Gene Mapping Of Fruit Surface And Seed Coat Color In Melon(Cucumis Melo L.)

Melon is one of the major horticultural crops in the world.As one of the important public fruits,melon occupies a certain position in Chinese agricultural industry.Rind color is important since the appearance is one of the main determinants for consumer preference in the market.Starting from the ovary development,fruit rind color remain green until maturity.The organic matter can be synthesized by photosynthesis during the whole fruit development,which can increase the nutritional content of fruit and improve its nutritional value.In addition,green rind is more acceptable as a healthy color.As the carrier of plant life,seed traits are related to the growth and development of plants.Although seed traits are not the main agronomic traits of melon,they are also related to seed germination and morphological formation of seedlings.The research group previously found a rare trait in melon--red seed coat.Although this trait did not significantly affect the seed germination and seedling development of melon,it can be used as a marker to control the purity of melon hybrids.As close relatives of melon,the seed traits of watermelon and pumpkin are very important,seed coat color is an important trait highly affecting the seed quality and appearance for them.In order to identify the genetic analysis of melon green rind and red seed coat,the melon inbred line HNA24 with white rind and yellow seed coat and inbred line 418 with green rind and red seed coat were used as the parent materials to construct a six-generation population（P₁、P₂、F₁、F₂、BC₁P₁ and BC₁P₂）for genetic analysis and gene mapping of rind color and seed coat color traits.Genome-wide association analysis of natural populations was used to verify the results.The main results are as follows:1.Genetic analysis of melon rind color and seed coat color.By the descriptive statistics of the phenotype data of six-generation population,the results showed:there were significant segregation in rind color and seed coat color in populations（F₂,BC₁P₁ and BC₁P₂）.Statistical analysis showed that green rind was dominant relative to white rind,and red seed coat color was recessive relative to yellow.All the segregation results suggested that rind color and seed coat color were controlled by a single nuclear gene with complete dominance in the population.2.Genetic mapping by BSA-Seq analysis and linkage analysis.Next generation sequencing aided bulked-segregant analysis（BSA-seq）of the green rind pool and white rind pool selected from an F2 population revealed that the green rind gene was located in the range of 0.64 Mb～1.38 Mb of chromosome 4,and the red seed coat color pool and yellow seed coat color pool revealed that the red seed coat color gene was located in the range of 3.45 Mb-33.24 Mb on chromosome 6,with the interval of 0.7 Mb and 29.79 Mb,respectively.3.Fine mapping and candidate gene identification.The two parents were used in re-sequencing for genome-wide detection of SNPs and Indels.16 InDel markers were developed in the candidate region on chromosome 4 to screen all F2 individuals for polymorphic analysis.Finally,the rind color candidate gene was delimited to a 23 kb（0.66 Mb～0.69 Mb）region,there were 3 candidate genes in this region.39 markers（26 InDel loci,11 SNP loci,and 2 SSR loci）were developed in the candidate region on chromosome 6 to screen all F2 individuals for polymorphic analysis.Finally,the seed coat color candidate gene was delimited to a 116 kb（12.99 Mb～13.115 Mb）region,and there were a total of 7 genes in this region.4.GWAS analysis.The genome-wide association study was obtained from previous studies in our laboratory,we present the genome resequencing of 200 diverse accessions to conduct the SNP correlation analysis.There was no significant association loci between green and non-green rind,indicating that the genetic basis of this trait was complex.A highly significant[-log10（P）>7.16]locus for seed coat red/non-red trait was found on chromosome 6,with a range of 12,926,065-14,439,327 bp,which was consistent with the results of BSA-seq mapping,and the strongly associated SNP was located at 13,841,088 bp on chromosome 6.5.Expression analysis of candidate genes.The sequence alignment of the 3 candidate genes on chromosome 4（Melo3C003374,Melo3C030358,Melo3C003375）revealed the parent had non-synonymous mutations.qRT-PCR analysis showed that only Melo3C003375 had expression differences between the two parents,which shared 85%sequence identity with APRR2 in Arabidopsis thaliana,this gene was probably the candidate gene for green rind traits.Among 7 genes（Melo3C019553,Melo3C031999,Melo3C019554,Melo3C032001,Melo3C032000,Melo3C019558,Melo3C032005）on chromosome 6,4 genes（Melo3C031999,Melo3C019554,Melo3C032001,Melo3C032005）have non-synonymous mutations.qRT-PCR analysis showed that only Melo3C031999 and Melo3C019554 had expression differences in expression after seed coat turning stage.Melo3C031999 has highly homologous to Arabidopsis thaliana At2G15000,which is an unknown protein.Melo3C019554 shares 47.2%homology sequences with At3G19510 in Arabidopsis thaliana and contains zinc finger protein domain.Both Melo3C031999 and Melo3C019554 may be candidate genes for the red seed coat trait.