Development of a cucumis sativus tilling platform for forward and reverse genetics

Background: Cucumber (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 Findings: A 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 G(33) may affect the protein function, whereas mutations at S-238 and S-249 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. Conclusions: We 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

Characterisation of alleles of tomato light signalling genes generated by TILLING

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699International audienceTargeting Induced Local Lesions IN Genomes (TILLING) combines chemical mutagenesis with high throughput screening to allow the generation of alleles of selected genes. In this study, TILLING has been applied to produce a series of mutations in genes encoding essential components of the tomato light signal transduction pathway in an attempt to enhance fruit nutritional quality. Point mutations to DEETIO-LATED1 (DET1), which is responsible for the high pigment2 (hp2) tomato mutant, resulted in elevated levels of both carotenoid and phenylpropanoid phytonutrients in ripe fruit, whilst immature fruit showed increased chlorophyll content, photosynthetic capacity and altered fruit morphology. Furthermore, genotypes with mutations to the UV-DAMAGED DNA BINDING PROTEIN 1 (DDB1), COP1 and COP1 like were also characterised. These genotypes largely did not display phenotypes characteristic of mutation to light signalling components but their characterisation has enabled interrogation of structure function relationships of the mutated genes. (C) 2012 Elsevier Ltd. All rights reserved

Development of a Cucumis sativus TILLinG Platform for Forward and Reverse Genetics

Background: [br/] Cucumber (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. [br/] Principal Findings: [br/] A 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 G(33) may affect the protein function, whereas mutations at S-238 and S-249 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. [br/] Conclusions: [br/] We 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

Impact of EMS concentration on seed germination.

<p>Impact of EMS concentration on seed germination.</p

The induced mutation leading to andromonoecy is located in the active site of CsACS2.

<p>(<b>A</b>) Amino acid alignments of CsACS2 and homologous proteins from <i>Cucumis melo</i> (<i>Cm</i>), <i>Vitis vinifera</i> (<i>Vv</i>), <i>Arabidopsis thaliana</i> (<i>At</i>), <i>Oryza sativa</i> (<i>Os</i>), <i>Lycopersicon esculentum</i> (<i>Le</i>), <i>Petunia hybrida</i> (<i>Ph</i>), <i>Antirrhinum majus</i> (<i>Am</i>), <i>Zea mays</i> (<i>Zm</i>), <i>Medicago truncatula</i> (<i>Mt</i>) and <i>Picea glauca</i> (<i>Pg</i>). Numbers above the alignment indicate the amino acid positions along the CsACS2 protein. CsACS2 induced missense mutations, G33C, S238F and S249F are shown above the alignment. (<b>B</b>) 3D structure model of CsACS2. Superposition of the tomato ACS structure determined by x-ray crystallography <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097963#pone.0097963-Huai1" target="_blank">[34]</a>, indicated in blue and the 3D model of CsACS2 indicated in grey. CsACS2 model was determined using the Geno3D server (<a href="http://geno3d-pbil.ibcp.fr" target="_blank">http://geno3d-pbil.ibcp.fr</a>). Ball and stick representations show the competitive inhibitor AVG in yellow, and the amino acids G³³ in red, S²³⁸ in green and S²⁴⁹ in magenta. (<b>C–D</b>) Sexual flower types from monoecious (<b>C</b>, WT) and the TILLinG andromonoecious mutant (<b>D</b>, G33C). The male flowers, identical in all the cucumber types are not shown. Ov, ovary; Sg, stigma; St, stamen.</p

Nature of mutations found in the cucumber TILLinG population.

<p>Nature of mutations found in the cucumber TILLinG population.</p

Tilled genes and mutation frequency in the cucumber mutant population.

<p>The size of the tilled amplicons, the GC content, the number and type of induced alleles and the mutation frequency per amplicon are shown. The mutation frequency for each amplicon is calculated as follows: {(size of the amplicon) × (total number of samples screened)} / (total number of identified mutants). The average mutation frequency was estimated to be one mutation per 1147 kb.</p