5′-3′ Primer sequence
CAD external Forward primer
CAD external Reverse primer
C3H external Forward primer
C3H external Reverse primer
CAD internal Forward primer with M13 tail
CAD internal Reverse primer with M13 tail
C3H internal Forward primer with M13 tail
C3H internal Reverse primer with M13 tail
M13 Forward primer
M13 Reverse primer
Bio-informatic sequence analyses
Phylogenetic analyses of CAD proteins was performed using the clustalW program with default parameters (http://www.clustal.org/clustal2/). The choice of gene regions to be TILLed and amplification primers were designed respectively using the CODDLE tool (Codons Optimized to Discover Deleterious Lesions; http://www.proweb.org/coddle/), primer 3 and OligoCalc system (http://www.basic.northwestern.edu/biotools/oligocalc.html). Potential effects of missense mutations were evaluated using the SIFT software (Sorting Intolerant From Tolerant; http://sift.jcvi.org/) and the PARSESNP software (Project Aligned Related Sequences and Evaluate SNPs; http://www.proweb.org/parsesnp/).
The flax phenotypic data is publically available on the UTILLdb (http://urgv.evry.inra.fr/UTILLdb).
Max. Chant. Gratefully acknowledges financial support of the Région Nord-Pas-de-Calais and the University of Lille 1 for the Ph.D. grant. SH gratefully acknowledges the financial support of the Agence Nationale de la Recherche (ANR) for the PT-Flax project (ANR-09-GENM-020-005).
Man. Chat. would like to thank Dr S.K. Samanta, Prof Charles Spillane (NUIG) and Anish Kumar for general advice on flax, mutant population creation and pilot EMS dose experiments.
The authors would like to thank all the different people who contributed to the collection and phenotyping of the flax plants (Stephane Fénart, Anne-Sophie Blervacq, Rudy Huis, Guillaume Beaumont, Charles-Henri Biard, Laurent Rigolle, Géraldine Dambrey, Godfrey Neutelings, Michel Burtin, Romain Boucly, Valérie Devillers, Clarisse Toitot, Sébastien Acket, Melha Lazouk, Thi Mai Huong To, Elis elkassis, Pascal Boulnois).
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