| Variegated leaf mutant is an important material for the study of chloroplast development.In this study,a variegated leaf mutant(Csvl)was screened from the EMS-induced mutant library in cucumber.The variegation trait can be inherited steadily through mutant multi-generation self-pollination.The leaves of Csv1 mutant showed variegated phenotype which was different from that of wild type.In other words,yellow,white,light green and normal green areas were randomly distributed on one leaf,and the degree of the variegation varied with light intensity and development.In this research,variegated leaf mutant Csv1 and its original parent were used as experimental materials,observation of main agronomic traits,genetic analysis,identification of the candidate gene for Csvl mutant,were conducted in this study with genome re-sequencing,KASP genotyping and related bioinformatics techniques.In addition,the differentially expressed genes of Csvl mutant and wild type plants were compared by RNA-seq,and the genes related to the variegated leaves were screened.The main results are as follows:(1)Phenotypic identification1)Differences in leaf characteristics,whole plant characteristics and flower morphology between Csvl mutant and wild type plantsThe color of each leaf appeared normal green in wild type plant,while it become variegated in Csvl mutant when the leaves grew to 3 cm.The leaf length,leaf width and leaf area of Csvl mutants were all reduced significantly compared with those of wild type plants.Additionally,the plant height,stem diameter,node number,internode length and branching of Csvl mutant were significantly reduced as well as,and the morphology of female and male flowers was abnormal.2)Differences in chloroplast structure and chlorophyll content between Csvl mutant and wild type plantsThe mesophyll cells of Csv1 mutant and its wild type were observed by transmission electron microscopy.The result showed that the chloroplast of leaf in wild type included well-organized thylakoids and starch grain.The mesophyll cell of the Csvl mutant contained a visible chloroplast but no structural feature of normal chloroplast.The chlorophyll content of Csvl mutant and its wild type leaves was determined with acetone extraction,the results showed that the chlorophyll content(chlorophyll a,chlorophyll b and carotenoid)of Csvl mutant was lower significantly than that of wild type.The photosynthetic parameters of Csvl mutant and its wild type leaves were measured and theresults showed that the net photosynthetic rate of leaves in Csvl mutant were significantly lower than those of wild type leaves,while stomatal conductance,intercellular CO2 concentration and transpiration rate were higher obviously than those of wild type leaves.3)Photooxidation stress in leaves of Csvl mutant and wild type plantsThe chlorophyll fluorescence parameters of leaves were analyzed between Csvl mutant and wild type,and the results indicated that the actual photosynthetic capacity,photosynthetic activity and electron transfer rate of photosystem II in the yellow sector of Csvl mutant leaves were lower than those of its wild type.It was speculated that the leaves of Csvl mutant might be subjected to photooxidation stress.It was inferred that the formation of variegated leaves was induced by natural light through shading and filtering ultraviolet light treatment of the Csvl plants without variegated leaves,and ultraviolet light may be the main factor inducing the formation of variegated leaves.(2)Genetic analysisThe leaves of F1 generation showed the same normal green as those of wild type plants,however,the significant phenotypic segregation was occurred in 158 F2 plants.The result of ?2 test(? 2 = 0.135 < ? 2 0.05,1 = 3.84)showed that the ratio of wild type phenotype and variegation phenotype plants in F2 population was 3 : 1(wild type : Csvl mutant = 116 : 42),which revealed that the phenotype of Csvl mutant was regulated by a single recessive gene.(3)Candidate gene identificationMut Map and KASP genotyping results revealed that Csa6G405290(Cscs)located on chromosome 6 of cucumber,encoding chorismate synthase,was the candidate gene for variegated leaf mutant in cucumber.Bioinformatics analysis of candidate gene showed that the Cscs sequence was 5392 bp in length,consisting of 13 exons and 12 introns.A transition from proline(Pro)to serine(Ser)was occurred in the ninth exon of Cscs gene due to a C-to-T substitution,and the mutation of amino acid(P-S)occurred at the FMN binding site of CS in cucumber.The expression of candidate genes was analyzed by q RT-PCR.The results showed that there was no difference in the expression level of Cscs between Csvl mutant and wild type leaves.In wild type plants,the expression of Cscs was the highest in stem,followed by tendrils,leaves,roots,male flowers and female flowers.(4)Transcriptome sequencing analysisA total of 183 differentially expressed genes were then identified in Csvl mutant and wild type leaves by RNA-Seq.Based on the annotation and analysis of these genes,heat shock protein,zinc finger protein,ethylene responsive factor and WRKY transcription factor associated genes were identified as the candidate genes involved in the formation of leaf variegation in cucumber.These genes were closely related to chloroplast development and chlorophyll synthesis.Gene Ontology analysis showed that differentially expressed genes of Csvl mutant and wild type leaves were mainly referred to developmental process,nucleic acid binding transcription factor activity,cellular process,catalytic activity,metabolic process,biological regulation,and so on.Pathway analysis revealed that differentially expressed genes were mainly concentrated in sesquiterpene and triterpene biosynthesis,protein processing in endoplasmic reticulum,plant-pathogen interaction,antigen processing and presentation and MAPK signaling pathway.In summary,the mechanism of variegated leaf formation in cucumber has been preliminarily speculated,which provides a theoretical basis for further exploring the molecular mechanism of the formation of leaf variegation in cucumber. |
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