The olive tree (Olea europaea L.) is one of the most cultivated crops in the Mediterranean basin. Its economic importance is mainly due to the intense production of table olives and oil. Cultivated varieties are characterized by high morphological and genetic variability and present a large number of synonyms and homonyms. This necessitates the introduction of a rapid and accurate system for varietal identification. In the past, the recognition of olive cultivars was based solely on analysis of the morphological traits, however, these are highly influenced by environmental conditions. Therefore, over the years, several methods based on DNA analysis were developed, allowing a more accurate and reliable varietal identification. This review aims to investigate the evolving history of olive tree characterization approaches, starting from the earlier morphological methods to the latest technologies based on molecular markers, focusing on the main applications of each approach. Furthermore, we discuss the impact of the advent of next generation sequencing and the recent sequencing of the olive genome on the strategies used for the development of new molecular markers.

Olive is one of the oldest cultivated species in the Mediterranean Basin, including Tunisia, where it has a wide diversity, with more than 200 cultivars, of both wild and feral forms. Many minor cultivars are still present in marginal areas of Tunisia, where they are maintained by farmers in small local groves, but they are poorly characterized and evaluated. In order to recover this neglected germplasm, surveys were conducted in different areas, and 31 genotypes were collected, molecularly characterized with 12 nuclear microsatellite (simple sequence repeat (SSR)) markers, and compared with 26 reference cultivars present in the Tunisian National Olive collection. The analysis revealed an overall high genetic diversity of this olive’s germplasm, but also discovered the presence of synonymies and homonymies among the commercialized varieties. The structure analysis showed the presence of different gene pools in the analyzed germplasm. In particular, the marginal germplasm from Ras Jbal and Azmour is characterized by gene pools not present in commercial (Nurseries) varieties, pointing out the very narrow genetic base of the commercialized olive material in Tunisia, and the need to broaden it to avoid the risk of genetic erosion of this species in this country.