Fine Mapping And Cloning Of Gene ClLL1 (Citrullus Lanatus Lobed Leaf 1) In Watermelon (Citrullus Lanatus)

Lobed-leaf is a kind of leaf morphology to adapt the variable environment?especially adversity?,has many advantages in production,such as improving drought resistance,cold resistance and strong wind resistance.Watermelon is an important economic crop of cucurbitaceae,which leaf shape is lobed-leaf.Mapping and cloning the genes that related to the development of leaf shape will provide an insight into mechanism of leaf shape development and improving the drought resistant varieties of watermelon.In this study,the material was obtained from the isolated group of cultivar"1101"by self-delivery.The morphological characteristics of the leaf shape were observed,and the genetic analysis showed that the leaf margin of watermelon was a dominant trait controlled by a single gene,named ClLL1?Citrullus lanatus Lobed Leaf 1?.Furthermore,the precise localization of ClLL1 gene was studied by BSR-seq technology.The candidate gene ORF5?Cla018360?was determined,then cloning the gene and analyzed by bioinformatics methods.The major results listed as follows:1.The statistical results showed that the leaf shapes can be visibly distinguished after the emergence of the third true leaf and differentiated at the sixth adult leaf stage.In addition,the symmetrical marginal protrusions can clearly be observed on the sixth true leaf from non-lobed leaf plants.2.The F_2:3:3 plants were self-pollinated to produce a sufficiently sized F_3:4 population.A small proportion of F_3:4 progenies was planted to analyze the genetic inheritance of the lobed leaf trait.As a result,93 F_3:4 individuals presented two phenotypes,including 69 lobed and 24non-lobed plants,with a 3:1 Mendelian ratio??²=0.03,p=0.86?.Furthermore,leaves of seedlings derived from nonlobed F2:3 individuals had a leaf form consistent with the parent plant.In summary,we inferred that a dominant allele,designated as ClLL1,controlled the lobed leaf trait in watermelon.3.A total of 5966 reliable SNPs and indels were identified genome-wide via a combination of BSA and RNA-seq and used in subsequent studies to develop markers.4.To locate gene ClLL1 on a chromosome,13 markers were designed per chromosome and used to screen the F_3:4 population?93 individuals?.Linkage analysis revealed that marker W01144 on chromosome 4 was linked to the lobed leaf trait,new markers were designed to screen the population,the gene ClLL1 to be roughly delimited within a 2.64 Mb region between markers W03041 and W01214.5.To precisely identify the genomic region surrounding gene ClLL1,a larger segregating population consisting of 2455 individuals was utilized,the primary flanking markers were utilized to screen the population and identified the recombinants,a series of new markers were developed based on reliable SNPs and indels,combined with margin phenotype,finally,the location of ClLL1 was narrowed down between markers W07164 and W07061,the physical distance is 29.14-kb,the genetic distance is 0.48 c M.6.There are 5 open reading frames?ORF?in the fine mapping interval.Based on gene annotation and expression analysis,ORF5?encoding a homeobox-leucine zipper-like protein?were considered as most likely candidate genes.7.The gene expression profile of Cla018360 was studied.Using real-time fluorescence quantitative PCR to analysis the relationship between the gene and leaf shape.The results showed that the lobed leaf expression of Cla018360 was more than 9 times higher than non-lobed leaf,indicated that Cla018360 gene plays an important role in the development of lobed leaf.The gene expression of different tissues was further examined.The results showed that genes were expressed in these tissues.8.After PCR detection and sequencing verification,the cDNA of Cla018360 was successfully cloned.Through bioinformation analysis and protein subcellular localization come to the conclusion that Cla018360 is a member of transcription factor family of HD-zip?.It is a homologue of the gene LMI1,localizes to the nucleus.9.Connected the candidate gene Cla018360 to the pCAMBIA2300 construct expression vectors,and then,transfer to agrobacterium to verify the function of the gene.