Deciphering the Fine Anthocyanin Regulation in Eggplant: a multi –omics approach

Anthocyanins are responsible for the purple pigmentation of eggplant fruits. Structural genes and major transcription factors controlling their biosynthesis are well characterized. However, the molecular mechanisms underlying the specific loss of pigmentation in the fruit peel remain poorly understood. This thesis investigated the regulation of anthocyanin biosynthesis in the purple-fruited eggplant cultivar Black Beauty (BB) and its naturally occurring green-fruited mutant Green Beauty (GB) through genomic, transcriptomic, metabolomic, and functional genomics approaches, aiming to identify candidate genes and regulatory mechanisms responsible for fruit color variation. Chapter I: Genomics. Genomic analysis was performed using the Bulk Segregant analysis and by a high-quality genome assemblies generated from PacBio HiFi sequencing. Comparative analysis revealed extensive collinearity between BB and GB and no sequence differences in the known structural genes or major transcriptional regulators of the anthocyanin pathway. However, two major genomic alterations were identified in GB: a deletion affecting SUVH5, a histone H3 lysine-9 methyltransferase potentially involved in epigenetic regulation, and a homozygous deletion in the promoter region of MYB1, the principal regulator of anthocyanin biosynthesis in eggplant fruits. Chapter II: Transcriptomics and Metabolomics. Transcriptomic and metabolomic analyses were conducted to investigate gene expression, alternative splicing, long non-coding RNAs (lncRNAs), and anthocyanin profiles in flowers and fruits. In BB, the activation of structural and regulatory genes was confirmed, and several lncRNAs associated with anthocyanin biosynthesis were identified, alongside alternative splicing events in genes directly or indirectly involved in the pathway. These findings supported a potential regulatory model involving lncRNAs and alternative splicing of MYB75, a major floral activator, and Ethylene Insensitive3-Like (EIL), a regulator associated with ethylene biosynthesis. Metabolomic profiles also suggested that different copies of biosynthetic genes may contribute to anthocyanin diversity during fruit development. In contrast, GB fruits showed a marked reduction in anthocyanin content and complete absence of MYB1 expression, accompanied by suppression of downstream biosynthetic genes. Weighted gene co-expression network analysis (WGCNA) identified a fruit-associated module enriched in lncRNAs, including two transcripts strongly and positively correlated with both MYB1 expression and anthocyanin accumulation. Chapter III: Functional Genomics. Functional validation was performed availing of the CRISPR/Cas9-mediated genome editing to generate knockout lines for SUVH5 and the two candidate lncRNAs. The preliminary results indicated that SUVH5 does not appear to play a major role in anthocyanin biosynthesis. Conversely, phenotypic observations in T0 lines suggested a positive association between the two lncRNAs and pigment accumulation, although further validation is required. These findings provide new insights into the transcriptional and post-transcriptional mechanisms underlying fruit peel pigmentation in eggplant and establish a foundation for further functional characterization of its regulatory network