Mapping Of Csf Gene Of Cucumber Short Fruit Mutant And Prediction Of Candidate Genes

Cucumber(Cucumissativus L.)is an important vegetable crop that is cultivated globally and has high economic value.The fruit is the product organ of cucumber,and the length of the fruit is an important factor affecting its yield and appearance quality of the product,and is directly related to economic benefits.The excavation of fruit length-related genes is of significance for guiding the selection of varieties with the target fruit length.The traits related to the length of cucumber fruit in nature are quantitative traits(QTL)regulated by multiple genes.The performance of single gene loci is often interfered by other loci,the accuracy of phenotypic identification is low,and the research on gene mapping and excavation is difficult.Up to now,in the related research on cucumber fruit size,only some QTL mapping reports on fruit length and fruit thickness have been reported.Compared with wild-type plants,mutant plants generally only change at a single locus,so that interference from other loci can be excluded,so it can be efficiently applied to the in-depth analysis of gene function.There have been few reports on the use of mutant materials to successfully map and predict genes that control the length of cucumber fruit.In this paper,the excavation research of related genes based on cucumber short fruit mutants is of great significance for further research on the molecular regulation mechanism of cucumber fruit length and the breeding of varieties related to cucumber fruit length traits.In this study,based on the‘Changchunmici’ cucumber mutant library obtained by EMS mutagenesis,a stable genetic short fruit mutant was screened.The short fruit mutant was used as a parent,and it was crossed with the wild type ’Changchunmici’ cucumber,and self-crossed to construct the F2 population.Based on physiological and genetic analysis,the bulk segregant analysis(BSA)and high throughput sequencing were combined(BSA-seq).The mutant was rapidly genetically mapped by bioinformatics,and finally the short fruit gene controlling the length of cucumber fruit was mapped on the chromosome.The results are as follows:1.Phenotypic and genetic analysis of cucumber short fruit mutantsThe multi-generation and multi-season observations of cucumber short-fruit mutants showed that the fruit became shorter and thicker than the wild-type ’Changchunmici’cucumber,and the tail of the fruit was shortened,and the middle part was slightly raised,and the ratio of the diameter(L)to the cross diameter(D)was significantly smaller,and the tendril was slow to develop.The fruits of different growth stages of the mutants were analyzed by paraffin section and scanning electron microscopy.The results showed that the main factor leading to the difference in cucumber fruit length was the difference in the number of cells inside the fruit.The mutant was hybridized with ’Changchunmici’ to obtain the F1 generation,and the F2 generation was obtained by self-crossing,and the traits were statistically analyzed.Field observations revealed that the fruit phenotypes of the F1 population were all wild-type phenotypes.Of the 120 F2 populations,88 showed wild type and 32 showed short fruit phenotype.The χ2 text showed that the short fruit mutant was a single gene-controlled recessive mutatio.2.Mapping candidate genes for cucumber short fruit mutants based on sequencingUsing the bulk segregant analysis(BSA),the extreme phenotype individual plant from F2 segregating population constructed by hybridizing mutants with wild type were separately extracted DNA,mixed the DNA in equal volumes and sequenced(30 F2 individuals in each pool).The sequencing results were then compared to the reference genomic sequence to obtain a distribution curve of the SNP-index on all chromosomes.The BSA-seq analysis mapped the candidate gene to 22.8 Mb-23.8 Mb on chromosome 1,12.8 Mb-16.9 Mb and 16.9 Mb-18.8 Mb on chromosome 4.Seven SNP loci were screened in the target region by bio informatics.3.Candidate gene prediction and quantitative real-time PCR analysisThe candidate SNP loci were screened according to the results of BSA-seq,and the targeted mutations in the region were analyzed.Four candidate genes were screened,which were Csa4G441080,Csa4G439050,Csa4G363450,and Csa4G439060.Quantitative real-time PCR analysis revealed that Csa4G439060 differed significantly between the two parents during different stages of fruit growth.During the rapid growth period of fruit(0-8 d after flowering),the expression level of this gene in the mutant fruit was significantly higher than wild type fruit.The wild type and mutant materials were used as templates,and the primers were designed according to the CDS sequence of Csa4G439060 for cloning.The two transcripts obtained only had a single base change,which confirmed that the gene in the mutant did mutate at this position.Sequence alignment analysis revealed that the 1546 bp cytosine(C)of the base sequence of this gene was replaced by thymine(T),and the encoded amino acid was mutated from arginine(Arg)to lysine(Lys).Comprehensive analysis predicted that Csa4G439060 is a candidate gene for controlling the length of cucumber fruit.In this study,the BSA-seq method was used to successfully map and predict a cucumber short fruit candidate gene,which laid a foundation for molecular breeding related to cucumber fruit length.