Genetic Regulation, Environmental Cues, and Extraction Methods for Higher Yield of Secondary Metabolites in Capsicum.

Capsicum (chili pepper) is a widely popular and highly consumed fruit crop with beneficial secondary metabolites such as capsaicinoids, carotenoids, flavonoids, and polyphenols, among others. Interestingly, the secondary metabolite profile is a dynamic function of biosynthetic enzymes, regulatory transcription factors, developmental stage, abiotic and biotic environment, and extraction methods. We propose active manipulable genetic, environmental, and extraction controls for the modulation of quality and quantity of desired secondary metabolites in Capsicum species. Specific biosynthetic genes such as Pun (AT3) and AMT in the capsaicinoids pathway and PSY, LCY, and CCS in the carotenoid pathway can be genetically engineered for enhanced production of capsaicinoids and carotenoids, respectively. Generally, secondary metabolites increase with the ripening of the fruit; however, transcriptional regulators such as MYB, bHLH, and ERF control the extent of accumulation in specific tissues. The precise tuning of biotic and abiotic factors such as light, temperature, and chemical elicitors can maximize the accumulation and retention of secondary metabolites in pre- and postharvest settings. Finally, optimized extraction methods such as ultrasonication and supercritical fluid method can lead to a higher yield of secondary metabolites. Together, the integrated understanding of the genetic regulation of biosynthesis, elicitation treatments, and optimization of extraction methods can maximize the industrial production of secondary metabolites in Capsicum.

Integrated analysis of DNA methylation, transcriptome, and global metabolites in interspecific heterotic Capsicum F1 hybrid.

Hybrid breeding is one of the efficacious methods of crop improvement. Here, we report our work towards understanding the molecular basis of F1 hybrid heterosis from Capsicum chinense and C. frutescens cross. Bisulfite sequencing identified a total of 70597 CG, 108797 CHG, and 38418 CHH differentially methylated regions (DMRs) across F1 hybrid and parents, and of these, 4891 DMRs showed higher methylation in F1 compared to the mid-parental methylation values (MPMV). Transcriptome analysis showed higher expression of 46-55% differentially expressed genes (DE-Gs) in the F1 hybrid. The qRT-PCR analysis of 24 DE-Gs with negative promoter methylation revealed 91.66% expression similarity with the transcriptome data. A few metabolites and 65-72% enriched genes in metabolite biosynthetic pathways showed overall increased expression in the F1 hybrid compared to parents. These findings, taken together, provided insights into the integrated role of DNA methylation, and genes and metabolites expression in the manifestation of heterosis in Capsicum.

Capsicum chinense MYB Transcription Factor Genes: Identification, Expression Analysis, and Their Conservation and Diversification With Other Solanaceae Genomes.

Myeloblastosis (MYB) genes are important transcriptional regulators of plant growth, development, and secondary metabolic biosynthesis pathways, such as capsaicinoid biosynthesis in Capsicum. Although MYB genes have been identified in Capsicum annuum, no comprehensive study has been conducted on other Capsicum species. We identified a total of 251 and 240 MYB encoding genes in Capsicum chinense MYBs (CcMYBs) and Capsicum baccatum MYBs (CbMYBs). The observation of twenty tandem and 41 segmental duplication events indicated expansion of the MYB gene family in the C. chinense genome. Five CcMYB genes, i.e., CcMYB101, CcMYB46, CcMYB6, CcPHR8, and CcRVE5, and two CaMYBs, i.e., CaMYB3 and CaHHO1, were found within the previously reported capsaicinoid biosynthesis quantitative trait loci. Based on phylogenetic analysis with tomato MYB proteins, the Capsicum MYBs were classified into 24 subgroups supported by conserved amino acid motifs and gene structures. Also, a total of 241 CcMYBs were homologous with 225 C. annuum, 213 C. baccatum, 125 potato, 79 tomato, and 23 Arabidopsis MYBs. Synteny analysis showed that all 251 CcMYBs were collinear with C. annuum, C. baccatum, tomato, potato, and Arabidopsis MYBs spanning over 717 conserved syntenic segments. Using transcriptome data from three fruit developmental stages, a total of 54 CcMYBs and 81 CaMYBs showed significant differential expression patterns. Furthermore, the expression of 24 CcMYBs from the transcriptome data was validated by quantitative real-time (qRT) PCR analysis. Eight out of the 24 CcMYBs validated by the qRT-PCR were highly expressed in fiery hot C. chinense than in the lowly pungent C. annuum. Furthermore, the co-expression analysis revealed several MYB genes clustered with genes from the capsaicinoid, anthocyanin, phenylpropanoid, carotenoid, and flavonoids biosynthesis pathways, and related to determining fruit shape and size. The homology modeling of 126 R2R3 CcMYBs showed high similarity with that of the Arabidopsis R2R3 MYB domain template, suggesting their potential functional similarity at the proteome level. Furthermore, we have identified simple sequence repeat (SSR) motifs in the CcMYB genes, which could be used in Capsicum breeding programs. The functional roles of the identified CcMYBs could be studied further so that they can be manipulated for Capsicum trait improvement.

Development and characterization of non-coding RNA based simple sequence repeat markers in Capsicum species.

Plant growth and development are largely regulated by non-coding RNAs (ncRNA); thus ncRNA based markers would be rewarding in molecular breeding. In the present study, for the first time we developed total 623 ncRNA based SSRs including 119 microRNASSRs (miRNASSRs) and 504 long non-coding RNASSRs (lncRNASSRs) distributed across 12 Capsicum chromosomes. Out of 623 ncRNASSRs, 120 (including 60 each miRNASSRs and lncRNASSRs) were used for genotyping of 96 Capsicum accessions belonging to C. annuum, C. chinense and C. frutescens; and 75% SSRs were polymorphic. Model-based and distance-based cluster analyses identified three species specific clusters, i.e. cluster-I (C. annuum), cluster-II (C. frutescens) and cluster-III (C. chinense); therefore, these SSRs may have a potential role to play in interspecific Capsicum breeding. Tissue specific expression of SSR containing ncRNAs and versatile functions of their targets suggested the usefulness of SSRs for mapping of genes/QTLs and breeding of wide range of traits in Capsicum.

Identification of genes involved in fruit development/ripening in Capsicum and development of functional markers.

The molecular mechanism of the underlying genes involved in the process of fruit ripening in Capsicum (family Solanaceae) is not clearly known. In the present study, we identified orthologs of 32 fruit development/ripening genes of tomato in Capsicum, and validated their expression in fruit development stages in C. annuum, C. frutescens, and C. chinense. In silico expression analysis using transcriptome data identified a total of 12 out of 32 genes showing differential expression during different stages of fruit development in Capsicum. Real time expression identified gene LOC107847473 (ortholog of MADS-RIN) had substantially higher expression (>500 folds) in breaker and mature fruits, which suggested the non-climacteric ripening behaviour of Capsicum. However, differential expression of Ehtylene receptor 2-like (LOC107873245) gene during fruit maturity supported the climacteric behaviour of only C. frutescens (hot pepper). Furthermore, development of 49 gene based simple sequence repeat (SSR) markers would help in selection of identified genes in Capsicum breeding.