Genetic Mapping Of A Variegated Leaf (vg) Nutant In Tomato

Tomato is the most important vegetable.Photosynthetic efficiency has a very important effect on tomato yield and quality,while leaves are the main place for plant photosynthesis to accumulate organic matter.The phenotypes of these mutants are common and easy to observe,the formation of mutants is due to chlorophyll content,chloroplast development and photosynthesis and other processes are abnormal,thus affecting crop quality or yield.Therefore,the study of leaf color mutants is conducive to the study of related mechanisms,as well as assisted breeding to improve crop yield.In this study,a tomato variegated leaf mutant?vg?was found in the transgenic process.The phenotypic characteristic,mutagenesis,genetic analysis,mapping and function of mutated gene were studied to explore the molecular mechanism of vg mutant phenotype,and it would provide theoretical basis for tomato photosynthetic pathways.The main findings are as follows:1.The vg mutant showed normal seedling stage and leaf color change began about one month after sowing.The bleach first occurs on part of new mature leaves showing whitening,and further to immature leaves showing whitening/pale yellow respectively.In the autumn,the growth potential of vg mutant was weaker at high light condition.2.We observed the chloroplast ultra-structure of AC and vg mutant with TEM and found the chloroplasts of vg mutant in pale yellow leaves lacked the common thylakoid membranes and normal structure.Although loosely thylakoid membranes seem to form in chloroplasts of vg mutant in green area,there are no normal grana stacks and little or only a few starch granules formed Chloroplasts of vg mutant in whitening areas are intermediate state.In pale yellow areas,the contents of Chlorophyll a,Chlorophyll b and total Chlorophyll significantly decreased.However,there was no significant differences in green areas?Fig.4a,b,c?.The Chlorophyll b content was slightly reduced in the whitening areas.And the soluble sugar and starch contents of green leaves were significantly reduced.3.The co-segregation and genetic analysis showed that the phenotype of vg mutant was not related to the target gene transferred,but not the mutation caused by T-DNA insertion,and the mutation were caused by transgenic process.From the F₂ population derived from the cross of LA1589× vg,we suggest that leaf variegated phenotype is a recessive nuclear gene controlled.4.We mapped vg on chromosome 7 with 45 recessive individuals and two In Del markers in each of the 12 chromosome.Further,15 In Del markers were developed,and the vg gene is located at a genetic distance of 756 kb between 7-59980 and 7-60636.A total of 182 individuals with mutant phenotypes were isolated and used for fine mapping.The vg gene was mapped on chromosome 7 between s6027 and Ba6040 and the genetic distance is about 128 kb according to the tomato genome sequence by using 1 In Del and 8 CAPS molecular markers.5.We got 21 candidate ORFs in this region through prediction software and gene analysis.ORF10,ORF11,ORF12 and ORF13 were associated with chloroplast development or chlorophyll biosynthesis related genes.It has been shown that homologous genes of ORF10 is related to thylakoid formation in Arabidopsis Formation.The coding sequences of 4 genes and promoter sequence of ORF10 were amplified in the mutant and AC,and there were no differences in the nucleotide sequence.6.The expression of 12 genes in AC and mutants was analyzed by qRT-PCR.The result was obvious.It showed that the expression levels of ORF9,ORRF10 and ORF13 decreased significantly.And on the contrary,the expression of ORF11 increased significantly.Therefore,it is speculated that ORF10 may be the target gene that leads to the mutant phenotype.7.The pTRV2 virus expression vector of ORF10 and ORF13 was constructed by VIGS technique,and the receptor used AC.The results showed that plant with ORF13 silenced has no phenotype alteration on leaves.And the leaves of plant with ORF10 silenced showed bleaching phenotype similar to the mutant.Thus,ORF10 is the target gene that leads to the mutant phenotype.