Amplification and DNA Sequencing of the Nuclear Ribosomal Internal Transcribed Spacer Region from Dried Black Pepper Samples for Traceability

Abstract

Traceability is the ability to follow the circulation of a product through all steps in the supply chain. Traceability will ensure transparency of product origin and quality. Traceability involves product records and labeling. For biological samples, in addition to the commonly used management parameters, a number of specific markers will be used to control the product, such as nucleic acid (DNA), protein, carbonhydrates, etc. Pepper is a product with economic value and is grown in many different countries around the world. Pepper products from different growing regions will have different quality and prices. Tracing the origin of pepper also faces many challenges. Therefore, in this study, appropriate conditions for DNA extraction from dried pepper were determined, including extraction buffer, sample grinding method, and total DNA precipitation conditions. Appropriate conditions to carry out amplification of the internal transcribed spacer (ITS) region were also determined, including the amount of DNA template (100 ng), primer concentration (0.3 μM), primer annealing temperature (55 oC) and number of reaction cycles (35 cycles). The optimal procedure was applied to amplify the ITS region of 19 pepper samples. The ITS region nucleotide sequences of 19 samples were determined and used to build a phylogenetic tree of pepper samples from different growing regions in Vietnam. The genetic diversity of the ITS region of Vietnamese pepper was also evaluated and compared with data originating from some countries, such as Thailand, China, and the US.