Mapping And Cloning Of ClSC,a Gene For Watermelon Fruit Stripe Color

Watermelon(Citrullus lanatus)is a kind of Cucurbit crop with high economic and nutritional value.With the improvement of people’s living standard,the demand for watermelon quality and diversity is also increased.As an important appearance quality,rind stripe has always been the focus of consumers and breeders.At present,the research on watermelon stripe mainly focuses on genetic analysis and gene localization,and the functional analysis of key genes about stripe color is rarely reported.In the present study,watermelon materials with dark green stripe WM114 and with light green stripe WM60 were studied.With F2 population,the inheritance of rind stripe color was analyzed,and the gene Citrullus lantus stripe color(CISC)controlling stripe color was finely mapped,which laid a foundation for the subsequent functional verification and breeding application for stripe color gene.The main research results are as follows:1.Dark green stripe material WM114 and light green stripe material WM60 were used to construct the F1 and F2 populations,The rind stripe color of F2 population was observed at 0 d,7 d,14 d,21 d and 28 d after pollination.The results indicated that the light green stripe in watermelon was controlled by a pair of recessive genes,which were named Citrullus lanatus stripe color(CISC)2.The chlorophyll content and carotenoid content in light green fruit stripes were significantly lower than that in dark green fruit stripes,indicating that the lower chlorophyll content in light green fruit stripes is responsible for the light green fruit stripes.The chloroplast structure in the light green stripe was abnormal by transmission electron microscopy.3.Gene CISC was initially located on chromosome 9 according to BSA-based analysis.New Snp markers and Indel markers were developed according to parental resequencing results.4.The sequence analysis of genes in the candidate interval revealed that there was a single base non-synonymous mutation at the junction between the 7th intron and the 8th exon of Cla97C09G175170 gene,which led the premature codon termination and protein abnormality.Expression analysis showed that Cla97C09G175170 gene expression level in dark green stripe was significantly higher than that in light green stripe,which further verified Cla97C09G175170 as a candidate gene for the stripe color of watermelon fruits.5.According to Cla97C09G175170 gene single base mutation,a new dCAPS marker was designed and verified in 28 watermelon materials.It was found that the genotype and phenotype matched correctly in all 28 watermelon materials,which can provide an effective marker for molecular marking-assisted breeding of watermelon.6.Two conserved domains were found in the prediction of protein domains.Through gene annotation,it was found that ClSC encodes a two-component response regulatory protein APRR2.Phylogenetic analysis of CISC and homologous genes showed that although CISC and Arabidopsis Thaliana APRR2 are both APRR2 genes,they are not located in the same clade.CISC is located in the same clade with melon and cucumber,and has the closest genetic distance.Subcellular localization prediction of CISC protein showed that CISC was located in the nucleus.7.Cis-element prediction analysis was conducted on CISC promoter,and it was found that there were a variety of cis-elements on the promoter,such as light response element,core promoter element,and involved in methyl jasmonate,gibber,abscisic acid reaction,etc.The prediction results showed that a variety of factors affected the activity of CISC gene promoter.8.In order to analyze the regulation mechanism of CISC in fruit stripe color,transcriptome sequencing was performed on WM114 and WM60 fruit stripes 21 days after pollination,and 3298 differentially expressed genes were found between WM114 and WM60,among which 1888 genes were up-regulated and 1410 genes were down-regulated.KEGG enrichment analysis showed that differentially expressed genes were mainly enriched in photosynthetic antenna proteins,phenylpropane biosynthesis,porphyrin and other metabolic pathways.CISC may influence chlorophyll content by regulating chloroplast development and photosynthesis.