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Pun1 gene has an important role in determining the pungency in chilli pepper. Induction of mutation in chilli pepper using Ethyl Methane Sulfonate (EMS) may produce variation in the first exon of the Pun1 gene and associate with pungency level. In this study, variations were analysed based on the partial sequence of Pun1 gene (exon-1) and capsaicinoid content of chilli pepper mutants. The genomic DNA of the Ethyl Methane Sulfonate (EMS) mutant plants, i.e. 3 local chilli peppers genotype 2 (G2), genotype 7 (G7), and genotype 11 (G11) were isolated from the young leaf by using CTAB method, and then amplified with a pair of Pun1 primers using Polymerase Chain Reaction (PCR) technique. Chilli pepper fruits harvested at 20 Days Post Anthesis (DPA) were used for extraction and analysed for total capsaicinoid using spectrophotometer with the wavelength of 280 nm. The presence of Single Nucleotide Polymorphism (SNP) in chilli peppers genotype 7 induced by EMS 0.01% (G7K1) and genotype 11 induced by EMS 0.01% (G11K1) and 0.04% (G11K3). The SNP was identified as substitution resulted in a change from cytosine (C) to thymin (T) and produced similar amino acid (silent mutation). The total capsaicinoid content in chilli pepper mutants was lower than the control, except in chilli pepper genotype 2 induced by EMS 0.01% (G2K1). There is no correlation between the polymorphism identified in the first exon of Pun1 gene and the total capsaicinoid content, which might be due to the possibility that EMS influence to another gene in capsaicinoid biosynthesis.
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