Fine Genetic Mapping And Candidate Gene Identification Of The Mottled Rind Color(morc) On Immature Fruit Of Cucumber

The mottled skin color of immature cucumber fruit is one of the important appearance quality traits,which is an important factor to determine the commercial value of cucumber,and directly affects consumers’choice.However,the research on the formation and development regulation of cucumber mottled skin is very limited.Therefore,it is of great theoretical and practical significance for understanding the formation mechanism of cucumber mottled skin,promoting cucumber quality breeding,and improving the economic value of cucumber to study the fine mapping and function of genes regulating cucumber variegated skin color.In this study,green mottled skin germplasm WI7632 and white unmottled skin germplasm XNS16-1 were used as parents to construct F₂ segregation population,combined with phenotypic data to reveal the genetic rule of mottled immature fruit rind color in cucumber,and to explore the internal physiological mechanism of mottled skin color formation through chlorophyll content determination and transmission electron microscope observation between parents.The fine mapping of the mottled immature fruit rind color gene Morc was delimited by the bulked segregant analysis（BSA）combined with molecular marker technology,and the candidate genes in the mapping interval were predicted;at the same time,combined with transcriptome data,the gene regulatory network of mottled immature fruit rind color formation that Morc gene may participate in was explored,which laid a foundation for further study of the biological function of Morc gene.The results are as follows:1.Plants of F₁ generation were all mottled skin color,and the proportion of mottled and unmottled skin color was 90:32 in F₂ population planted in spring of 2020,55:18 in F₂population planted in autumn of 2020,and 109:31 in F₂ population planted in spring of 2021,which conformed to the genetic segregation ratio of 3:1,indicating that the mottled skin color of immature cucumber fruit is regulated by dominant single gene Morc.2.Through the determination of chlorophyll content and observation of chloroplast microstructure of their parents,it was found that the chlorophyll content and carotenoid content in WI7632 were significantly higher than those in XNS16-1,and the pigment content in WI7632 white peel was significantly lower than that in green peel,and there were obvious defects in chloroplast development in XNS16-1 and WI7632.XNS16-1>WI7632 white peel>WI7632 green peel;the more abnormal the chloroplast development,the lower the chlorophyll content.It is speculated that the mottled skin color of cucumber fruit may be caused by the decrease of chlorophyll synthesis caused by abnormal chloroplast development at the local position of the peel.3.In the initial mapping stage,Morc gene was located between UW083747 and SSR16068 on chromosome 5 with a physical distance of 3.38 Mb by BSA method.In the fine mapping stage,through the continuous development of new molecular markers in the initial mapping interval and screening of new recombinant plants by expanding the mapping population,the Morc gene was finally located in the 42.9 kb region between Scindel18 and morcindel15.Compared with the cucumber reference genome,it was found that the mapping interval contained two candidate genes Csa V3＿5G028870 and Csa V3＿5G028880.Interestingly,the expression level of gene Csa V3＿5G028870 was significantly different between mottled traits and unmottled traits,while another gene Csa V3＿5G028880 exhibited similar expression levels in both traits.Based on the gene functional annotation information and the gene differential expression data in the transcriptome data,the most possible candidate gene Csa V3＿5G028870 was selected for PCR cloning and sequencing analysis.The results showed that the gene was completely deleted in XNS16-1.The length of the deletion fragment is that 4895 bp contains the promoter sequence of 3272 bp and the 985 bp sequence after terminator code of the gene.It is predicted that Csa V3＿5G028870 is the best candidate for Morc gene.4.Transcriptome sequencing analysis based on mixed pool（BSR-seq）showed that there were 2982 differentially expressed genes between mottled and non-mottled skin color gene pools,including 1119 up-regulated genes and 1863 down-regulated genes.GO and KEGG analysis of differentially expressed genes revealed phenylalanine metabolic pathways related to skin color development,and differentially expressed genes involved in oxidoreductase activity（GO:0016491）,ammonia lyase activity（GO:0016841）,heme binding（GO:0020037）,tetrapyrrole binding（GO:0046906）and iron ion binding（GO:0005506）.And the differentially expressed genes encoding ZFP,ERF,MYB,b HLH and WRKY transcription factors which are closely related to pigment synthesis and photosynthesis may be involved in the formation of mottled skin color of cucumber tender fruit regulated by Morc gene.