MutMap-based Gene Mapping Of Cucumber Fruit Length And Verification Of Candidate Genes

Cucumber(Cucumis sativus L.,2n=14)is a economically significant vegetable crop,belonging to Curcurbitaceae family,which commercial consumption is huge in worldwide.Cucumber fruit length is the main basis for market consumption and a number of genetic and molecular studies about the length of cucumber fruits have been carried out.As quantitative characters,the characters of fruit length dependent on gene expression,hormone level,and environmental conditions and so on,and a high narrow-sense heritability is usually observed in it.Nonetheless,molecular mechanism underlying cucumber fruit size and shape is still not clear.Cucumber has a narrow genetic base,and it is not easy to construct high saturation genetic map and clone some important genes of agronomic traits based on map-based cloning method.The trait for fruit size is usually controlled by QTL loci.It is very difficult to clone the QTL loci using the natural population.EMS mutation provides a valuable method to obtain a specific material with the target locus mutation compared with wild type,which could be used for mapping and cloning of specific locus conveniently.In this study,a mutant of cucumber with shortened fruit length was screened from a mutant library constructed by materials changchunmici,which is mutated by EMS.Through the bulked sequencing of segregation population of F2 generation from the crossing of mutant and wild type,and the analysis of MutMap,we found the mutation site of cucumber fruit length rapidly and accurately,thus candidate gene of cucumber fruit length was found.Research results were as follows:1.Observed phenotype and inheritance of cucumber fruit length mutantThrough the multiple observations of cucumber fruit length mutant from different seasons and generations,the phenotypes of cucumber were described as follows:sharply shortened length of fruit,small and round seed with only half size of the wild type,significantly decreased height of plant,significantly receded growth potential and fertility.Meanwhile,smaller,deeply green leaves and shorter tendrils could be observed in mutant.Through the observation of paraffin section of ovary,commodity fruits and mature fruits from mutant,middle type and wild type,the results showed that the difference of cell numbers is the main reason for cucumber fruit length.Through the SEM observation of the stem and leaves,the results showed that the cell in stem of mutant aligned irregularly,squeezed each other.The number of stomas in leaves of mutant reduced,thus photosynthesis ability became weaken,which leading to a lower growth potential.Through the x2 text,it is concluded that the mutation of fruit length is a recessive mutation controlled by single gene.2.Location of candidate gene FL7.1 of cucumber fruit length mutant through MutMapMutant-pool(aa)and wild type-pool(AA)from the F2 generation using mutant and wild type as parents,were sequenced based on BSA method..Through the analysis of MutMap,the gene of FL(Fruit Length)was located on the region of 14.11-17.63 Mb at chromosome 7.Based on the mutation principle of A(SNP-index)?0.8 and EMS mutation,the candidate gene for FL was finally identified as the gene of Csa7M435510.1,and called FL7.1.The cytosine(C)located in base sequence 1058 bp site in this gene was replaced by thymine(T),and 353 site serine in amino acid sequence coded by this gene was mutated into phenylalanine,and the protein coded by this gene was called At-p60 katanin.3.The validation and expression analysis of candidate gene of cucumber fruit length mutantThe mutant and wild type from F2 generation population as template,cloning the CDS sequence of FL7.1.Only single-base changed in two transcripts from CDS sequence,,which demonstrated that the gene FL7.1 of mutant mutated in this site.Through the bioinformatics analysis of CDS sequence from mutant and wild type,the results showed that the base change lead to the change of molecular mass of protein,thus the percentage of amino acid forming this protein also changed.Sequence alignment with At-p60 katanin in other plants and analysis of phylogenetic tree,it is found that protein sequence of this gene from cucumber has high homology with At-p60 katanin in crops from Leguminosae,Solanaceae,Gramineae.The similarity is highest between cucumber and melon,which reached 93.99%.Subcellular localization demonstrated that At-p60 katanin coded FL7.1 exists in cytoplasm and cell nucleus.Quantitative expression results showed that this protein could express a specificity in cucumber tissues,which was observed the highest in leaves,the second in ovary,and the lowest expression in stem,and the relative expression level was decreased in mutant.These results all above could hypothesize that the function of gene FL7.1 is weak after mutation.