MSc

Byadagi chilli is famous for its deep red colour and negligible or zero pungency. Demand for Byadagi chilli has increased enormously as a source of natural red colour in food industry, confectionaries, cosmetics, beverages, pharmaceuticals and even as a dye in textile industries. Byadagi chilli is mainly exported as oleoresin which serves as a substitute for paprika oleoresin.The red color of chilli fruits is due to several related carotenoid pigments. The most important pigments are capsanthin and its isomer capsorubin.
The present study was conducted to analyze Capsanthin-capsorubin synthase gene (Ccs) in Byadagi chilli and to elucidate the carotenoid metabolic pathway for production of capsanthin and capsorubin. The studies were focused on seven genetically distinct chilli varieties /accessions of three different Capsicum spp. based on colour at fully ripe fruit stage. The accessions selected were ByadagiKaddi, Byadagi Dabbi, Ujwala, Anugraha (Capsicum annuum), Vellayani Samrudhi (Capsicum frutescens), Vellayani Thejus and CC8-1 (Capsicum chinense).
Genomic DNA was isolated from tender leaves of one month old plants by CTAB method. Two chilli Ccs gene specific SSR primers viz. Ccs Cds and Ccs promoter were used to amplify the Ccs gene.The amplified PCR products obtained with Ccs Cds and Ccs promoter were sequenced by outsourcing and sequence data analyzed using bioinformatics tools.
The Ccs gene was found amplified in all the genotypes including the yellow fruitedaccession CC8-1. Size of amplified product was 1.5kb with Ccs cds primer in all the genotypes. For Ccs promoter, amplified product was920bp inByadagiKaddi, Byadagi Dabbi, Ujwala, Anugraha, Vellayani Samrudhi and 1200bp in Vellayani Thejus and CC8-1
BLASTn analysis of the Ccs gene amplified with Ccs cds primer showed 99 -100 per cent similarity with the reference nucleotide sequence in all the genotypes. BLASTx analysis of Ccs gene sequence amplified with Ccs cds primer showed 99-100 per cent similarity with the reference amino acid sequence in the seven genotypes studied. Analysis of conserved domains revealed that lycopene beta cyclase was the conserved domain in Capsicum annuum and C. chinense genotypes while in C. frutescens NADB super family protein was the conserved domain. The number of ORFs in the Ccs sequence amplified with Ccs cds primer ranged from six to seven in the genotypes studied and the number of amino acids coded ranged from 463-469 in C. annuum, 298 in C. frutescens and 217 in C. chinense.
Multiple sequence alignment of the sequences revealed SNP variations in the genotypes studied and SNP variation caused change in amino acid coded. SNP variations were observed in five genotypes viz. Byadagi Kaddi, Byadagi Dabbi, Vellayani Samrudhi, Vellayani Thejus and CC8-1 while no SNP variations were seen in the varities Ujwala and Anugraha
Byadgi kaddi had two SNPs leading to change in amino acids at 43rd and 425th position of Capsanthin capsorubin synthase peptide. Tyrosine (Y) was found replaced by Phenyl alanine (F) in the 43rd position and Lysine (K) was found replaced by Glutamic acid (E) in the 425th position. Byadagi dabbi also had the same amino acid change at 425th position, Lysine (K) was replaced by Glutamic acid (E). PrematureStop codon UAG was observed in yellow fruited variety CC8-1 at 200th position
BLASTn analysis of Ccs gene sequence amplified with Ccs promoter primer in seven genotypes showed 90-99 per cent similarity with the reference nucleotide sequence. Multiple sequence alignment of the promoter region could see structural changes in the sequences. Several SNPs in the sequences, a tandem repeat structure, insertion, deletions and various cis regulatory elements like heat stress related cis-elements (HSE), Myb binding site (MYBPZM) and light responsive elements, TATA box, and CAAT box could be observed in the promoter region.
Ccs gene was located in Chromosome six of Capsicum annuum and in the genome map of chilli it was seen in between 9497216 – 9500911kb. Ccs cds gene specific primer was seen to bind 18bp downstream region of the sequence. The Ccs promoter was seenupstream of the protein coding region.
Elucidation of carotenoid metabolic pathway in Capsicum annuum revealed that 17 enzymes were present in the carotenoid biosynthesis pathway. Gene regulatory network analysis, using cytoscape showed that network contained 94 nodes and many of the genes were associated with carotenoid biosynthesis processes. The main seventeen carotenoid metabolic pathway genes, some transcription factors and transferase/transport proteins were densely connected. Among the pathway genes, Phytoene synthase had the highest number (30 No.) of interactive proteins.
The identified SNPs in the present study have to be further characterized and validated, transcriptome analysis of Ccs gene in the different genotypes, homology modeling the Ccs enzyme and prediction of active sites could derive more information on the identified SNPs