Mapping And Functional Analysis Of Organ Development Gene Odd1 And Thermo-sensitive Leaf Coloration Gene Hs1 In Cucumis Sativus L.

Cucumber（Cucumis sativus L.）is an economically important vegetable crop worldwide,which has a unique growth habit and shoot architecture of Cucurbitaceae.The ability to regulate shoot architecture is very useful to improve planting density and productivity in cucumber breeding.However,our knowledge of the mechanisms controlling shoot architecture in cucumber is still limited.Therefore,it is necessary to elucidate the regulatory molecular mechanisms of cucumber growth and development.Therefore,using developmental mutants,to locate and clone the genes related to organ development of cucumber,and to study their molecular mechanisms,is of great significance for the generation of ideotypes with defined architectural features.In the meanwhile,the frequent occurrence of extreme high temperature weather is seriously threatening the productivity of cucumber.It is urgent to uncover the mechanism of heat tolerance for accelerating the breeding process of new heat-tolerant varieties.In this study,we identified a spontaneous cucumber mutant,organ development defective 1（odd1）.Genetic analysis demonstrated that the phenotype of odd1 mutant is controlled by a single recessive nuclear gene.The Mut Map method was used for the mapping of odd1 gene.And In Del markers were used to confirm the mapping result.In the meantime,with an integrated profiling of transcriptome and proteome,the molecular mechanism of the growth and development of odd1 were analyzed,and the regulatory network of genes and proteins involved in cucumber growth and development was mapped.A spontaneous cucumber mutant,heat sensitive 1（hs1）,whose leaves showed partial or complete albinism under high temperature of 35℃but appeared green under normal temperature（18℃-26℃）,was used to isolate hs1 using the map-based cloning technology.Gene silencing and the subcellular localization were performed to preliminarily confirm the biological function of the Cshs1 candidate gene.The main results are as follows:1.Phenotypic and genetic analysis of odd1.The results showed that comparing with the wild type,the root system of odd1 became longer.The mutant has much smaller plant stature.The internode length and the stem diameter of the odd1 are drastically decreased.The stem of odd1 is solid.Further observation revealed that the mutant has dark green and curled-edge cotyledons.Its true leaves with abnormal veins are wrinkled and dark green.Not only the leaf size of odd1 is smaller,but also the leaf shape became like ginkgo leaf.The petiole of odd1 is cylindrical.Comparing with the wild type at the same growth period,odd1 has more leaves,lower chlorophyll and carotenoid content,and lower photosynthetic capacity.The petals of male and female flowers of mutant are deeply split and smaller.The anthers of odd1 split into three parts.Not only the stigma of odd1 become larger and valgus,but also the shape became like cauliflower.The style of odd1 is obviously thicker.The node position of first female and male flower of odd1 are significantly increased.Flowering was delayed in odd1.The mutant plants can produce normal pollen,but the female flowers are sterile and the pollen survival rate and number are reduced.The parthenocarpy characteristics of the mutant is disappeared.The cell size and cell number of cotyledon in odd1 is significantly smaller than that in WT,and similar differences are observed in functional leaf and petal of WT and odd1.The shoot apical meristem（SAM）of odd1 became smaller.The waterlogging tolerance of odd1 was enhanced.The odd1 mutant was crossed with Chinese long 9930 that has similar phenotype with the WT to produce F₁.All F₁progenies present WT phenotype.The F₂and F_t（F₁ was crossed with odd1 again）populations fit the segregation ratios of 3:1 and 1:1,respectively.These results demonstrate that the phenotype of odd1 mutant is controlled by a single recessive nuclear gene.2.Mapping of odd1.Using Mut Map strategy,the odd1 pool in F₂ population was directly subjected to genome resequencing,and then the genomic resequencing data were compared with the genome sequence of Chinese long 9930.SNP-index correlation analysis was performed,and odd1 was successfully located between Cs In Del1 and Cs In Del2 on chromosome 5 with a physical distance of about 3.78Mb.Fine mapping and candidate gene identification are currently ongoing.3.Female infertility in odd1.Through a series of comprehensive analysis,such as anatomical observation,pollen vigor identification,cross test,observation of pollen germination in vivo,it was found that the fertility of pollen grains in odd1 plants was normal,but the females were infertile.Further studies have shown that the main reason for female infertility of odd1 is due to the absence of ovules in the mutant and the inability to complete fertilization.4.Combined transcriptomic and proteomic analysis was performed using apical buds with growth points and apical leaves from mutant and WT groups.We found that the significantly decreased expression of the genes and proteins related to growth and development in the SAM of odd1,were the main reasons for the abnormal growth and development of organs in odd1.5.CsaV3＿1G044470.1 is the best candidate gene of hs1.Through map-based cloning technology,two SNP markers（Cs SNP4 and Cs SNP6）were used to narrow down the localization region,and hs1 was successfully located in the39.7kb physical region of chromosome 1.There were three predicted genes in this region.Cs SNP5 co-segregated with hs1 phenotype,and Cs SNP5 was located in the second exon of CsaV3＿1G044470.1.Through full-length gene cloning and sequencing,it was found that only the G-A mutation occurred in the second exon of CsaV3＿1G044470.1,which caused the threonine at position 292 change to methionine（T292M）,other genes in the mapping interval had no difference.This indicated that CsaV3＿1G044470.1 is the best candidate gene.6.Preliminary analysis of the function of Cshs1.CsaV3＿1G044470.1encodes the riboflavin（vitamin B2）biosynthesis protein Rib D（riboflavin deaminase/reductase）.Through the phenotypic identification of Cshs1 silencing plants,it was found that the leaves of Cshs1 silencing plants showed partial or complete albinism.This indicated that the riboflavin pathway plays an important role in protecting cucumber from high temperature damage.This study not only expand ed and deepened our insight into the molecular regulation of organ growth and development in cucumber,but also successfully isolated and identified the thermo-sensitive leaf coloration gene Cshs1 in cucumber,providing a theoretical basis and candidate target genes for the generation of ideotype and molecular breeding of heat tolerance in cucumber.