Analysis Of Pepper Fruit Color Formation Mechanism Based On Transcriptomics And Targeted Metabolomics

Fruit colour,as one of the essential characteristics of pepper,was widely concerned by breeders and consumers.Bright colours not only increase the commercial value of the fruit,but also serve as an essential indicator for fruit ripeness.More and more studies show that chlorophyll,flavonoids and carotenoids are closely related to the formation of pepper fruit colour.Therefore,the analysis of molecular mechanisms related to the synthesis of chlorophyll,flavonoids,and carotenoids is of great significance for improving the brightness of pepper fruits and increasing the economic value of pepper.In the present study,genes related to chlorophyll and carotenoid synthesis were mapped using bulked segregant analysis（BSA）combined with RNA sequencing（BSR-seq）.Furthermore,four pepper varieties with different fruit colours were identified as research materials,and the synthesis and regulation mechanism of flavonoids and carotenoids in pepper fruits were explored based on the targeted metabolome and transcriptome sequencing technology.The main content and results of this study are as follows:（1）Fine mapping and identifying candidate genes involved in stay-green of pepper fruit.In this study,a pepper mutant with stay-green fruit named TNX348was classified from our germplasm resource bank,and the genetic analysis of TNX348 indicated that a single recessive nuclear gene controlled its stay-green trait.The F₂population constructed with TNX348 was utilized for bulked segregant analysis combined with RNA sequencing and linkage analyses.The causal gene of the stay-green character was mapped to an approximately 131-kb region between the two markers of Caps6649159 and Indel6780656.The sequencing analysis of genes in the candidate region revealed that there was a single base mutation in the coding region of a senescence-induced chloroplast protein gene Ca SGR1（Capana01g000359）in the mutant,and the SNP was co-separated with the stay-green phenotype in the F₂population.Furthermore,the expression of Ca SGR1 in TNX348 was significantly inhibited during fruit development.Therefore,Ca SGR1 was identified as a candidate gene for controlling stay-green phenotype.（2）Expression pattern analysis of gene in TNX348 and HJ10-1 fruits during fruit development.To analyze the degradation and regulation mechanism of chlorophyll in pepper fruits,the transcriptome of TNX348 and HJ10-1 were sequenced at green maturity,bresker,and physiological maturity stages.2295,6728 and 4506 differentially expressed genes between TNX348 and HJ10-1 were identified in the three fruit development stages,respectively.Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analyses showed that differentially expressed genes were significantly enriched in chlorophyll metabolism and plant hormone signal transduction pathways,indicating that structural genes related to chlorophyll metabolism and plant hormone signaling factors are involved in chlorophyll degradation.The expression pattern of hormone signaling factors showed that 11 hormone signaling factors such as ABI（ABA insensitive）,ABF（ABA responsive element-binding factor）,and NAC transcription factors have the same or opposite expression patterns as chlorophyll metabolism structural genes such as Ca SGR1.It indicates that ABI,ABF,and NAC hormone signaling factors may be involved in chlorophyll degradation by regulating the expression of Ca SGR1.（3）Genetic Analysis and Mapping of the causal gene of red fruit trait in pepper.To map the causal gene of red fruit trait,an F₂separation population of fruit colour was constructed.The genetic analysis indicated that a single dominant nuclear gene controlled red fruit trait.The causal gene of red fruit trait was mapped to an approximately 739-kb region between the two markers of Indel9224993 and Caps9964566.There is a capsanthin synthase（CCS）gene of the carotenoid metabolic pathway in the candidate region.Sequencing analysis revealed that CCS gene was deleted,and no capsanthin accumulated in the yellow fruit inbred line TLIUS2F-1.Therefore,CCS was identified as a candidate gene for controlling red fruit phenotype.（4）Accumulation of flavonoids and expression analysis of genes related to flavonoid synthesis in the fruits of 4 different colour pepper varieties.Qualitative and quantitative analysis of flavonoids was performed in fruits of 4 different colour pepper varieties using LC-MS technology.188 kinds of flavonoids were detected in the fruits of four pepper varieties at two fruit development stages.Among them,the content of anthocyanins,flavonols,and flavonoids in purple varieties CJ12-17-1 were significantly higher than that in other types.The expression pattern of flavonoid synthesis-related genes indicates that the high expression of PAL（phenylalanine lyase）,C4H（cinnamic acid hydroxylase）,4CL（coumadin Co A ligase）in the phenylpropane synthesis pathway and DFR（dihydroflavonol 4-reductase）,UGT（UDP-glucosyltransferase）,and GST2（glutathione S-transferase）in the flavonoid metabolic pathway is responsible for the high accumulation of anthocyanins,flavonols,and flavones in the fruit of CJ12-17-1.（5）Integrative analysis of metabolome and transcriptome in pepper fruit.To obtain the regulatory network of flavonoid synthesis in pepper fruit,a correlation analysis between differentially expressed genes and differentially accumulated flavonoids was performed.The 4308 differentially expressed genes are highly correlated with the flavonoid accumulation in two fruit developmental stages.Based on the weighted gene co-expression network analysis（WGCNA）of 4308differentially expressed genes,the two modules were determined to be positively correlated with flavonoid synthesis.In these two modules,26 and 23 genes,such as Capana10g001281（SLC40）,Capana10g001433（MYB113）and Capana03g001813（WDR68）,were predicted to encode transporters and transcription factors,respectively.Moreover,these transcription proteins and transcription factors are in the core position of the regulatory network,which indicates that these transporter and transcription factors may regulate the synthesis and transport of flavonoids.（6）Accumulation of carotenoids and expression analysis of genes related to carotenoid synthesis in the fruits of 4 different color pepper varieties.To reveal the synthesis and regulation mechanism of carotenoids in pepper fruits,quantitative analysis of carotenoids from four different fruit colour pepper varieties was performed using LC-MS technology.The analysis results showed that the carotenoid accumulation showed significant colour specificity at the physiological maturity stage.Yellow or orange carotenoids such asα-carotene,lutein,and violaxanthin accumulate significantly in yellow and orange fruits,while red capsanthin is highly expressed in red fruits.The expression pattern of carotenoid structural genes showed that lycopeneε-cyclase（LCYE）and lycopeneβ-cyclase 1（LCYB1）were highly expressed in yellow material.In contrast,lycopeneβ-cyclase 2（LYCB2）had higher transcriptional abundance in red material.It indicated that（LCYE and LCYB1）and LYCB2 mediate the flux into theβ,εcarotene branch andβ,βcarotene branch,respectively,which ultimately affects the accumulation of carotenoids such asα-carotene,lutein and capsaicin.Moreover,CCS genes were significantly up-regulated in the four pepper varieties during fruit development,but capsanthin was only detected in the red fruit,indicating that the accumulation of capsanthin is not exclusively dependent on the expression of the CCS gene,but may also be related to the activity of CCS enzymes after translation.