Research On Molecular Mechanism Of Defective Coloration In Tomato Mutant Eg During Fruit Ripening By BSA And RNA-seq

Tomato（Solanum lycopersicum Mill）is a respiratory-climacteric fruit.In the ripening process,degradation of chlorophyll and synthesis of lycopene result in the fruit color changing from green to red.The mechanisms of chlorophyll degradation and lycopene synthesis pathway in tomato fruit have been extensively studied,but their regulation mechanisms are still unclear.In this paper,Solanum lycopersicum,cv.Ailsa Craig（AC）tomato fruit,a red-ripe cultivar,and ever green（eg）fruit,a green-ripe mutant,were used as materials to study the molecular mechanism of the defective coloration in tomato during fruit ripening by BSA and RNA-seq.The results are as follows:1.Ripening of the wild-type AC and mutant eg tomato fruits were initiated by exogenous ethylene.After 9 days of ethylene treatment,fruit firmness and soluble solid content showed that AC and eg fruits ripened normally.AC fruit became red,along with degradation of chlorophyll and synthesis of lycopene during the ripening process.Besides,the chloroplast of AC fruit transformed into chromoplast.However,the fruit of eg remained green,and chlorophyll degradation and lycopene synthesis were blocked.Besides,eg fruit could not form chromoplast.The results indicate that the mutant eg is a C-type stay-green mutant.2.Wild-type AC（male parent）was hybridized to the mutant eg（female parent）,to construct the F₂segregating population.The F₂generation group presented four fruit types:red fruit,red-green fruit,yellow fruit and green fruit,with the character separation ratio about 9:3:3:1,indicating that tomato mutant eg is a two-genes recessive mutant.The chlorophyll degradation pathway was blocked in red-green fruit and green fruit.The chlorophyll degradation pathway was normal in red fruit and yellow fruit.Besides,the lycopene synthesis pathway was normal and the chloroplast was transformed into chromoplast in red fruit and red-green fruit.The lycopene synthesis pathway was blocked and the chromoplast could not form in yellow fruit and green fruit.3.After the Bulked Segregant Analysis（BSA）sequencing,mutant eg showed extremely significant peaks on chromosome 3 and chromosome 8,indicating that eg was a two-sites mutation,which was consistent with the trait separation（9:3:3:1）of F₂generation population.4.Through the analysis of candidate genes,there was a gene associated with chlorophyll degradation in the chromosome 8 interval,namely stay-green 1（SGR1）.This gene had a SNP mutation site,at which the second base at the 5’end of intron 3 was mutated from T to C,resulting in a change in the splice site of intron 3.Together with RNA-seq sequencing,it was found that the transcript of SGR1 gene had been changed.A sequence of 32 bp containing the termination codon TAA was inserted between exon 3 and exon 4,leading to early termination of its encoded protein.Validation of the SNP site of the SGR1 gene was performed using 50 red-green fruit plants and 35 green fruit plants in the F₂generation population,yet all of these 85 recessive plants mutated from T to C at the SNP site,which proved that SGR1 was the target gene in chromosome 8 and the gene controlled the chlorophyll degradation in tomato eg fruit.5.5 pairs of polymorphic In Del markers in chromosome 3 were successfully developed for interval localization of chromosome 3 candidate genes.Finally,the candidate interval of chromosome 3 was reduced to within 8 Mb.6.After analysis of the differentially expressed genes during fruit ripening by RNA-seq,compared with AC fruit,the gene expression levels of Solyc01g088090（PPH）,Solyc08g080090（SGR1）,Solyc03g031860（PSY1）,Solyc02g081330（PSY2）,Solyc06g074240（CYC-B）,Solyc02g081170（CHRC）,Solyc03g093830（OR）,Solyc03g082420（HSP21-1）and Solyc05g014280（HSP21-2）were lower in eg fruit during the ripening process.In addition,related genes of the light-harvesting complexes（LHCI and LHCII）and the light system（PSI and PSII）in AC and eg tomato fruits were all down regulated,but the expression levels of these genes were lower in AC fruit.In summary,eg is a two-sites mutation.During fruit ripening,chlorophyll degradation was blocked in mutant eg due to mutation of SGR1 gene in chromosome 8.Mutant eg could not form chromoplasts due to the mutant gene in chromosome 3.It was speculated that lycopene synthesis and the formation of chromoplast which was formed as a storage structure,were both blocked,resulting in eg fruit failing in synthesizing lycopene during the ripening process.