Molecular studies for disclosing the genetic identity of local cultivars in olive and understanding the stilbene synthase pathway in grapevine

This study has been focused on two main fruit trees, olive and grapevine, from the genetic and genomic point of view. Olive (Olea europaea L.) is one of the oldest tree crops of the Mediterranean regions. In spite of the increasing economic importance and wider diffusion of monovarietal virgin olive oils, very few studies have focused on the genetic characterization of olive cultivars locally grown in the Veneto region, North-Eastern Italy. Since the genotype mainly affects the composition and sensory properties of extra virgin olive oils, the characterization of autochthonous olive cultivars is a key step for the production, marketing and traceability of high quality olive oils. Here we have performed a survey of mapped SSR marker loci for the analysis of nuclear DNA polymorphisms to distinguish olive cultivars and cultivar groups within the entire olive cultivation area in Veneto, from the Garda Lake to the Euganean and Trevisan hills, in order to obtain a systematic examination of the Veneto regional olive germplasm patrimony. The final aim of the study was to resolve cases of homonymy and synonymy, and to warrant the genetic authenticity of olive cultivars and of their clones in case of mislabelling. A total of 204 unknown olive tree accessions along with 25 olive reference cultivars were subjected to molecular analyses using ten highly polymorphic nuclear SSR markers. The overall molecular data were used for the computation of genetic similarity and diversity statistics, for the construction of ordination dendrograms and the determination of centroids of local accessions, along with reference cultivars. A genetic structure analysis of the olive population as a whole was also performed for the definition of cultivar groups. Based on multi-locus SSR genotypes and pair-wise SM coefficients, we found that on one hand most of the olive samples displayed genetic identity with the reference cultivars “Casaliva” (60 samples) while several others showed identity with different national and local varieties: “Miniol” (21 samples), “Grignano” (20 samples), “Tonda di Villa” (19 samples), “Leccino” and “Capolga” (17 samples each), “Gargna”, “Compostaro” and “Oblica” (9 samples each), “Rossanel” (8 samples), “Fort” and “Baia” (5 samples each), “Grappolo” and “Favarol” (3 samples each) having bootstrap values greater than 85%. As few as 2 samples were attributable to “Moraiolo”, “Carnica” and “Maurino” each. The uppermost hierarchical level of population structure suggested a clear maximum of cultivar groups for K=15. It is worth noting that more than 98% of individual trees revealed a pure ancestry and only 13 accessions were admixed showing a membership to the associated cultivar group <70%. The total value of observed heterozygosity was equal to Ho=91.6% (st. dev. 1.9%). and the mean expected heterozygosity was He=74.1% (st. dev. 2.9%). Moreover, the Nei’s unbiased genetic diversity was as equal to uHe=74.3%, whereas the total estimate of the Shannon’s information index of phenotypic diversity was equal to I=1.612. The fixation index, equal to Fst=0.247, revealed that only less than 25% of the genetic variation was found among cultivar groups and that approximately as much as 75% of the total polymorphism was scored among trees within cultivar groups. The narrow genetic differentiation among subgroups over the investigated multi-locus marker loci was conﬁrmed by the gene ﬂow estimate that was as low as Nm=0.412. It is worth mentioning that all Wright’s inbreeding coefficients were negative and on average equal to Fis=–0.863 and Fit=–0.159, indicating a significant excess of heterozygosity for the olive accessions of the Veneto germplasm. Overall, the SSR markers used in this study have shown their efficacy to distinguish cases of homonymy, synonymy and mislabelling, providing the first systematic analysis of olive germplasm for horticultural valorisation of Veneto cultivars in the regional areas most suited for typical monovarietal virgin olive oil productions. For what concerns grapevine, plant stilbenes are a small group of phenylpropanoid (PP) compounds that have been detected in a limited number of unrelated plant species of at least 72 including grapevine. Plants accumulate it in leaves, roots and fruits in response to both biotic and abiotic stresses such as mechanical wounding, UV-C exposure, pathogen infection, and chemicals treatment. In the last decade stilbenes had raised the interest of many scientists not only because of their role in the plant protection against environmental stresses, but also because of their pharmacological properties. The biosynthetic pathway leading to their production is a side branch of the general PP biosynthetic pathway and can be considered as an extension of the flavonoid pathway. Stilbene synthase (STS) is the key enzyme leading to the biosynthesis of resveratrol which is the basic unit of the biosynthesis of plant stilbenes. Recently we reported the identification and functional characterization of two grapevine R2R3-MYB transcription factors (TFs), which appear to regulate the stilbene biosynthetic pathway. These TFs, designated VvMYB14 and VvMYB15, strongly co-express with VvSTS genes under a range of stress-induced conditions and in developmentally regulated tissues, including leaves exposed to wounding treatment, UV-C irradiation and downy mildew infection, and several stages of seed and berry development. In transient gene reporter assays, these VvMYB TFs were demonstrated to specifically activate the promoters of VvSTS genes, and the ectopic expression of VvMYB15 in grapevine hairy roots resulted in increased VvSTSs expression and in the accumulation of glycosylated stilbenes in planta. However, despite these significant advances, many question remain to be answered regarding this grapevine biosynthetic pathway. Based on the notion that genes involved in similar or related processes may exhibit similar expression patterns over a range of experimental conditions, we performed a co-expression network analysis on different grapevine gene expression datasets identifying candidate TFs belonging to the WRKY family possibly involved in the regulation of the stilbene biosynthetic pathway. These genes show co-expression correlation values even higher than those observed for VvMYB14/15 and are strongly induced in response to the same abiotic stress treatments which lead to VvSTS induction. The expression of candidate WRKY TFs, namely WRKY03, WRKY43 and WRKY53 together with the expression of the two MYBs already characterized (MYB14 and MYB15) and VvSTSs genes was monitored in different stress conditions, including wounding and UV-C treatment, in order to confirm data obtained from co-expression analyses in whole transcriptome datasets. Results confirmed that there is a strong correlation between the expression of candidate WRKY genes and VvSTSs suggesting a role for these TFs in the regulation of the stilbene pathway. In order to confirm this hypothesis, we performed several functional studies. Amongst them, of particular interest are those results obtained from dual reporter luciferase assays. These assays were aimed at measuring the activity of the WRKY TFs on the VvSTS promoters by mean of transfection experiments in grapevine liquid cell cultures. Interestingly our results indicate a role for VvWRKY43 and WRKY53 in the regulation of VvSTSs. Nevertheless, these TFs seem to act exclusively in combination with MYB14 and MYB15, since, whenever co-transfected alone, they did not show any significant effect on the VvSTS promoter. Differing from WRKY43 and WRKY53, WRKY03 did not show any effect on the VvSTS promoter activity neither alone nor in combination with R2R3-MYB TFs, but was demonstrated to induce the promoter of VvMYB14. Then, our results indicate that WRKY03 probably act upstream R2R3-MYB factors in the regulation of the grapevine STS pathway whereas WRKY43 and WRKY53 have a role as co-factors with VvMYB14 and VvMYB15 TFs.