Assessment of Gene Polymorphism in Terpene Synthase among Local Variety of Pineapple through Gene Sequence Analysis

Pineapple or Ananas comosus is an important horticultural industry throughout the area of the tropical and subtropical. Studies on pineapple compounds revealed more than 280 aroma compounds found the nature of pineapple flavors. Pineapple is widely consumed as fresh and canned fruit as processed juices and also use as ingredient in food due to its unique sweet flavor. In pineapple, the most abundant aroma volatiles that contributes to its flavour of pineapple are mainly from ester and terpene. Terpene synthase is an enzyme that responsible for the synthesis of sesquiterpenoid in aromatic plant. Identification of numerous variation in genes and analysis of their effect may lead to a better understanding of their impact on the gene function. In this study, optimization of extraction method using two different extraction methods produced different yield of DNA. SDS method produced higher amount of DNA yield as compared to the CTAB method. PCR amplification by using primer for Exon 2 and genomic DNA extracted using SDS method produced all specific single band as expected. While genomic DNA extracted using CTAB method failed PCR amplification for the same primer for two pineapple varieties, despite of its apparent band in gel electrophoresis. A total 15 bands were amplified using primer Exon 2, Exon 3 and Exon 6 for all five pineapple varieties. The bands amplified were thick and intense which indicates its integrity and specificity of the primer. Effort to sequences all fifteen amplification products were failed due to problem during transportation that cause DNA to be degraded. Only two samples Moris for Exon 2 and Sarawak for Exon 3 were successfully sequenced after troubleshooting. These sequences were compared with the reference terpene synthase gene, with respect to its exon, individually. Gene sequence analysis revealed low polymorphism for Exon 2 in Moris but high single sequence polymorphism for Sarawak Exon 3. Several single base variations detected caused non-synonymous mutation at the protein level that indicates their potentially significant change at protein level. Nevertheless, these variations detected required further investigation and characterization for its significance to cause major variation at the functional level of the protein terpene synthase