Map-based Cloning And Function Analysis Of A Yellow Gene Clyp In Watermelon(Citrullus Lanatus)

Watermelon(Citrullus lanatus)is a kind of annual herbaceous belonging to the genus Citrullus in the Cucurbitaceae family.It is known as the king of summer fruits because its flesh,crispy,juicy,and rich in mineral salts and vitamins.Yellow plant mutants are ubiquitous in plants,they are ideal materials for studying chloroplast development and high light efficiency.A new yellow plant mutant,WM103,was found in our watermelon germplasm collection.WM103 is different from other conventional yellow plant mutants,the appearance period and the severity of yellow phenotype are significantly affected by the growth environment.In this study,using genetic analysis,we found the yellow plant phenotype of WM103 was controlled by a single recession gene(named Clyp).Clyp was further fine mapped,and the gene Cla97C10G186360,which encode a BCM protein,was proved as its candidate gene.Then the function of Clyp in regulating the yellow phenotype was explored by RT-PCR and VIGS.We also did the RNA-Seq to preliminary reveal the regulation network of Clyp in yellow plant.Those results layed a solid foundation for further exhaustive study of the molecular mechanism and regulation network of Clyp.The main results were listed as follows:1.The phenotypic observation of mutant material WM103.When the plants grow in appropriate conditions,compared to the wild type material WT2,the leaves of WM103 are slightly yellow at the seedling stage.Then,at the later stage of growth,the plants of WM103 begin to appear obvious yellow phenotypes from the bottom to the top,until the entire plant are yellow.However,when the plants of WM103 suffer some stress at any stage of their growth period,the plants will exhibit the yellow phenotypes earlier.Chlorophyll contents were measured and the cell structures were observed using the leaves of two parent materials,the results showed that,compared with WT2,the chlorophyll content and the number of chloroplasts in WM103 were decreased singnificantly,the defective thylakoids and degraded starch granules were found in WM103.2.A F2 population with 1373 strains were constructed by WM103 and WT2.The color of those plants after 45 days of planting were observed and analysised,the segregation ratio of green and yellow was 3:1,indicated that the yellow plant phenotype of WM103 was controlled by a recessive single gene,named Clyp.Using BSA analysis of watermelon SNP liquid chip,Clyp was initially located on chromosome 10 between 407610-2493883 bp;SSR markers and a small F2 population were also used to map Clyp,and Clyp still was located on chromosome 10 between 1332738-2723000 bp.3.In order to fine mapping Clyp,the two parents were deeply re-sequenced and more molecular markers within the candidate interval were developed.Using a F2 population with 1373 strains,Clyp was finally located on chromosome 10,within 83.63 kb.In this interval,there are nine genes,but only one gene(Cla97C10G186360)has a 90bp insertion/deletion between WM103 and WT2.Based on this insertion/deletion,a molecular marker,InDel 18,were developed to scan in the WM103 × WT2 F2 population and a watermelon natural population,the results showed that InDel 18 was totally linkage with the yellow plant trait.So Cla97C10G186360 gene was preliminarily identified as a candidate gene of Clyp.4.The functional prediction of Cla97C10G186360 shows that it encodes a chlorophyll metabolism balance protein BCM,containing six transmembrane domains.The deletion of 90 bp in WM103 destroyed the transmembrane structure,possibly affected the stability and function of the protein.Expression analysis of Cla97C10G186360 in different parts of two parents showed that the expression of Clyp in WM103 were significantly reduced.Then the VIGS technology was used to verify the function of Clyp,all the Clyp silenced lines were showed yellow plant phenotypes,further clarifyed that Cla97C10G186360 is the candidate gene of Clyp.5.In order to preliminary reveal the regulation network of ClYP in yellow plant,Transcriptome sequencing was performed using the middle green leaves of WT2,Totally,2113 differentially expressed genes(DEGs),which have different expression levels among WM103Y,WM103G,and WT2,were found.The GO and KEGG enrichment showed that those DEGs were the enrichment were mainly related to photosynthesis in biological processes and chlorophyll in molecular function.Analyzing the expression levels of differentially expressed genes in the category,it was found that in WM103Y,the expression levels of genes related to chlorophyll synthesis and photosynthesis were down regulated,while genes related to chlorophyll degradation and aging were up regulated.