Eight microsatellite primers were used to distinguish 23 olive cultivars that were parents, or potential parents, in a Spanish breeding program. Four of these microsatellites were particularly informative and were used to check the paternity of 11 olive progenies thought to come from selfings or controlled crosses involving nonemasculated flowers. Seven progenies were found to be highly contaminated, i.e. many seedlings had unexpected alleles, and only four were found to be pure or almost pure. Almost all the contamination detected came from outcrossing, indicating that placing the pollination bags well before anthesis is important and that emasculation to avoid selfing is unnecessary. More than two nonparental alleles per primer were found in each contaminated progeny, showing that more than one cultivar caused contamination. However, the allele data are consistent with `Picual' (the main commercial cultivar growing in the area where the crosses were made) being the contaminant in 48% of the nontrue seedlings (excluding `Picual' self progenies). Some other cultivars planted near the female trees were also found to be sources of contamination. The results obtained show that microsatellite analysis is a convenient technique to assess routinely the crosses made in breeding programs and to check self-incompatibility in olive. The pure progenies identified will be useful for reliable inheritance studies in olive, which have rarely been reported in the literature.
Raul de la Rosa, Celia M. James and Kenneth R. Tobutt
A. Belaj, I. Trujillo, R. de la Rosa, L. Rallo and M.J. Giménez
Random amplified polymorphic DNA (RAPD) analysis was performed on the main Mediterranean cultivars of olive (Olea europaea L.) from the Germplasm Bank of the Centro de Investigación y Formación Agraria “Alameda del Obispo” in Cordoba, Spain. One hundred and ninety reproducible amplification fragments were identified using 46 random primers followed by agarose gel electrophoresis. Some 63.2% of the amplification products were polymorphic, with an average of 2.6 RAPD markers obtained for each primer. The combination of polymorphic markers resulted in 244 banding patterns. The high degree of polymorphism detected made identification of all the cultivars (51) possible by combining the RAPD banding patterns of just only four primers: OPA-01, OPK-08, OPX-01, and OPX-03. Cultivar-specific RAPD markers and banding patterns were also found. A dendrogram based on unweighted pair-group method cluster analysis was constructed using a similarity matrix derived from the RAPD amplification products generated by the 46 primers. Three major groups of cultivars could be distinguished by RAPD analysis: 1) cultivars from east and northeast Spain, 2) Turkish, Syrian, and Tunisian cultivars, and 3) the majority of common olive cultivars in Spain. The dendrogram thus showed a good correlation between the banding patterns of olive cultivars and their geographic origin. A higher level of polymorphism was observed when polyacrylamide gel electrophoresis was used to separate the amplification products. Thus, adequate use of RAPD technology offers a valuable tool to distinguish between olive cultivars.
Raúl De la Rosa, Angjelina Belaj, Antonio Muñoz-Mérida, Oswaldo Trelles, Inmaculada Ortíz-Martín, Juan José González-Plaza, Victoriano Valpuesta and Carmen R. Beuzón
In the present work, a set of eight new hexa-nucleotide simple sequence repeats (SSRs) is reported in olive (Olea europaea L). These SSRs loci were generated on the basis of expressed sequence tag (EST) sequences in the frame of an olive genomic project. The markers showed a high level of polymorphism when tested on a set of cultivars used as genitors in the olive breeding program of Córdoba, Spain. The long-core repeat motif of these markers allows a wider separation among alleles, thus permitting an accurate genotyping. Besides, these markers showed comparable levels of polymorphism to di-nucleotide SSRs, the only ones so far reported in olive. Selected on the basis of their discrimination capacity, four of the eight SSRs were used to test their ability for paternity testing in a total of 81 seedlings coming from 12 crosses. The paternity testing showed that seven crosses matched the alleged paternity and the remaining five were products of illicit pollinations. These results exactly matched with previous paternity testing performed with di-nucleotide SSR markers. These results demonstrate the usefulness of the developed hexa-nucleotide repeated motifs for checking the paternity of breeding progenies and suggest their use on variability studies.