Self-incompatibility of olive.

Abstract

The olive (Olea europaea L.) is one of the most ancient fruit trees and has been cultivated for its oil in the Mediterranean area for thousands of years. Today, the consumption of olive oil and table olives is increasing both in traditional producing countries and the entire world. Most olive cultivars are self-incompatible and do not produce a commercial yield after self pollination. In this thesis, inflorescence architecture and sexual compatibility relationships of some olive cultivars, and gene expression in olive pistils during flowering were studied. To study the inflorescence architecture of olive, 45 inflorescences in each of the cultivars Manzanillo, Mission, and Frantoio were checked every morning from flower opening to petal fall. The flower position on the inflorescence had a highly significant effect on the opening day in all cultivars. Terminal flowers and the flowers located on the primary branches opened earlier than flowers located on the secondary branches. Flower position also had a highly significant effect on gender in Manzanillo and Mission. In Manzanillo, the secondary branches had fewer perfect flowers than the primary branches. In Mission, the secondary branches had no perfect flowers at all. In Manzanillo, perfect flowers had significantly longer petal persistence than staminate flowers. To study flower competition within the inflorescence, the distal halves, on which the flowers tend to be perfect, of 120 inflorescences in three trees of Manzanillo were removed about one month before full bloom. This resulted in a highly significant increase in the percentage of perfect flowers on the proximal halves. The effects of shoot orientation and inflorescence location on inflorescence characteristics in the cultivars Frantoio, Kalamata, and Koroneiki were also studied. For each cultivar, inflorescence characteristics in three sections of shoots (top, middle, and base) and four sides of the three selected trees (north, south, east, and west) were recorded. The statistical analysis showed that basal inflorescences were shorter and with fewer flowers but with the same percentage of perfect flowers. Shoot orientation did not have any influence on these characteristics in any of the cultivars. Sexual compatibility was assessed using two methods. In the first method, controlled crossings were performed in the cultivars Frantoio, Koroneiki, and Kalamata. The pistils were harvested one week after hand pollination and stained with 0.1% aniline blue. The styles and ovules were separated, mounted in 80% glycerol, and observed under a fluorescence microscope. In Frantoio and Koroneiki, the number of ovules penetrated by a pollen tube was used to estimate the level of sexual compatibility. In Kalamata, the numbers of ovules penetrated by pollen tubes were not significantly different between treatments; therefore, the number of pollen tubes in the lower style was used. All the cultivars studied were self- incompatible. Frantoio (as a host) was incompatible with Koroneiki and Barnea but partially compatible with Mission. Koroneiki (as a host) was incompatible with Barnea but partially compatible with Frantoio and Mission. Kalamata (as a host) was compatible with Barnea, incompatible with Mission and Koroneiki in 2004, but partially compatible with them in 2005. In the second method, eight microsatellite markers were used for genotyping three Kalamata mother trees, 40 embryos per mother tree, and all the potential pollen donors. Genotyping data were analysed using FaMoz software, and the number of embryos assigned to each putative pollen donor was determined. Paternity analysis showed that Kalamata (as a host) was self-incompatible, compatible with Barnea, Benito, and Katsourela, but incompatible with Arbequina, Azapa, and Picual. To study the gene expression in olive pistils during flowering, a genomic approach was initiated using cDNA subtractive array analysis. Total RNA was isolated from olive pistils at two developmental stages, where self-incompatibility (SI) genes are expected to be differentially expressed: 1) small green flower buds (expression of SI genes not expected) and 2) large white flower buds containing receptive pistils just prior to opening (expression of SI genes expected). From each stage, cDNA libraries were prepared and put through forward and reverse subtractive hybridisations to enrich for differentially expressed cDNAs in stage 2. Macroarrays were prepared by printing 2304 differentially expressed cDNAs onto nylon membranes and hybridised with forwardand reverse-subtracted probes. The analysis identified 90 up-regulated cDNA clones highly expressed in receptive pistils. Further subtracted and unsubtracted hybridisations confirmed up-regulation of the majority of these cDNAs. Gene expression profiles across different tissues showed that most of the genes were pistil-specific. The expression pattern of the genes showed high similarity in Kalamata, Frantoio, Barnea, and Pendolino. All the screened genes were sequenced and their similarities were searched in the NCBI database. The most redundant and interesting up-regulated clones were those similar to a receptor protein kinase-like protein. Some versions of this protein play a role in the sporophytic SI system of Brassica and the gametophytic SI system of Papaver and rye.