Fine Mapping And Establishment Of Molecular Marker-assisted Selection System For Bisexual Flowers In Watermelon(Citrullus Lanatus L.)

Watermelon（Citrullus lanatus）,Cucurbitaceae family,Citrullus genus（2n=2x=22）,annual trailing herb,also known as water melon,cold melon.It is an important worldwide horticultural cash crop.Chinese watermelon production and sales account for more than 60%of the world.Watermelon cultivars are mostly monoecious,and some of them are andromonoecious.Andromonoecious watermelon cultivars produce male and bisexual flowers,and bisexual flowers are easy to self-pollinate.In hybrid seed production,hermaphrodite flowers as the mother need to be emasculated,which is time-consuming and laborious,and also easy to lead to false hybrids seeds due to incomplete emasculation.Therefore,fine mapping of the key genes of watermelon bisexual flowers and establishment of marker-assisted selection technology system are of great significance for the study of watermelon sex differentiation and breeding.In the previous analysis,we used ZXG1555（maternal）,COS（paternal）,F₁and F₂mapping population developed during the spring and autumn.We found that a single dominant gene regulated watermelon bisexual flowers.By using BSA-seq sequencing and F₂generation genetic linkage analysis,the key genes of bisexual flowers were preliminary located on chromosome 3,with the interval of 29,252,650bp-30,021,268 bp（about 768 kb）.Based on the above studies,expanding ZXG1555×COS-F₂generation population,we used the preliminary mapping of flanking markers to screen the recombinant plants and continued to develop polymorphic molecular markers.The key genes controlling the bisexual flowers traits of watermelon were fine mapping by combining the genotype and phenotype of the recombinant plant,and the candidate genes within the interval were screened and analyzed.To establish and verify the molecular marker-assisted selection technology system of watermelon bisexual flowers,the main results of this paper are as follows:（1）CAPS markers were developed within the initial positioning interval,and the six CAPS markers were used to genotype in the F₂mapping population developed during the spring and autumn seasons of 2021.QTL analysis of bisexual flowers traits was performed based on the genotyping results of 18 pairs of molecular markers（12 pairs in 2021 and 6 pairs in 2022）and the phenotypic data of F₂population in spring and autumn.The results showed that the stable interval was obtained in both seasons（Chr3:29,252,650bp-30,021,268 bp）,the Chr3＿29540662 marker has an obvious peak in the interval,Chr3＿29540662 marker phenotype and genotype analysis found that the genotype and phenotype of Chr3＿29540662 marker were consistent,which can be used to distinguish bisexual flowers and unisexual flowers watermelon.（2）The recombinant plants was screened from 710 F₂generation population by the preliminary location-flanking markers Chr3＿29252650 and Chr3＿30021268,and the recombinant plants was genotyped by the newly developed molecular markers in the preliminary mapping range.Finally,the key genes of bisexual flowers were located within the interval of chromosome 3 of29,488,876 bp-29,570,980 bp（82.104 kb）,containing a total of 5 candidate genes.The 5 candidate genes were annotated in the Cucurbit Genomics Database.Integrative Genomic Viewer（IGV）,a software integrative Genomic Viewer,was used to analyze the sequences and structural variation sites of the gene coding region and promoter region in the two parents.It was found that four candidate genes had non-synonymous mutations in coding region,and the SNPS on the promoters of the five candidate genes were all in the cis-acting elements.Based on the studies on bisexual flowers of other cucurbit plants,it is preliminatively suggested that Cla97C03G066110（1-aminocyclopropane-1-carboxylate synthase 7）,which is related to ethylene synthesis,may be a key candidate gene for regulating bisexual floral traits in watermelon.（3）The molecular marker Chr03＿29539131,which was closely linked to the bisexual flowers traits of watermelon in the fine mapping interval,was used for genotype analysis of 32 watermelon natural populations（2 andromonoecious,30 monoecious）,and the phenotypic data were combined to analyze the genotype and phenotypic compatibility.The results show that Chr03＿29539131 can distinguish between bisexual flowers and monoecious watermelon plants and that this molecular marker can be used for watermelon hermaphroditic trait screening and molecular marker-assisted breeding.