Establishment of a reliable protocol for gDNA extraction from olive oil.

Abstract

With no possibility to reach in quantity the production of countries with large areas of olive orchards, as a small country, Portugal needs to define a strategy for valuing the high quality and specificities of its olive oil. No doubt the focus must be on the valorization of the Portuguese cultivars, the key factor in determining the singularity of the produced olive oils. Fraud detection, as the use of non-Portuguese varieties, is the main aim of varietal and DOP olive oil producers. In this sense, it is mandatory to have tools allowing the control of the varietie(s) giving rise to the olive oil, both in quality and in quantity. One objective of the project Por3O running at University of Évora is the establishment of a molecular tool that identifies the varieties used to produce a given olive oil. Ideally, this tool could be further proposed for screening for frauds and to support olive oil certification. However, as PCR-based tool, it requires the availability of genomic DNA (gDNA) with quality enough to be used on fragment amplification. The step related with extraction of high quality gDNA from olive oil samples, which considers gDNA integrity and absence of inhibitory compounds, is often a limiting step in the development of a PCR-based genotyping tools. The establishment of a robust and efficient extraction protocol is thus crucial. In theory, the use of DNA commercial kits has the advantage of a higher reproductivity greatly removing technician expertise biases. Several DNA commercial kits were tested on its capacity to extract gDNA from a commercial blend olive oil and they were compared with an in-house method based on CTAB-based protocol previously published [1]. The different methods were compared in terms of starting volume of oil sample required for extraction, average gDNA concentration, total gDNA extraction yield and efficiency in Single-Sequence Repeats (SSRs) markers amplification. Considering the results achieved, we propose a workflow regarding gDNA extraction to further molecular analysis.