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.
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).
- Oomah BD: Flaxseed as a functional food source. J Sci Food Agric. 2001, 81: 889-894. 10.1002/jsfa.898.View ArticleGoogle Scholar
- Baley C: Analysis of the flax fibres tensile behaviour and analysis of the tensile stiffness increase. Compos Part Appl Sci Manuf. 2002, 33: 939-948. 10.1016/S1359-835X(02)00040-4.View ArticleGoogle Scholar
- Day A, Ruel K, Neutelings G, Crônier D, David H, Hawkins S, Chabbert B: Lignification in the flax stem: evidence for an unusual lignin in bast fibers. Planta. 2005, 222: 234-245. 10.1007/s00425-005-1537-1.PubMedView ArticleGoogle Scholar
- Roach MJ, Deyholos MK: Microarray analysis of flax (Linum usitatissimum L.) stems identifies transcripts enriched in fibre-bearing phloem tissues. Mol Genet Genomics MGG. 2007, 278: 149-165. 10.1007/s00438-007-0241-1.PubMedView ArticleGoogle Scholar
- Roach MJ, Mokshina NY, Badhan A, Snegireva AV, Hobson N, Deyholos MK, Gorshkova TA: Development of Cellulosic Secondary Walls in Flax Fibers Requires B-Galactosidase. Plant Physiol. 2011, 156: 1351-1363. 10.1104/pp.111.172676.PubMedPubMed CentralView ArticleGoogle Scholar
- Huis R, Morreel K, Fliniaux O, Lucau-Danila A, Fénart S, Grec S, Neutelings G, Chabbert B, Mesnard F, Boerjan W, Hawkins S: Natural hypolignification is associated with extensive oligolignol accumulation in flax stems. Plant Physiol. 2012, 158: 1893-1915. 10.1104/pp.111.192328.PubMedPubMed CentralView ArticleGoogle Scholar
- Fenart S, Ndong Y-PA, Duarte J, Rivière N, Wilmer J, Wuytswinkel O, Van Lucau A, Cariou E, Neutelings G, Gutierrez L, Chabbert B, Guillot X, Tavernier R, Hawkins S, Thomasset B: Development and validation of a flax (Linum usitatissimum L.) gene expression oligo microarray. BMC Genomics. 2010, 11: 592-10.1186/1471-2164-11-592.PubMedPubMed CentralView ArticleGoogle Scholar
- Ragupathy R, Rathinavelu R, Cloutier S: Physical mapping and BAC-end sequence analysis provide initial insights into the flax (Linum usitatissimum L.) genome. BMC Genomics. 2011, 12: 217-10.1186/1471-2164-12-217.PubMedPubMed CentralView ArticleGoogle Scholar
- Venglat P, Xiang D, Qiu S, Stone SL, Tibiche C, Cram D, Alting-Mees M, Nowak J, Cloutier S, Deyholos M, Bekkaoui F, Sharpe A, Wang E, Rowland G, Selvaraj G, Datla R: Gene expression analysis of flax seed development. BMC Plant Biol. 2011, 11: 74-10.1186/1471-2229-11-74.PubMedPubMed CentralView ArticleGoogle Scholar
- Cloutier S, Ragupathy R, Miranda E, Radovanovic N, Reimer E, Walichnowski A, Ward K, Rowland G, Duguid S, Banik M: Integrated consensus genetic and physical maps of flax (Linum usitatissimum L.). TAG Theor Appl Genet Theor Angew Genet. 2012, 125: 1783-1795. 10.1007/s00122-012-1953-0.View ArticleGoogle Scholar
- Kumar S, You FM, Cloutier S: Genome wide SNP discovery in flax through next generation sequencing of reduced representation libraries. BMC Genomics. 2012, 13: 684-10.1186/1471-2164-13-684.PubMedPubMed CentralView ArticleGoogle Scholar
- Day A, Fénart S, Neutelings G, Hawkins S, Rolando C, Tokarski C: Identification of cell wall proteins in the flax (Linum usitatissimum) stem. Proteomics. 2013, 13: 812-825. 10.1002/pmic.201200257.PubMedView ArticleGoogle Scholar
- Wang Z, Hobson N, Galindo L, Zhu S, Shi D, McDill J, Yang L, Hawkins S, Neutelings G, Datla R, Lambert G, Galbraith DW, Grassa CJ, Geraldes A, Cronk QC, Cullis C, Dash PK, Kumar PA, Cloutier S, Sharpe AG, Wong GK-S, Wang J, Deyholos MK: The genome of flax (Linum usitatissimum) assembled de novo from short shotgun sequence reads. Plant J Cell Mol Biol. 2012, 72: 461-473. 10.1111/j.1365-313X.2012.05093.x.View ArticleGoogle Scholar
- Barvkar VT, Pardeshi VC, Kale SM, Kadoo NY, Gupta VS: Phylogenomic analysis of UDP glycosyltransferase 1 multigene family in Linum usitatissimum identified genes with varied expression patterns. BMC Genomics. 2012, 13: 175-10.1186/1471-2164-13-175.PubMedPubMed CentralView ArticleGoogle Scholar
- Babu PR, Rao KV, Reddy VD: Structural organization and classification of cytochrome P450 genes in flax (Linum usitatissimum L.). Gene. 2013, 513: 156-162. 10.1016/j.gene.2012.10.040.PubMedView ArticleGoogle Scholar
- Wróbel-Kwiatkowska M, Starzycki M, Zebrowski J, Oszmiański J, Szopa J: Lignin deficiency in transgenic flax resulted in plants with improved mechanical properties. J Biotechnol. 2007, 128: 919-934. 10.1016/j.jbiotec.2006.12.030.PubMedView ArticleGoogle Scholar
- Day A, Neutelings G, Nolin F, Grec S, Habrant A, Crônier D, Maher B, Rolando C, David H, Chabbert B, Hawkins S: Caffeoyl coenzyme A O-methyltransferase down-regulation is associated with modifications in lignin and cell-wall architecture in flax secondary xylem. Plant Physiol Biochem PPB Société Française Physiol Végétale. 2009, 47: 9-19.Google Scholar
- Henikoff S, Till BJ, Comai L: TILLING. Traditional Mutagenesis Meets Functional Genomics Plant. Physiol. 2004, 135: 630-636.Google Scholar
- Wang TL, Uauy C, Robson F, Till B: TILLING in extremis. Plant Biotechnol J. 2012, 10: 761-772. 10.1111/j.1467-7652.2012.00708.x.PubMedView ArticleGoogle Scholar
- An G, Lee S, Kim S-H, Kim S-R: Molecular genetics using T-DNA in rice. Plant Cell Physiol. 2005, 46: 14-22. 10.1093/pcp/pci502.PubMedView ArticleGoogle Scholar
- Busov V, Fladung M, Groover A, Strauss S: Insertional mutagenesis in Populus: relevance and feasibility. Tree Genet Genomes Vol. 2005, 1: 135-142. 10.1007/s11295-005-0019-8.View ArticleGoogle Scholar
- Nath Radhamony R, Mohan Prasad A, Srinivasan R: T-DNA insertional mutagenesis in Arabidopsis: a tool for functional genomics. Electron J Biotechnol. 2005, 8 (22): 82-106.Google Scholar
- Berenschot AS, Zucchi MI, Tulmann-Neto A, Quecini V: Mutagenesis in Petunia x hybrida Vilm. and isolation of a novel morphological mutant. Braz J Plant Physiol. 2008, 20: 95-103.View ArticleGoogle Scholar
- Morita R, Kusaba M, Iida S, Yamaguchi H, Nishio T, Nishimura M: Molecular characterization of mutations induced by gamma irradiation in rice. Genes Genet Syst. 2009, 84: 361-370. 10.1266/ggs.84.361.PubMedView ArticleGoogle Scholar
- Triques K, Sturbois B, Gallais S, Dalmais M, Chauvin S, Clepet C, Aubourg S, Rameau C, Caboche M, Bendahmane A: Characterization of Arabidopsis thaliana mismatch specific endonucleases: application to mutation discovery by TILLING in pea. Plant J Cell Mol Biol. 2007, 51: 1116-1125. 10.1111/j.1365-313X.2007.03201.x.View ArticleGoogle Scholar
- Tsai H, Howell T, Nitcher R, Missirian V, Watson B, Ngo KJ, Lieberman M, Fass J, Uauy C, Tran RK, Khan AA, Filkov V, Tai TH, Dubcovsky J, Comai L: Discovery of Rare Mutations in Populations: TILLING by Sequencing. Plant Physiol. 2011, 156: 1257-1268. 10.1104/pp.110.169748.PubMedPubMed CentralView ArticleGoogle Scholar
- Caillot S, Rosiau E, Laplace C, Thomasset B: Influence of light intensity and selection scheme on regeneration time of transgenic flax plants. Plant Cell Rep. 2009, 28: 359-371. 10.1007/s00299-008-0638-2.PubMedView ArticleGoogle Scholar
- Rowland GG: An EMS-induced low-linolenic-acid mutant in McGregor flax (Linum usitatissimum L.). Can J Plant Sci. 1991, 71: 393-396. 10.4141/cjps91-054.View ArticleGoogle Scholar
- Harloff H-J, Lemcke S, Mittasch J, Frolov A, Wu JG, Dreyer F, Leckband G, Jung C: A mutation screening platform for rapeseed (Brassica napus L.) and the detection of sinapine biosynthesis mutants. Theor Appl Genet. 2011, 124: 957-969.PubMedView ArticleGoogle Scholar
- Okabe Y, Asamizu E, Saito T, Matsukura C, Ariizumi T, Brès C, Rothan C, Mizoguchi T, Ezura H: Tomato TILLING Technology: Development of a Reverse Genetics Tool for the Efficient Isolation of Mutants from Micro-Tom Mutant Libraries. Plant Cell Physiol. 2011, 52: 1994-2005. 10.1093/pcp/pcr134.PubMedPubMed CentralView ArticleGoogle Scholar
- Chen L, Huang L, Min D, Phillips A, Wang S, Madgwick PJ, Parry MAJ, Hu Y-G: Development and Characterization of a New TILLING Population of Common Bread Wheat (Triticum aestivum L.). PLoS ONE. 2012, 7: e41570-10.1371/journal.pone.0041570.PubMedPubMed CentralView ArticleGoogle Scholar
- Rawat N, Sehgal SK, Joshi A, Rothe N, Wilson DL, McGraw N, Vadlani PV, Li W, Gill BS: A diploid wheat TILLING resource for wheat functional genomics. BMC Plant Biol. 2012, 12: 205-10.1186/1471-2229-12-205.PubMedPubMed CentralView ArticleGoogle Scholar
- Fraser CM, Chapple C: The phenylpropanoid pathway in Arabidopsis. Arab Book. 2011, 9: e0152-View ArticleGoogle Scholar
- Abdulrazzak N, Pollet B, Ehlting J, Larsen K, Asnaghi C, Ronseau S, Proux C, Erhardt M, Seltzer V, Renou J-P, Ullmann P, Pauly M, Lapierre C, Werck-Reichhart D: A coumaroyl-ester-3-hydroxylase Insertion Mutant Reveals the Existence of Nonredundant meta-Hydroxylation Pathways and Essential Roles for Phenolic Precursors in Cell Expansion and Plant Growth. Plant Physiol. 2006, 140: 30-48.PubMedPubMed CentralView ArticleGoogle Scholar
- Eudes A, Pollet B, Sibout R, Do C-T, Séguin A, Lapierre C, Jouanin L: Evidence for a role of AtCAD 1 in lignification of elongating stems of Arabidopsis thaliana. Planta. 2006, 225: 23-39. 10.1007/s00425-006-0326-9.PubMedView ArticleGoogle Scholar
- Youn B, Camacho R, Moinuddin SGA, Lee C, Davin LB, Lewis NG, Kang C: Crystal structures and catalytic mechanism of the Arabidopsis cinnamyl alcohol dehydrogenases AtCAD5 and AtCAD4. Org Biomol Chem. 2006, 4: 1687-1697. 10.1039/b601672c.PubMedView ArticleGoogle Scholar
- Dalmais M, Schmidt J, Le Signor C, Moussy F, Burstin J, Savois V, Aubert G, Brunaud V, de Oliveira Y, Guichard C, Thompson R, Bendahmane A: UTILLdb, a Pisum sativum in silico forward and reverse genetics tool. Genome Biol. 2008, 9: R43-10.1186/gb-2008-9-2-r43.PubMedPubMed CentralView ArticleGoogle Scholar
- Sikora P, Chawade A, Larsson M, Olsson J, Olsson O: Mutagenesis as a Tool in Plant Genetics, Functional Genomics, and Breeding. Int J Plant Genomics. 2011, 2011: 1-13.View ArticleGoogle Scholar
- Krieg DR: Ethyl Methanesulfonate-Induced Reversion of Bacteriophage T4rii Mutants. Genet. 1963, 48: 561-580.Google Scholar
- Greene EA, Codomo CA, Taylor NE, Henikoff JG, Till BJ, Reynolds SH, Enns LC, Burtner C, Johnson JE, Odden AR, Comai L, Henikoff S: Spectrum of Chemically Induced Mutations From a Large-Scale Reverse-Genetic Screen in Arabidopsis. Genet. 2003, 164: 731-740.Google Scholar
- Le Signor C, Savois V, Aubert G, Verdier J, Nicolas M, Pagny G, Moussy F, Sanchez M, Baker D, Clarke J, Thompson R: Optimizing TILLING populations for reverse genetics in Medicago truncatula. Plant Biotechnol J. 2009, 7: 430-441. 10.1111/j.1467-7652.2009.00410.x.PubMedView ArticleGoogle Scholar
- Sibout R, Eudes A, Mouille G, Pollet B, Lapierre C, Jouanin L, Séguin A: CINNAMYL ALCOHOL DEHYDROGENASE-C and -D Are the Primary Genes Involved in Lignin Biosynthesis in the Floral Stem of Arabidopsis. Plant Cell Online. 2005, 17: 2059-2076. 10.1105/tpc.105.030767.View ArticleGoogle Scholar
- Baucher M, Chabbert B, Pilate G, Doorsselaere JV, Tollier MT, Petit-Conil M, Cornu D, Monties B, Montagu MV, Inze D, Jouanin L, Boerjan W: Red Xylem and Higher Lignin Extractability by Down-Regulating a Cinnamyl Alcohol Dehydrogenase in Poplar. Plant Physiol. 1996, 112: 1479-1490.PubMedPubMed CentralGoogle Scholar
- Baucher M, Bernard-Vailhé MA, Chabbert B, Besle JM, Opsomer C, Van Montagu M, Botterman J: Down-regulation of cinnamyl alcohol dehydrogenase in transgenic alfalfa (Medicago sativa L.) and the effect on lignin composition and digestibility. Plant Mol Biol. 1999, 39: 437-447. 10.1023/A:1006182925584.PubMedView ArticleGoogle Scholar
- MacKay JJ, O’Malley DM, Presnell T, Booker FL, Campbell MM, Whetten RW, Sederoff RR: Inheritance, gene expression, and lignin characterization in a mutant pine deficient in cinnamyl alcohol dehydrogenase. Proc Natl Acad Sci U S A. 1997, 94: 8255-8260. 10.1073/pnas.94.15.8255.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhang K, Qian Q, Huang Z, Wang Y, Li M, Hong L, Zeng D, Gu M, Chu C, Cheng Z: GOLD HULL AND INTERNODE2 Encodes a Primarily Multifunctional Cinnamyl-Alcohol Dehydrogenase in Rice. Plant Physiol. 2006, 140: 972-983. 10.1104/pp.105.073007.PubMedPubMed CentralView ArticleGoogle Scholar
- Guillaumie S, Pichon M, Martinant J-P, Bosio M, Goffner D, Barrière Y: Differential expression of phenylpropanoid and related genes in brown-midrib bm1, bm2, bm3, and bm4 young near-isogenic maize plants. Planta. 2007, 226: 235-250. 10.1007/s00425-006-0468-9.PubMedView ArticleGoogle Scholar
- Jourdes M, Cardenas CL, Laskar DD, Moinuddin SGA, Davin LB, Lewis NG: Plant cell walls are enfeebled when attempting to preserve native lignin configuration with poly-p-hydroxycinnamaldehydes: Evolutionary implications. Phytochemistry. 2007, 68: 1932-1956. 10.1016/j.phytochem.2007.03.044.PubMedView ArticleGoogle Scholar
- Ralph J, Akiyama T, Kim H, Lu F, Schatz PF, Marita JM, Ralph SA, Reddy MSS, Chen F, Dixon RA: Effects of Coumarate 3-Hydroxylase Down-regulation on Lignin Structure. J Biol Chem. 2006, 281: 8843-8853. 10.1074/jbc.M511598200.PubMedView ArticleGoogle Scholar
- Fenart S, Chabi M, Gallina S, Huis R, Neutelings G, Riviere N, Thomasset B, Hawkins S, Lucau-Danila A: Intra-platform comparison of 25-mer and 60-mer oligonucleotide Nimblegen DNA microarrays. BMC Res Notes. 2013, 6: 43-10.1186/1756-0500-6-43.PubMedPubMed CentralView ArticleGoogle Scholar
- Barvkar VT, Pardeshi VC, Kale SM, Kadoo NY, Giri AP, Gupta VS: Proteome profiling of flax (Linum usitatissimum) seed: characterization of functional metabolic pathways operating during seed development. J Proteome Res. 2012, 11: 6264-6276.PubMedGoogle Scholar
- Brkljacic J, Grotewold E, Scholl R, Mockler T, Garvin DF, Vain P, Brutnell T, Sibout R, Bevan M, Budak H, Caicedo AL, Gao C, Gu Y, Hazen SP, Holt BF, Hong S-Y, Jordan M, Manzaneda AJ, Mitchell-Olds T, Mochida K, Mur LAJ, Park C-M, Sedbrook J, Watt M, Zheng SJ, Vogel JP: Brachypodium as a Model for the Grasses: Today and the Future. Plant Physiol. 2011, 157: 3-13. 10.1104/pp.111.179531.PubMedPubMed CentralView ArticleGoogle Scholar
- Minoia S, Petrozza A, D’Onofrio O, Piron F, Mosca G, Sozio G, Cellini F, Bendahmane A, Carriero F: A new mutant genetic resource for tomato crop improvement by TILLING technology. BMC Res Notes. 2010, 3: 69-10.1186/1756-0500-3-69.PubMedPubMed CentralView ArticleGoogle Scholar
- Till BJ, Reynolds SH, Greene EA, Codomo CA, Enns LC, Johnson JE, Burtner C, Odden AR, Young K, Taylor NE, Henikoff JG, Comai L, Henikoff S: Large-Scale Discovery of Induced Point Mutations With High-Throughput TILLING. Genome Res. 2003, 13: 524-530. 10.1101/gr.977903.PubMedPubMed CentralView ArticleGoogle Scholar
- Martín B, Ramiro M, Martínez-Zapater JM, Alonso-Blanco C: A high-density collection of EMS-induced mutations for TILLING in Landsberg erecta genetic background of Arabidopsis. BMC Plant Biol. 2009, 9: 147-10.1186/1471-2229-9-147.PubMedPubMed CentralView ArticleGoogle Scholar
- Sestili F, Botticella E, Bedo Z, Phillips A, Lafiandra D: Production of novel allelic variation for genes involved in starch biosynthesis through mutagenesis. Mol Breed. 2010, 25: 145-154. 10.1007/s11032-009-9314-7.View ArticleGoogle Scholar
- Uauy C, Paraiso F, Colasuonno P, Tran RK, Tsai H, Berardi S, Comai L, Dubcovsky J: A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat. BMC Plant Biol. 2009, 9: 115-10.1186/1471-2229-9-115.PubMedPubMed CentralView ArticleGoogle Scholar
- Dong C, Dalton-Morgan J, Vincent K, Sharp P: A Modified TILLING Method for Wheat Breeding. Plant Genome J. 2009, 2: 39-10.3835/plantgenome2008.10.0012.View ArticleGoogle Scholar
- Slade AJ, Fuerstenberg SI, Loeffler D, Steine MN, Facciotti D: A reverse genetic, nontransgenic approach to wheat crop improvement by TILLING. Nat Biotechnol. 2005, 23: 75-81. 10.1038/nbt1043.PubMedView ArticleGoogle Scholar
- Harloff H-J, Lemcke S, Mittasch J, Frolov A, Wu JG, Dreyer F, Leckband G, Jung C: A mutation screening platform for rapeseed (Brassica napus L.) and the detection of sinapine biosynthesis mutants. Theor Appl Genet. 2012, 124: 957-969. 10.1007/s00122-011-1760-z.PubMedView ArticleGoogle Scholar
- Wang N, Wang Y, Tian F, King GJ, Zhang C, Long Y, Shi L, Meng J: A functional genomics resource for Brassica napus: development of an EMS mutagenized population and discovery of FAE1 point mutations by TILLING. New Phytol. 2008, 180: 751-765. 10.1111/j.1469-8137.2008.02619.x.PubMedView ArticleGoogle Scholar
- Stephenson P, Baker D, Girin T, Perez A, Amoah S, King GJ, Østergaard L: A rich TILLING resource for studying gene function in Brassica rapa. BMC Plant Biol. 2010, 10: 62-10.1186/1471-2229-10-62.PubMedPubMed CentralView ArticleGoogle Scholar
- Himelblau E, Gilchrist EJ, Buono K, Bizzell C, Mentzer L, Vogelzang R, Osborn T, Amasino RM, Parkin IAP, Haughn GW: Forward and reverse genetics of rapid-cycling Brassica oleracea. TAG Theor Appl Genet Theor Angew Genet. 2009, 118: 953-961. 10.1007/s00122-008-0952-7.View ArticleGoogle Scholar
- Gady AL, Hermans FW, Van de Wal MH, van Loo EN, Visser RG, Bachem CW: Implementation of two high through-put techniques in a novel application: detecting point mutations in large EMS mutated plant populations. Plant Methods. 2009, 5: 13-10.1186/1746-4811-5-13.PubMedPubMed CentralView ArticleGoogle Scholar
- González M, Xu M, Esteras C, Roig C, Monforte AJ, Troadec C, Pujol M, Nuez F, Bendahmane A, Garcia-Mas J, Picó B: Towards a TILLING platform for functional genomics in Piel de Sapo melons. BMC Res. Notes. 2011, 4: 289-10.1186/1756-0500-4-289.PubMedPubMed CentralView ArticleGoogle Scholar
- Dahmani-Mardas F, Troadec C, Boualem A, Lévêque S, Alsadon AA, Aldoss AA, Dogimont C, Bendahmane A: Engineering melon plants with improved fruit shelf life using the TILLING approach. PLoS One. 2010, 5: e15776-10.1371/journal.pone.0015776.PubMedPubMed CentralView ArticleGoogle Scholar
- Sabetta W, Alba V, Blanco A: Montemurro C: sunTILL: a TILLING resource for gene function analysis in sunflower. Plant Methods. 2011, 7: 20-10.1186/1746-4811-7-20.PubMedPubMed CentralView ArticleGoogle Scholar
- Knoll JE, Ramos ML, Zeng Y, Holbrook CC, Chow M, Chen S, Maleki S, Bhattacharya A, Ozias-Akins P: TILLING for allergen reduction and improvement of quality traits in peanut (Arachis hypogaea L.). BMC Plant Biol. 2011, 11: 81-10.1186/1471-2229-11-81.PubMedPubMed CentralView ArticleGoogle Scholar
- Bush SM, Krysan PJ: iTILLING: a personalized approach to the identification of induced mutations in Arabidopsis. Plant Physiol. 2010, 154: 25-35. 10.1104/pp.110.159897.PubMedPubMed CentralView ArticleGoogle Scholar
- Chawade A, Sikora P, Bräutigam M, Larsson M, Vivekanand V, Nakash MA, Chen T, Olsson O: Development and characterization of an oat TILLING-population and identification of mutations in lignin and beta-glucan biosynthesis genes. BMC Plant Biol. 2010, 10: 86-10.1186/1471-2229-10-86.PubMedPubMed CentralView ArticleGoogle Scholar
- Perry J, Brachmann A, Welham T, Binder A, Charpentier M, Groth M, Haage K, Markmann K, Wang TL, Parniske M: TILLING in Lotus japonicus identified large allelic series for symbiosis genes and revealed a bias in functionally defective ethyl methanesulfonate alleles toward glycine replacements. Plant Physiol. 2009, 151: 1281-1291. 10.1104/pp.109.142190.PubMedPubMed CentralView ArticleGoogle Scholar
- Gottwald S, Bauer P, Komatsuda T, Lundqvist U, Stein N: TILLING in the two-rowed barley cultivar “Barke” reveals preferred sites of functional diversity in the gene HvHox1. BMC Res Notes. 2009, 2: 258-10.1186/1756-0500-2-258.PubMedPubMed CentralView ArticleGoogle Scholar
- Porceddu A, Panara F, Calderini O, Molinari L, Taviani P, Lanfaloni L, Scotti C, Carelli M, Scaramelli L, Bruschi G, Cosson V, Ratet P, Larembergue H, De Duc G, Piano E, Arcioni S: An Italian functional genomic resource for Medicago truncatula. BMC Res Notes. 2008, 1: 129-10.1186/1756-0500-1-129.PubMedPubMed CentralView ArticleGoogle Scholar
- Xin Z, Li Wang M, Barkley NA, Burow G, Franks C, Pederson G, Burke J: Applying genotyping (TILLING) and phenotyping analyses to elucidate gene function in a chemically induced sorghum mutant population. BMC Plant Biol. 2008, 8: 103-10.1186/1471-2229-8-103.PubMedPubMed CentralView ArticleGoogle Scholar
- Till BJ, Cooper J, Tai TH, Colowit P, Greene EA, Henikoff S, Comai L: Discovery of chemically induced mutations in rice by TILLING. BMC Plant Biol. 2007, 7: 19-10.1186/1471-2229-7-19.PubMedPubMed CentralView ArticleGoogle Scholar
- Wu J-L, Wu C, Lei C, Baraoidan M, Bordeos A, Madamba MRS, Ramos-Pamplona M, Mauleon R, Portugal A, Ulat VJ, Bruskiewich R, Wang G, Leach J, Khush G, Leung H: Chemical- and Irradiation-induced Mutants of Indica Rice IR64 for Forward and Reverse Genetics. Plant Mol Biol. 2005, 59: 85-97. 10.1007/s11103-004-5112-0.PubMedView ArticleGoogle Scholar
- Caldwell DG, McCallum N, Shaw P, Muehlbauer GJ, Marshall DF, Waugh R: A structured mutant population for forward and reverse genetics in Barley (Hordeum vulgare L.). Plant J Cell Mol Biol. 2004, 40: 143-150. 10.1111/j.1365-313X.2004.02190.x.View ArticleGoogle Scholar
- Vanholme R, Morreel K, Ralph J, Boerjan W: Lignin engineering. Curr Opin Plant Biol. 2008, 11: 278-285. 10.1016/j.pbi.2008.03.005.PubMedView ArticleGoogle Scholar
- Carrier G, Santoni S, Rodier-Goud M, Canaguier A, de Kochko A, Dubreuil-Tranchant C, This P, Boursiquot J-M, Le Cunff L: An efficient and rapid protocol for plant nuclear DNA preparation suitable for next generation sequencing methods. Am J Bot. 2011, 98: e13-15. 10.3732/ajb.1000371.PubMedView ArticleGoogle Scholar
- Wienholds E, van Eeden F, Kosters M, Mudde J, Plasterk RHA, Cuppen E: Efficient target-selected mutagenesis in zebrafish. Genome Res. 2003, 13: 2700-2707. 10.1101/gr.1725103.PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.