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 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. 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.

Assessing the impact of transgenerational epigenetic variation on complex traits.

Loss or gain of DNA methylation can affect gene expression and is sometimes transmitted across generations. Such epigenetic alterations are thus a possible source of heritable phenotypic variation in the absence of DNA sequence change. However, attempts to assess the prevalence of stable epigenetic variation in natural and experimental populations and to quantify its impact on complex traits have been hampered by the confounding effects of DNA sequence polymorphisms. To overcome this problem as much as possible, two parents with little DNA sequence differences, but contrasting DNA methylation profiles, were used to derive a panel of epigenetic Recombinant Inbred Lines (epiRILs) in the reference plant Arabidopsis thaliana. The epiRILs showed variation and high heritability for flowering time and plant height ( approximately 30%), as well as stable inheritance of multiple parental DNA methylation variants (epialleles) over at least eight generations. These findings provide a first rationale to identify epiallelic variants that contribute to heritable variation in complex traits using linkage or association studies. More generally, the demonstration that numerous epialleles across the genome can be stable over many generations in the absence of selection or extensive DNA sequence variation highlights the need to integrate epigenetic information into population genetics studies.