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.


RESULTS
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.
INRA-URGV, UMR1165, Unité de Recherche en Génomique Végétale, Saclay Plant Sciences, Evry, France.
Adnane
A
Boualem
INRA-URGV, UMR1165, Unité de Recherche en Génomique Végétale, Saclay Plant Sciences, Evry, France.
Sebastien
S
Fleurier
INRA-URGV, UMR1165, Unité de Recherche en Génomique Végétale, Saclay Plant Sciences, Evry, France.
Christelle
C
Troadec
INRA-URGV, UMR1165, Unité de Recherche en Génomique Végétale, Saclay Plant Sciences, Evry, France.
Pascal
P
Audigier
Bench Bio Pvt Ltd., c/o Jai Research Foundation, Vapi, Gujarat, India.
Anish P K
AP
Kumar
Bench Bio Pvt Ltd., c/o Jai Research Foundation, Vapi, Gujarat, India; Plant and AgriBiosciences Research Centre (PABC), Botany and Plant Science, National University of Ireland Galway, University Road, Galway, Ireland.
Manash
M
Chatterjee
Department of Plant Production, College of Food and Agricultural Sciences, King Saud University, Riyadh, Saudi Arabia.
Abdullah A
AA
Alsadon
Department of Plant Production, College of Food and Agricultural Sciences, King Saud University, Riyadh, Saudi Arabia.
Monther T
MT
Sadder
Department of Plant Production, College of Food and Agricultural Sciences, King Saud University, Riyadh, Saudi Arabia.
Mahmoud A
MA
Wahb-Allah
Department of Plant Production, College of Food and Agricultural Sciences, King Saud University, Riyadh, Saudi Arabia.
Abdullah A
AA
Al-Doss
INRA-URGV, UMR1165, Unité de Recherche en Génomique Végétale, Saclay Plant Sciences, Evry, France.
Abdelhafid
A
Bendahmane
D000595:Amino Acid Sequence; D018553:Cucumis sativus / Q000235:genetics*; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D010641:Phenotype; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D059386:Reverse Genetics / Q000379:methods
D010940:Plant Proteins