Abstract
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.
Highlights
Cucumber (Cucumis sativus) belongs to the Cucurbitaceae family, known as cucurbits and gourds, that includes more than 800 species distributed across tropical and subtropical regions [1]
We have developed a cucumber mutant population that can be used as an efficient reverse genetics tool
The cucumber Targeting Induced Local Lesions in Genomes (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
Summary
Cucumber (Cucumis sativus) belongs to the Cucurbitaceae family, known as cucurbits and gourds, that includes more than 800 species distributed across tropical and subtropical regions [1]. The most common techniques to produce altered or loss of function mutations are based on insertional mutagenesis [10] and RNA interference [11] Because these methods are mainly based on the ability of a given plant to be transformed, their usefulness as general reverse genetics methods is limited to very few plant species and are unconceivable for species recalcitrant to plant transformation, such as cucumber. TILLinG produces a broad range of mutations including nonsense, missense and splicing mutations which can be used for protein domain annotation This technique has been successfully applied to a large variety of organisms including plants and animals [15] and has become the method of choice for gene functional analysis in crop species. 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
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