On chloroplast DNA variations in the olive (Olea europaea L.) complex: comparison of RFLP and PCR polymorphisms

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Abstract
Previous papers have dealt with olive chloroplastic DNA (cpDNA) variation revealed using several methods (RFLPs, PCR-RFLPs and microsatellites) and have led to different conclusions. This paper aims to reconsider these divergences. A Southern approach was applied to reveal polymorphism. We used chloroplast DNA of Phillyrea media as a probe. Based on these data, only four chlorotypes were identified in the olive complex. The number of detected lineages was lower than reported in the literature using a direct cpDNA RFLP approach, and was insufficient to distinguish the North African subspecies europaea, maroccana, guanchica and laperrinei. Furthermore, one individual considered belonging to the subspecies laperrinei was questionable. Using other cpDNA and mitochondrial DNA (mtDNA) polymorphisms – based on PCR and RFLP methods, respectively – we showed that this individual displays the cytoplasmic lineage CE1-ME1 characteristic of most Eastern mediterranean cultivars and of Olea europaea subsp. laperrinei from Hoggar. However, based on RAPDs, this individual appeared as mislabelled and probably corresponded to a Mediterranean cultivar or a feral form. In addition, we checked O. e. subsp. laperrinei herbarium samples using two cpDNA microsatellites, which revealed polymorphisms. These also supported that both populations from Niger and Algeria displayed a chlorotype related to CE1. Consequently, based on cpDNA, the relationships of O. e. subsp. laperrinei from Hoggar with a Mediterranean lineage appeared well supported, whereas the South West Moroccan and Macaronesian olives appeared in a different clade using both mtDNA and cpDNA polymorphisms. We conclude that methods based on PCR reveal more polymorphisms in the cpDNA and lead to more-reliable results that the classical RFLP method.