Genetic analysis of olive (Olea europea L.) varietes in the Maltese islands using DNA markers: method development and technique evaluation

Abstract

The Maltese Islands have a long history of association with the cultivation of olive (Olea europaea L.). Being geographically isolated, the Islands host a number of indigenous varieties that show physical and agronomic traits that make the trees highly adapted to the local pedoclimatic conditions. Morphological marker identification of olive varieties is limited because it is subjective and influenced by the environment and farming practices. DNA molecular markers such as Random Polymorphic DNA (RAPD) and microsatellites embedded within the genome remain largely unaffected by the outside environment and therefore provide a more accurate method of identification. Locally, no previous genetic work making use of these markers has been reported for the olive. Therefore, the purpose of this study was to carry out a genetic diversity assessment focusing on three local cultivars of interest ("Bidni", "Bajda" and "Malti") and one form of the Maltese wild olive ("Oleaster") which were compared to two commercial Italian and Tunisian varieties ("Carolea" and "Tunezina" respectively). Development of the methods used for DNA extraction and marker amplification is discussed. The markers were chosen following strict selection criteria. A number of similarity indices, based on binary (presence-absence data) and allele frequencies, were used to provide estimates of the genetic similarity between any two varieties and to evaluate the performance of each. Cluster analysis was also carried out to generate dendrograms representing genetic relationships between the varieties. Additional tests that were carried out to determine the reliability and robustness of the methods are also discussed. A high inter-varietal genetic variability was found with both marker types. The total percentage polymorphic loci for RAPD markers was equal to 84.84% while an average of 4.86 alleles were detected per microsatellite locus. Indeed, one microsatellite locus generated six different allele combinations, allowing identification of all six varieties. Therefore, the local olive genome is composed of a very diverse group of cultivars, with "Bidni" and "Malti" standing out for their genetic uniqueness. This polymorphic content can be commercially useful in varietal identification for breeding programs, in obtaining certification of Maltese Olive Trees and in tracing the authenticity of Maltese olive oil. It has also been further demonstrated that the Maltese cultivars may exhibit significant genetic differences from other 0. europaea varieties because three out of seven microsatellite marker regions may not be conserved in the former. Tile marked differences generated in the two types of markers were attributed to the fact that they check for different parts of the genome. Cluster analysis has shown that similarity in the RAPD profile may not linked to geographic origin or fruit characteristics whereas a clear-cut separation is evident between Maltese varieties and foreign ones. Moreover, since "Oleaster", "Bidni" and "Malti" did not form any close clusters it is implied that perhaps their origins are not directly linked despite the fact that they are Maltese natives. In general, this work represented an important, preliminary step in setting up the methods for using DNA molecular markers to provide the very first insights regarding genetic diversity in local olives. It demonstrates that DNA markers can be used as a financially viable method of identification provided that the unique marker profiles are conserved by clone propagation.