IDENTIFICATION AND EXPRESSION ANALYSIS OF GENES INVOLVED IN VITAMINS BIOSYNTHESIS AND FRUIT RIPENING IN CAPSICUM SPECIES

Abstract

Chili peppers (Capsicum spp., family Solanaceae) are among the world‘s most economically important vegetable crops, valued for their rich nutritional and medicinal properties. These diploid plants (2n = 24; ~3.5 Gb genome) are believed to have originated in tropical Central and South America around 7500 BCE. Of the more than 38 reported species, five are widely cultivated: C. annuum, C. chinense, C. frutescens, C. baccatum, and C. pubescens. India, the world‘s leading producer (1.7 million tons annually; Ministry of Agriculture & Farmers' Welfare, 2022; FAO, 2022; USDA FAS, 2023), harbors remarkable genetic diversity, particularly in the Northeast, which is home to unique landraces such as the fiery hot Bhut jolokia (C. chinense). The genus is a biochemical powerhouse, rich in vitamins (both water- and fat-soluble), antioxidants (carotenoids, flavonoids, polyphenols), and capsaicinoids, which collectively contribute to numerous health benefits including anti-cancer, anti-microbial, and anti-inflammatory effects, as well as industrial applications such as food coloring and pest control. Although closely related to tomato- a well-established model for fruit crops the molecular mechanisms underlying important traits in Capsicum, such as fruit development, ripening, and the biosynthesis of essential vitamins (e.g., ascorbate and tocopherol), remain poorly understood. This knowledge gap has hindered targeted breeding efforts to improve desired fruit traits, quality and nutritional value in Capsicum. This study employed an integrative genomics approach to address these gaps. Orthology- based mining identified key genes involved in fruit development, ripening, and vitamin biosynthesis (Vitamin C and E) pathways in C. annuum, C. baccatum, and C. chinense genomes. Their expression profiles were analyzed using RNA-Seq data and quantitative real- time PCR (qRT-PCR), and correlated with phenotypic data, including vitamin quantification by High-Performance Liquid Chromatography HPLC at three distinct fruit developmental stages in contrasting genotypes. Furthermore, a set of functional molecular markers- Insertions/Deletions (InDels) and Simple Sequence Repeats (SSRs) were developed from the iv candidate genes associated with fruit development, ripening, and Vitamin C and E biosynthesis. Our investigation yielded several important findings. We identified 32 orthologs associated with fruit development and ripening, of which 12 showed significant differential expressions, further validated by qRT-PCR, six out 12 genes e.g., MADS- RIN, SGR1, ETR4, LeSPL, XTH5, MADS-protein1) shows almost similar expression in both qRT and transcriptome-MADS-RIN gene showed consistent, extreme upregulation (>500-fold) in ripening fruit across all species. SGR1 displayed consistently high expression in fruit, with the highest levels in C. frutescens in both datasets. ETR4, LeSPL, XTH5, MADS-protein1- Both methods captured the same pattern of higher expression in early stages (flower, Early fruit, breaker) and a decline upon maturity. Out of above some genes found with opposite/discordant expression, 2 genes GLK2 and hydroquinone glucosyltransferase, and NSGT1 significant differences in both qRT and transcriptome- GLK2 qRT-PCR showed upregulation in C. annuum but downregulation in others, while the transcriptome suggested downregulation in all three species. Hydroquinone glucosyltransferase showed opposing regulatory trends between the two methods. NSGT1 expression patterns differed significantly between the platforms. While in TAGL1, FUL1, and FUL2, both methods agreed on high expression, they showed minor differences in the precise level and tissue-specificity of expression. Significantly, the MADS-RIN ortholog (LOC107847473) exhibited a striking >500-fold upregulation in Mature fruit, underscoring its central regulatory role. Expression analyses of ethylene receptors revealed species-specific ripening behaviors, with C. frutescens displaying climacteric-like characteristics. These results demonstrate strong concordance between transcriptome profiling and qRT-PCR validation across developmental stages. Analysis of vitamins content across different fruit developmental stages in contrasting genotypes revealed the highest accumulation of Vitamin C in Capsicum species, in C. chinense, as in Cc-9 (38.41 mg/g) in C. frutescens as in Cf-7 (29.19 mg/g), and in C. annuum in Ca-8 (41.77 mg/g). while highest Vitamin E levels were recorded in C. v chinense in Cc-10 (109.39 mg/g), in C. frutescens in Cf-9 (112.44 mg/g), and in C. annuum in Ca-5 (111.43 mg/g). On average, C. chinense exhibited the highest Vitamin C content across stages, (25.33 mg/g), followed by C. annuum (24.25 mg/g) and C. frutescens (22.09 mg/g). Genotypes such as from C. chinense (Cc-1,Cc-2, Cc-3, Cc-5, Cc-6 and Cc-9), from C. annuum (Ca-3, Ca-4,Ca-7 and Ca-8) and from C. frutescens (Cf-4, Cf-5, Cf-7 and Cf-8) for Vitamin C, and genotypes from C. chinense (Cc-2, Cc-3, Cc-9, and Cc-10), from C. annuum (Ca-1, Ca-2 ,Ca-f and Ca-6) and from C. frutescens (Cf-2, Cf-4, Cf-7, Cf-9 and Cf-10) for Vitamin E, represent valuable genetic resources for breeding programs aimed at developing Capsicum varieties enriched in Vitamins C and E content. A comprehensive genome-wide analysis identified 29 and 81, 44 and 85, and 36 and 70 putative genes involved in Vitamin C and Vitamin E biosynthesis/degradation in the genomes of C. annuum, C. baccatum, and C. chinense, respectively. Among these, several genes associated with Vitamins C and E were found to be differentially expressed in RNA-Seq data generated from three fruit developmental stages of contrasting vitamins-rich species (C. annuum, C. chinense, and C. frutescens), including key regulators for Vitamin C such as GMP downregulated during fruit development in all species, GME shows same expression pattern in both qRT and transcriptome, AKR2 & GPI: both downregulated in all three species, GGP expression higher in C. annuum (qRT) vs higher in Early fruit (transcriptome), MIOX shows high in C. chinense (qRT) vs low in all species (transcriptome), AKR38 high in C. annuum (qRT) vs no expression in transcriptome, GPP upregulated in maturity in qRT and downregulated transcriptome, GalDH higher expression in qRT and very low expression in transcriptome and PMI gene expression higher in C. frutescens qRT and in transcriptome higher in C. annuum. Similarly for Vitamin E key regulators genes HPPD & IPI upregulated during fruit development in both qRT and transcriptome, VTE3 & VTE4: Nearly identical expression patterns in both methods, TAT shows species-specific expression in qRT and opposite pattern in transcriptome, TYRA expression high in C. frutescens qRT and low/no detection in transcriptome, VTE1 found in qRT and low/no expression in transcriptome, vi VTE2 expressed in all samples in qRT vs minimal expression transcriptome and VTE5 gene expressed across tissues in qRT vs no expression in transcriptome. qRT-PCR validation confirmed the transcriptome-based expression patterns in Vitamin C with Similar expression 4 genes were found (GMP, GME, AKR2, GPI, AKR1) in both qRT and transcriptome and Opposite/Discordant expression in 6 genes (GGP, MIOX, AKR38, GPP, GalDH, PMI) and in Vitamin E with similar expression 5 genes (GGDR, HPPD, IPI, VTE3, VTE4) and in opposite/discordant expression 5 genes (TAT, TYRA, VTE1, VTE2, VTE5) was found in both qRT and transcriptome. Correlation analysis between gene expression and fruit vitamin content revealed significant associations, suggesting that these genes represent strong candidates for functional validation through approaches such as overexpression or genome editing. To facilitate future genetic and breeding studies, we developed 49 gene-based SSR markers associated with fruit development and ripening traits, along with 24 SSR and 37 InDel markers linked to vitamin traits. These markers, after validation, could be deployed in breeding programs targeting improved fruit quality and nutritional content. This is the first comprehensive study to report genome-wide identification and expression profiling of genes involved in fruit development, ripening, and vitamin biosynthesis in Capsicum. The identified candidate genes, together with the novel SSR and InDel markers, represent valuable genomic resources that can accelerate molecular breeding of biofortified Capsicum varieties with enhanced vitamin content. These findings provide a foundational framework for researchers working on fruit crop improvement, particularly in the molecular dissection and manipulation of key nutritional and fruit traits.