Identification Of Cucumber Wrinkled And Compact Mutant C1056 And Cloning Of Mutant Gene WRC-1

Plant height is an important plant type trait of cucumber.Plant height variation can not only change the plant type,but also have a certain impact on cucumber yield,quality and light simplification cultivation of cucumber.The creation of new dwarf mutants not only enriches the cucumber mutant resources,but also provides new genetic material for the genetic improvement of cucumber plant type,and also provides valuable resources for the research of molecular heredity and regulation mechanism of cucumber plant height.The mapping and cloning of dwarf mutant genes can help to understand the dwarf mechanism and explore new dwarf genes,laying the foundation for its application in genetic breeding.In this study,a cucumber wrinkled and compact mutant C1056 was found from the EMS mutant library,and its phenotype and photosynthetic characteristics were identified.Based on the analysis of the genetic rules of dwarf traits,the map-based were used.The Mutmap method finely mapped,cloned and preliminarily verified the wrinkled and compact gene WRC-1.The main findings are as follows:1.Identification of wrinkled and compact mutant C1056The internode length of mutant C1056 was significantly shortened,and the plant height was up to about 2/3 of that of the wild type.The leaves became darker,the leaves shrunk.In some female flowers,the style and stigma of some pistils became larger,the petals became smaller and even disappeared,and the calyx disappeared.The male flowers and the female flowers with normal structure became smaller.Most of the fruits wilt and the ovary can be normally expanded after pollination.The diameter of vessel in mutant was reduced,and the length of internode cells was shorter,the cells became smaller.Compared with the wild type,the chlorophyll and carotenoids of the mutants decreased to different extents.The ratio of chlorophyll to carotenoids and chlorophyll a/b was significantly increased.The parameters of chlorophyll fluorescence and the net photosynthetic rate,stomatal conductance and transpiration rate did not change significantly,and the intercellular CO₂concentration was significantly higher than that of the wild-type.2.WRC-1 gene fine mapping and cloningGenetic analysis indicated that the mutant trait was a quality trait controlled by a recessive single gene.By combining map-based cloning and Mutmap,the WRC-1 candidate gene was map within on chromosome2（chr2）of cucumber,with a genetic distance of 0.2cM and a physical distance of 12.88Kb.There is only one complete candidate gene CsGME1（Csa2G011460）in this interval,which has only one copy in cucumber,encoding identical376 amino acid GDP-D-mannose-3’,5’-epimerase1.A non-synonymous mutation of C-T（C2041877T,chr2,9930）occurred in the last exon of WRC-1 in the mutant,leading to the replacement of CsGME1 protein P³⁵³（proline）with L³⁵³（leucine）.3.CsGME1 candidate gene identification and verificationA pair of CAPs labeled CAPs105601 designed according to the mutation site of candidate gene was found to be co-segregated with the trait.The CAPs marker was used to detect the loci uniqueness in 96 cucumber natural germplasm.The results showed that the synonymous mutation did not exist in 96 natural germplasm.The relative expression level of CsGME1 was the highest in male flowers and fruits,followed by the expression level in stems,leaves and female flowers,and the lowest in roots.There was no significant difference in the expression of CsGME1 gene between mutant and wild type.Phylogenetic tree analysis indicated that the cucumber gene was closely related to melon and farther away from Arabidopsis thaliana.The function of the mutant CsGME1 gene was preliminarily verified,and it was showed that the ascorbic acid content of the mutant decreased,the pollen vigor decreased,and the cell wall pectin component decreased.The mutant phenotype was partially recovered after spraying boric acid.