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

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.

Next generation sequencing and omics in cucumber ( Cucumis sativus L.) breeding directed research

“…As a reverse genetic strategy, this platform produces multiple alleles of a specific gene(s) like missense, nonsense, null alleles, splicing and cis-regulatory [102] which can be performed for altered protein(s) role [2]. The generated, stable and nontransgenic mutant alleles are useful in plant research and breeding for any species as markers or genetic sources [103].…”

“…Arabidopsis thaliana, Zea mays, Lotus japonicus and crop species-cereals, solanaceous, Brassica [100], Solanum lycopersicum, Pisum sativum, Hordeum vulgare and Cucurbitaceae [105]. Recently, five DNA mutant TILLinG libraries have been developed in different research groups for cucurbit species such as C. sativus monoecious line with mutation induced in CsACS2 sex determination gene [2], Poinsett76C. sativus [106], monoecious and climacteric line of C. melo [1], Piel de Sapo C. melo [67] and Cucurbita pepo [71].…”

“…Based on the TILLinG of 11 genes in melon, Dahmani-Mardas et al [1] estimated the overall mutation rate as one mutation for every 573 kb in CharMono mutant collection with eight alleles per tilled kilobase. In the C. sativus TILLinG platform, according to Boualem et al [2], the frequency of mutations in the five targeted genes is 1/1147 kb with an average of five alleles per gene. This mutation frequency obtained for C. sativus TILLinG population is twofold lower than the rate published for C. melo.…”

“…This mutation frequency obtained for C. sativus TILLinG population is twofold lower than the rate published for C. melo. In summary, the Cucurbitaceae TILLinG platforms can be suitable for functional genomics and plant breeding [2].…”

“…In cucumber, two independent TILLinG platforms established in 2014 by Boualem et al [2] and Fraenkel et al [106] for a mutant collection from monoecious cucumber line had three missense mutations in the CsACS2 sex determination gene. Only one mutation, namely G33C, showed sexual transition from monoecious to andromonoecy.…”