The Late-Ripening Mechanism Of A Jincheng Sweet Orange Mutant And The Identification Of Related Genes

Fruit ripening is a complex process that involves sugar and acid metabolism, cell wall metabolism, pigment metabolism, energy metabolism and so on. So far, the knowledge of the mechanism of fruit ripening is not sufficient, especially that of non-climacteric fruit as citrus. As a result, it is very important to research on the ripening mechanism in citrus in order to breed new citrus cultivars with earlier or later ripening time, or to change the ripening time by cultivating method in a targeted manner. The late-ripening mutant of ‘Jincheng’(C. sinensis L. Osbeck) sweet orange offers the best material for us to study the regulation of citrus ripening. In this research, we got a more comprehensive understanding of the fruit ripening process of Jincheng, and identified several ripening-related metabolic pathways and key genes, by comparative analysis of the differences between Jincheng and its mutant on physiology, biochemics, molecular and signal pathway during different maturity periods. The main results are as follows:1.Fruit Quality: During the fruit ripening process of Jincheng, the content of total soluble solids, sucrose, fructose, glucose, vitamin C, single fruit weight and the color index of fruit peel are all on a rise until the fruit is fully ripe; however, the content of the total titratable acidity, citric acid, quininic acid decrease all the way; the content of citric acid is apparently higher in the mutant than in the wild type; what’s special is that the content of the malic acid increases gradually from the beginning of fruit color change to the fruit fully ripe and then drops quickly.2.Endogenous Hormone: During the fruit ripening process of Jincheng, the content of abscisic acid(ABA) in fruit is the highest among the hormones we detected. The changing trend of the ABA content looks like a “M”, with two peaks at the beginning of fruit color changing and at fruit fully ripe. The content of jasmonic acid(JA) in fruit is lower than that of ABA and decreases gradually during fruit ripening. However, thecontent of JA in fruit is significantly higher in the mutant than the wild type during the early ripening stage. The content of auxin in fruit is lowest. It decreases gradually at first until the fruit is fully ripe and then increase slightly.3.Gene Expression: We focus on the analysis of gene expression involved in the sucrose, ABA, JA metabolism and signal transduction pathway during different fruit developmental and ripening stages. Cs SPS and Cs SUS are the key genes in sucrose synthesis pathway. Cs SPS function in the early stage of fruit ripening and lead to the sucrose accumulation after fruit pigmentation. Cs SUS function in the late stage of fruit ripening and is related to the conversion of glucose and fructose in fruit. The expression of most of the genes in the ABA signal transduction pathway increased during fruit ripening process, which suggests that ABA is a positive regulator in the fruit ripening process of sweet orange. The expression of genes in the JA synthesis pathway is not correspond to the JA content in the fruit, which shows the JA synthesis in Jincheng is affected by other factors. However, the expression of the genes in the JA signal transduction pathway increases in the late ripening stages, which proves that JA signal transduction related genes are involved in the fruit ripening regulation. Besides, many genes from the three pathways have similar expression pattern.4.Transcriptome: The overall transcriptome differences between Jincheng the wild type and the mutant at the fruit pigmentaion. 13,412 differentially expressed unigenes(DEG) were identified. Most of the DEGs are clustered into five pathways, metabolic pathways, plant-pathogen interaction, spliceosome, biosynthesis of plant hormones and biosynthesis of phenylpropanoids. Most of the transcription factors are belong to four families, Myb, AP2 / ERF, MADS-box and WRKY families. And found that the more mature orange fruit is, the lower the transcription levels is.5.SSR distribution in Jincheng transcriptome: Eleven percent of the all unigene detected contain SSR loci. The main repeat types are Mono-type, Tri-type and Di-type.And the main repeat motives are A/T, AG/CT and AAG/CTT.6.Development of the SSR markers: 52 pairs of the SSR primers are designed and synthesized according to the differential expressed genes containing SSR loci and with a5’-3’ direction. 33 pairs have clear products in orange, ponkan and tangerines, indicatingthat these SSR primers have transferability among related species. 18 pairs have polymorphic products among 16 citrus varieties with different ripening time. A TCP family member has the highest PIC value. And we also find that this gene has different expression levels during different ripening stages in citrus. The analysis of cis-acting element prediction shows that there are many plant hormone regulatory elements in the promoter region of this gene.In summary, the late-ripenign mutation in Jincheng causes many changes in the expression of the genes involved in the metabolic pathways, and many changes in the content of the metabolic products. This proves that the fruit ripening process is a complex process, and that this mutant related gene is an upstream gene or a node gene in the regulatory network. Although it is difficult to find the key gene, we identify many fruit ripening-related pathways and genes, which provide new basis and foundation for the study of fruit ripening. Meanwhile, the newly developed molecular markers provide new tools for the establishment of citrus genetic map, the analysis of citrus phylogenetic relationship, and the molecular marker assisted breeding in citrus.