Fine Mapping Of Major Gene ClScc Response For Seed Coat Crack In Watermelon

Watermelon（Citrullus lanatus L.）,as a worldwide horticultural crop,providing a variety of vitamins,carotenoids and other beneficial nutrients for human body.The appearance of seed coat an important agronomic trait.As the natural protective barrier of seeds,the seed coat protects the embryo.However,seed coat crack is easy to damage the watermelon seeds by pests and microorganisms,and also reduces the quality and quality of watermelon seeds,thus affecting the economic value of watermelon for seeds.The use of forward genetic strategy to clarify the key genes regulating the formation of watermelon seed coat crack,and establish a molecular marker-assisted selection technology system for great significance in theoretical research and breeding applications.In this study,genetic analysis was carried out on six generations of parents from Cream of Saskatchewan（COS,seed coat non-crack）and PI 192938（seed coat crack）,and determine the genetic rule of watermelon seed coat crack.The genome resequencing data of COS and PI 192938as parental materials and Bulked Segregant Analysis（BSA-seq）,obtained the chromosome region with seed coat crack,and developed molecular marker CAPS（Cleaved Amplified Polymorphic）on the target chromosome segment Sequence),In Del（Insertion-Deletion）,and LGC-KASP（Kompetitive Allele Specific PCR）were genotyped for the F₂generation population,and an initial mapping of the gene controlling seed coat crack was obtained.Expanding the F₂generation population,the two flanking markers were used to screen the recombinant plants,and the molecular markers were further developed to complete fine mapping of the seed coat crack gene.the candidate gene in the interval was screened and cloned,and the key candidate gene for regulating watermelon seed coat crack was finally determined by expression patterns analysis and promoter activity analysis.Finally,the major genes regulating watermelon seed coat crack were deduced.The results of this study are as following:（1）In both COS and PI 192938 genetic populations,The phenotype of F₁generation was seed coat non-crack without separation,similar to COS.The 290 F₂individuals in 2019 contained 219seed coat non-crack plants and 71 seed coat crack plants(χ²>χ^20.05=0.84).Among the 284 F₂plants in the year 2020,there were 210 seed coat non-crack and 74 seed coat crack individuals(χ²>χ^20.05=0.68),which the two groups fitted for the ratio of 3:1.The individual phenotypes in the F₁and COS backcross population BC₁P₁were all seed coat non-crack,and the ratio of seed coat non-crack to seed coat crack in the F₁and PI 192938 backcross population BC₁P₂was 1:1（χ²=1.894,p=0.168）.we concluded that the watermelon scc trait was controlled by a single recessive gene,and was named Cl Scc（Seed coat crack,Scc）.（2）By BSA-seq analysis,the watermelon seed coat crack control gene was mapped to chromosome 3,in the interval of about 11.21 Mb（1,790,000 bp-12,000,000 bp）.and 25 molecular markers were developed within this region.The recessive phenotypes in 2019 and 2020 were genotyped,and the candidate regions were reduced to 808.8kb（4,881,097bp-5,681,679bp）.（3）Expand the F₂generation population（1,152 strains）,screen recombinant plants,and screen a total of 29 recombinants.8 molecular markers were further developed within this interval to genotype the 29 recombinants.According to the genotyping results and phenotypic isolation of F₂and F_2:3families,the watermelon seed coat crack gene was located between the marker Chr03＿4946260 and Chr03＿5223372 with a physical distance of 277.11 kb,containing a total of37 annotated candidate genes.（4）Analyzed COS and PI 192938 resequencing data.All 37 candidate genes showed no polymorphic sites in the coding sequence（CDS）region between the parental lines.Further promoter sequences alignment of the candidate genes within these mapping intervals revealed that Cla97C03G056110,Cla97C03G056390,Cla97C03G056400,Cla97C03G056320,and Cla97C03G056460 exhibited SNP mutations between parental lines.Published transcriptome data showed that Cla97C03G056390 and Cla97C03G056400 were not expressed in watermelon seed.Cloning of the promoter sequences of Cla97C03G056110,Cla97C03G056320,and Cla97C03G056460.No SNPs were found in either Cla97C03G056320 or Cla97C03G056460.Three SNPs mutation in the Cla97C03G056110（CRIB domain）occurred in cis-acting elements（CAAT-box,MYC,P-box）.（5）To further shorten the interval,we compared the genome re-sequencing data of 21watermelon germplasm samples（8 seed coat crack materials and 13 seed coat non-crack materials）using in silico BSA.We calculated the conformity between the phenotype and genotype for each SNP locus and then found an 8.34 kb chromosome region（from 4,946,258 to 4,954,598 bp）containing four SNPs,with conformity>95%:SNP4,949,047,SNP4,955,945（95.23%conformity）,and SNP4,951,684,SNP4,951,703（100%conformity）.The compliance of other SNPs in the region ranged from 90.47%to 47.61%,and Cla97C03G056110 was the only one gene located in this region.Cloning the promoter sequence of Cla97C03G056110 from other ten watermelon accessions.The results showed that three SNPs mutation sites were coisolated from the seed coat crack character.（6）The flesh tissue and seed coat tissue at the same mature period of both parents were used as materials,and the expression patterns of candidate genes in different tissues were analyzed.Compared with flesh tissue,Cla97C03G056110 was specifically expressed in seed coat,and Cla97C03G056110 was expressed at a significantly higher level in COS than in PI 192938.The Dual-luciferase reporter assay showed that the promoter activity of COS was significantly higher than that of PI 192938.It is speculated that Cla97C03G056110 is a key candidate gene for Cl Scc.（7）Genotyping F₂isolated individuals and 14 watermelon accessions using the fine mapping interval the molecular marker Chr03＿4946260.The results showed that this marker enables molecular marker-assisted selection for seed coat crack materials and seed coat non-crack materials.