STUDIO DELLA DIVERSITA’ GENETICA IN OLIVO (Olea europea L.)
Imperato, Annalisa (2010) STUDIO DELLA DIVERSITA’ GENETICA IN OLIVO (Olea europea L.). [Tesi di dottorato] (Inedito)
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The olive (Olea europaea L.), is one of the most important cultivated tree in Italy and all around the eastern Mediterranean Basin. This species is characterized by a germplasm that includes a huge number of varieties, spread in different areas. Among the molecular markers available to study genetic variability in olive, Simple Sequence Repeats (SSRs) are among the most interesting for varietal identification, because they are codominant, informative and reproducible. In most of the collections varietal identification hasn’t been completed and really few agronomic feature have been methodically estimated. Cases of synonymy and homonimy are still very common, often because of mistakes in name transcription of a particular variety (Rao et al., 2009). Nevertheless at present for the olive we have available some methods, whose complementary use allow us a correct identification of cultivated vegetal material. Among several molecular markers used in olive, the microsatellits (SSR) are considered the markers by excellence, thanks to their codominant nature and to their discriminant capability and their being reproducible different labs. The main aim of the present thesis was to evaluate the genetic variability of olive varieties cultivated in Southern Italy using SSR markers. Twenty-six olive cultivars from Campania and twenty-one of nearby areas were analysed at six SSR loci. Campania is a region characterised by a morphologically diverse varieties able of producing selected and typical oils. The SSR loci were chosen from the literature for their high discriminating power and for their capability of supplying a high number of alleles. All SSR primer couples provided a clear amplification profile and turned out to be polymorphic in the population analysed. The two loci with the highest discriminating power were the DCA 3 and the DCA 16, in accordance with what had been reported in literature (Alba et al., 2009). High values of genetic diversity (He) and of discriminating power (D) were found in most of the SSR loci used, pointing out a high polymorphism among the olive cultivars analysed. A total of 81 different alleles were found, and both the mean heterozygosity and the discriminating power were on average high. All the cultivars had a unique profile, with the exception of the ‘Uova di Pavone’ and ‘Sant’ Agostino’ cultivars. These genotypes should be further analysed to define if they are a case of possible synonymy. The genetic relations among the examined varieties were studied through hierarchical analysis by UPGMA algorithm. The dendrogram indicated that most of the accessions from Campania are genetically different from those from neighbouring areas. The possible usefulness of the identified SSR profiles in the agro-food chain was also investigated. The DNA isolated from leaves, fruits or mono-varietal olive oils of three cultivars (‘Frantoio’, ‘Pisciottana’ and ‘Leccino’) was isolated and analysed with the six SSR primer pairs. The result indicated that SSR profile are conserved in the olive-food chain and therefore, SSR profiling is a valid tool for olive oil chain genetic traceability. However, some issues such as the sensitivity of the method and its ability of identifying varieties in complex mixtures, still need to be further investigated. The results of the genetic fingerprint of the here analysed cultivars underlined the richness and the possible specific nature of the olive from Campania. The SSRs’ profiles gave us valuable information that can be use to protect both the genetic material and mono-varietal oils. Furthermore, the data confirmed the genetic richness of regional germplasm, implying the necessity of measures to protect neglected traditional varieties. Our results pointed out that it’s possibile to identify DNA from two olive cultivars in a 1:1 DNA mixture, while it’s not possibile when DNA of each cultivar, which makes the blend, has a proportion lower than 50% , as demonstrated from previous literature (Breton et al., 2004). Although we should still check any problems connected with sensitivity and efficacy of method that is used for identification of varietal composition even in more complex oil blends, we can however state that DNA markers through PCR technology are a valid tool for assuring genetic traceability in any stage of the olive oil chain, their correspondence to DOP and IGP labels, and at last finished product quality, guaranteeing farmers, producers and consumers and even preventing them from any frauds. Thanks to increasing attention to the protection biodiversity, several olive germplasm ex-situ collections were developed to safeguard and to promote the use of genetic resources in farming. Management, evaluation and use of wide germplasm collections can be inefficient, mainly because the high number of genotypes present in collection and the unfeasibility of analysing with sufficient details all the accessions conserved. The establishment of a core set is a valid help for assessing, characterizing, and using a germplasm collections. Using SSR molecular markers data already available, five different core subset were built and evaluated using three different algorithms. This analysis was carried out on the World Olive Germplasm Bank (WOGB) of Cordoba. Among the six different strategies used for construction of five core subsets, only the sampling strategy based on Core Hunter algorithm, which simultaneously maximizes two genetic measures (the average modified Rogerss' distance and Nei's index) allowed to get the highest values of genetic parameters considered. This is in agreement with what literature reported (Thachuk et al., 2009). 133 and 151 genotypes were necessary to capture 99% of 236 alleles of the whole initial collection. A basic requirement for the use and preservation of olive germplasm is not only a careful genetic characterization, but also an accurate evaluation of plants sanitary conditions. It is essential to have a qualified nursery propagation equipment, in conformity with sanitary regulations that impose the genetic correspondence to a defined genotype as well as lack of those diseases, caused by pathogens and viruses, in young olive plants. For this reason, 174 WOGB accessions were analysed by molecular techniques to screen for the presence of 40 pathogens and 3 viruses. The olive fly Bactrocera oleae (Rossi) is the key pest of cultivated olive in the Mediterranean Basin. Infestation caused by the olive fruit fly caused direct and indirect damages. The direct damages are caused by active infestation, which consists in the destruction of the pulp fiction and as consequence, premature fruit drop. Furthermore, the fly also causes an alteration of olive oil’s qualitative parameters. The indirect damages are due to fungal and bacterial infestations, which start at the egg position and spread with the larval feeding tunnels. The control of olive fruit fly is essentially pursued by pesticide treatments, which are costly and have negative impact on the environment or human health. Alternative control methods are mass trapping, barrier films, combined treatments as mass trapping and application of kaolin clay or biological control. However such methods are not always effective as desired The availability of olive cultivars with low susceptibility to the fly could represent an important tool in strategies of integrated management of the B. oleae. It is known that morphological, physical, and chemical parameters, in addition to environmental conditions, are involved in the level of susceptibility of olive cultivars to fruit fly attacks. For example, some low susceptible olive cultivars have higher quantities of oleuropeine or cianidine compounds (Iannotta et al., 1999). It was proposed that the degree of cultivars susceptibility to fly infestation depends on the ability of the β-glucosidase enzyme to cleave oleuropeine and/or on the availability of oleuropeine to undergo the cleavage (Iannotta et al., 2007). On the contrary no information is available on the defence genes active against B oleae and on the molecular response of olive fruits to the fly infestation. It is known, that the molecular interaction between plants and herbivores originates both up and down regulation of a large number of genes encoding proteins involved in defense mechanisms and signaling, secondary metabolism, abiotic stress and photosynthesis, beside many genes with unknown functions. (Hermsmeier et al., 2001). Therefore, the study of the variation of the transcripts abundance occurring in infested olive fruit tissue, will provide novel information on olive sequences responding to B. oleae infestation and on their involvement in defence. In this frame an useful experimental approach, is the SSH (Suppression Subtractive Hybridization) technique. We used this strategy in order to isolate differentially expressed olive sequences occurring in infested tissue. In addition, we monitored the expression of up-regulated genes in different olive cultivars. For characterization of differential expressed genes in reaction to the fruit fly, a subtractive library was constructed through Suppression Subtractive Hybridization (SSH) method. The library was constructed subtracting mRNAs from un-attacked fruits of cultivar ‘Moraiolo’ from mRNAs isolated from with larval feeding tunnel. Among the 590 clones of the library, 219 were sequenced, having an insert size longer than 200 bp. In order to assign a putative function to the different transcripts, similarity analysis was carried out with tblastx algorithm. Approximately 70% of transcripts showed a significant similarity with coding sequences of different plant species (e-value <0.001) mainly Vitis vinifera; among them 113 sequences had similarity with transcripts coding for known protein and 106 with transcripts coding for unknown protein. Only 48% of the ESTs were classified into functional category (Biological process, Molecular Function, Cellular component) most likely as consequence of the very little information available on olive genome. The molecular functions of EST sequences, underline the complexity of olive molecular response to the fruit fly. The differential expression validation of the most interesting transcripts was performed by Real time PCR. Eight transcripts putatively involved in defence mechanisms, turned out to be overexpressed in olive fruits infested through larval feeding tunnel. Four transcripts showed a weak overexpression level, with RQ values variable from 1,29 to 2,28. Nevertheless differences in expression turned out to be statistically significant, when t-student test was performed. The transcripts most overexpressed in drupes with larval feeding tunnel, with RQ from 2,3 to 8,7, were similar to those coding a protein related to pathogenesis (Oe-PR27), to a lipoxygenase (Oe-LOX) and to a chitinase (Oe-Chitinase). We thought to evaluate the relative expression of the latter transcripts also in a different olive variety (‘Leccino’) and at two different stages (drupes with sign of oviposition stings and drupes with larval feeding tunnel). The overexpression of the three transcripts in the two attack biological stages was confirmed. Finally, in order to get the complete transcripts sequence RACE-PCR experiments were conducted. Two full lenght cDNA (Oe-Chitinase and Oe-PR27) were recovered by the 3’ and 5’ RACE-PCR. The amino acid sequences coded by these two genes was of 229 aa and 268 aa respectively. Our data demonstrated that the molecular response of olives drupes to B. oleae infestation is complex and involves numerous molecular functions. As expected, this molecular response implicates alterations of primary and secondary metabolism. We identified three genes that are up-regulated in infested olive fruits. Homologues of the identified sequences are involved in the molecular response of plant to biotic stress in different species. It will be interesting to verify if the expression profiles of the EST sequences generated in this study are associated to different level of susceptibility of olive cultivars to B. Oleae.
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