Dataset Information

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.

SUBMITTER: Chantreau M

PROVIDER: S-EPMC3853753 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Publications

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

Chantreau Maxime M Grec Sébastien S Gutierrez Laurent L Dalmais Marion M Pineau Christophe C Demailly Hervé H Paysant-Leroux Christine C Tavernier Reynald R Trouvé Jean-Paul JP Chatterjee Manash M Guillot Xavier X Brunaud Véronique V Chabbert Brigitte B van Wuytswinkel Olivier O Bendahmane Abdelhafid A Thomasset Brigitte B Hawkins Simon S

BMC plant biology 20131015

<h4>Background</h4>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 ...[more]

PMID: 24128060

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Project description:BackgroundCucumber (Cucumis sativus) belongs to the Cucurbitaceae family that includes more than 800 species. The cucumber genome has been recently sequenced and annotated. Transcriptomics and genome sequencing of many plant genomes are providing information on candidate genes potentially related to agronomically important traits. To accelerate functional characterization of these genes in cucumber we have generated an EMS mutant population that can be used as a TILLinG platform for reverse genetics.Principal findingsA population of 3,331 M2 mutant seed families was generated using two EMS concentrations (0.5% and 0.75%). Genomic DNA was extracted from M2 families and eight-fold pooled for mutation detection by ENDO1 nuclease. To assess the quality of the mutant collection, we screened for induced mutations in five genes and identified 26 mutations. The average mutation rate was calculated as 1/1147 Kb giving rise to approximately 320 mutations per genome. We focused our characterization on three missense mutations, G33C, S238F and S249F identified in the CsACS2 sex determination gene. Protein modeling and crystallography studies predicted that mutation at G33 may affect the protein function, whereas mutations at S238 and S249 may not impair the protein function. As predicted, detailed phenotypic evaluation showed that the S238F and the S249F mutant lines had no sexual phenotype. In contrast, plants homozygous for the G33C mutation showed a complete sexual transition from monoecy to andromonoecy. This result demonstrates that TILLinG is a valuable tool for functional validation of gene function in crops recalcitrant to transgenic transformation.ConclusionsWe have developed a cucumber mutant population that can be used as an efficient reverse genetics tool. The cucumber TILLinG collection as well as the previously described melon TILLinG collection will prove to be a valuable resource for both fundamental research and the identification of agronomically-important genes for crop improvement in cucurbits in general.

Project description:BackgroundFlaxseed oil is characterized by high content of essential polyunsaturated fatty acids (PUFA) promoted as a human dietary supplement protecting against atherosclerosis. The disadvantage of the high PUFA content in flax oil is high susceptibility to oxidation, which can result in carcinogenic compound formation. Linola flax cultivar is characterized by high linoleic acid content in comparison to traditional flax cultivars rich in linolenic acid. The changes in fatty acid proportions increase oxidative stability of Linola oil and broaden its use as an edible oil for cooking. However one of investigated transgenic lines has high ALA content making it suitable as omega-3 source. Protection of PUFA oxidation is a critical factor in oil quality. The aim of this study was to investigate the impact of phenylpropanoid contents on the oil properties important during the whole technological process from seed storage to grinding and oil pressing, which may influence health benefits as well as shelf-life, and to establish guidelines for the selection of new cultivars.MethodsThe composition of oils was determined by chromatographic (GS-FID and LC-PDA-MS) methods. Antioxidant properties of secondary metabolites were analyzed by DPPH method. The stability of oils was investigated: a) during regular storage by measuring acid value peroxide value p-anisidine value malondialdehyde, conjugated dienes and trienes; b) by using accelerated rancidity tests by TBARS reaction; c) by thermoanalytical - differential scanning calorimetry (DSC).ResultsIn one approach, in order to increase oil stability, exogenous substances added are mainly lipid soluble antioxidants from the isoprenoid pathway, such as tocopherol and carotene. The other approach is based on transgenic plant generation that accumulates water soluble compounds. Increased accumulation of phenolic compounds in flax seeds was achieved by three different strategies that modify genes coding for enzymes from the phenylpropanoid pathway. The three types of transgenic flax had different phenylpropanoid profiles detected in oil, highly increasing its stability.ConclusionsWe found that hydrophilic phenylpropanoids more than lipophilic isoprenoid compounds determine oil stability however they can work synergistically. Among phenolics the caffeic acid was most effective in increasing oil stability.

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Project description:BACKGROUND:A sustainable breeding program requires a minimum level of germplasm diversity to provide varied options for the selection of new breeding lines. To maximize genetic gain of the North Dakota State University (NDSU) flax breeding program, we aimed to increase the genetic diversity of its parental stocks by incorporating diverse genotypes. For this purpose, we analyzed the genetic diversity, linkage disequilibrium, and population sub-structure of 350 globally-distributed flax genotypes with 6200 SNP markers. RESULTS:All the genotypes tested clustered into seven sub-populations (P1 to P7) based on the admixture model and the output of neighbor-joining (NJ) tree analysis and principal coordinate analysis were in line with that of structure analysis. The largest sub-population separation arose from a cluster of NDSU/American genotypes with Turkish and Asian genotypes. All sub-populations showed moderate genetic diversity (average H?=?0.22 and I?=?0.34). The pairwise Fst comparison revealed a great degree of divergence (Fst?>?0.25) between most of the combinations. A whole collection mantel test showed significant positive correlation (r?=?0.30 and p?<?0.01) between genetic and geographic distances, whereas it was non-significant for all sub-populations except P4 and P5 (r?=?0.251, 0.349 respectively and p?<?0.05). In the entire collection, the mean linkage disequilibrium was 0.03 and it decayed to its half maximum within <?21?kb distance. CONCLUSIONS:To maximize genetic gain, hybridization between NDSU stock (P5) and Asian individuals (P6) are potentially the best option as genetic differentiation between them is highest (Fst?>?0.50). In contrast, low genetic differentiation between P5 and P2 may enhance the accumulation of favorable alleles for oil and fiber upon crossing to develop dual purpose varieties. As each sub-population consists of many genotypes, a Neighbor-Joining tree and kinship matrix assist to identify distantly related genotypes. These results also inform genotyping decisions for future association mapping studies to ensure the identification of a sufficient number of molecular markers to tag all linkage blocks.

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Dataset Information

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

Background

Results

Conclusions

Publications

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

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