Characterization and validation of olive FAD and SAD gene families: expression analysis in different tissues and during fruit development.

Stearoyl-ACP desaturases (SADs) and fatty acid desaturases (FADs) play a critical role in plant lipid metabolism and also affect oil fatty acid composition introducing double bonds into the hydrocarbon chains to produce unsaturated fatty acids. In the present study, the genomic sequences of three SAD and three FAD candidate genes were characterized in olive and their expression was evaluated in different plant tissues. OeSAD genes corresponded to olive SAD1 and SAD2 and to a newly identified OeSAD4, sharing the conserved protein structure with other plant species. On the other hand, the full-length genomic sequences of two microsomal OeFAD genes (FAD2-1 and FAD2-2) and the plastidial FAD6, were released. When the level of expression was tested on different tissues of cv. Leccino, OeSAD1 and OeSAD2 were mainly expressed in the fruits, while OeFAD genes showed the lowest expression in this tissue. The mRNA profiling of all genes was directly studied in fruits of Leccino and Coratina cultivars during fruit development. In both genotypes, the expression level of OeSAD1 and OeSAD2 had the highest value during and after the pit-hardening period, when oil accumulation in fruit mesocarp is intensively increasing. Furthermore, the expression level of both OeFAD2 genes, which were the main candidates for oleic acid desaturation, were almost negligible during fruit ripening. These results have made possible to define candidate genes of the machinery regulation of fatty acid composition in olive oil, providing information on their sequence, gene structure and chromosomal location.

The Paradox of Self-Fertile Varieties in the Context of Self-Incompatible Genotypes in Olive.

Olive, representing one of the most important fruit crops of the Mediterranean area, is characterized by a general low fruit yield, due to numerous constraints, including alternate bearing, low flower viability, male-sterility, inter-incompatibility, and self-incompatibility (SI). Early efforts to clarify the genetic control of SI in olive gave conflicting results, and only recently, the genetic control of SI has been disclosed, revealing that olive possesses an unconventional homomorphic sporophytic diallelic system of SI, dissimilar from other described plants. This system, characterized by the presence of two SI groups, prevents self-fertilization and regulates inter-compatibility between cultivars, such that cultivars bearing the same incompatibility group are incompatible. Despite the presence of a functional SI, some varieties, in particular conditions, are able to set seeds following self-fertilization, a mechanism known as pseudo-self-compatibility (PSC), as widely reported in previous literature. Here, we summarize the results of previous works on SI in olive, particularly focusing on the occurrence of self-fertility, and offer a new perspective in view of the recent elucidation of the genetic architecture of the SI system in olive. Recent advances in research aimed at unraveling the molecular bases of SI and its breakdown in olive are also presented. The clarification of these mechanisms may have a huge impact on orchard management and will provide fundamental information for the future of olive breeding programs.

Alcohol drinking after liver transplantation is associated with graft injury.

AIM: This was a single-center, mixed-design, cross-sectional and retrospective study to assess the performance of the 4-item, self-reported CAGE (Cut down, Annoyed, Guilty, Eye-opener) questionnaire in predicting histology-proven alcohol-related liver graft injury (ARLGI). METHODS: A total of 316 liver transplant (LT) patients between six months and five years were enrolled. Based on previous research, problem alcohol drinking (PAD) was defined as any score ≥ 1 on the CAGE, while a cut-off of 2 was assumed for alcohol dependence (AD). RESULTS: Responders were 195, 45 (23.1%) had a CAGE score ≥ 1 and 30 (15.3%) scored ≥ 2. After controlling for confounders, PAD was associated with hyperlipidemia (P=0.01), while AD with a male gender (P=0.01), hyperlipidemia (P=0.03) and alcohol as native diagnosis (P=0.03). PAD and AD were both associated with a significantly higher prevalence of ARLGI, i.e. 53.3% and 63.3%, respectively (P<0.0001). Hepatitis C virus (HCV) patients with PAD showed more steatosis (P=0.04), portal infiltrate (P=0.03), and pericellular/perivenular fibrosis (P=0.02). The likelihood ratios for CAGE scores ranging from 0 to 4 in predicting ARLGI were 0, 5.2, 7.8, 7.8, and 100, respectively. CONCLUSION: By use of a self-report instrument we found a 23.1% prevalence of PAD and a 15.3% prevalence of AD among LT patients between six months and five years. A variable degree of ARLGI was present in 53.3% of PAD and 63.3% of AD, respectively. HCV patients with PAD had more steatosis, portal inflammation, and pericellular fibrosis. Transplant physicians might improve their ability to predict the probability for ARLGI using the CAGE.

Patient satisfaction among liver transplant recipients: single-center survey.

A single-center survey using a semistructured questionnaire was conducted in liver transplantation recipients at discharge after the primary surgery. The objectives of the study were to assess patient satisfaction and to identify critical points that negatively affected their perception of the quality of care received, and to derive information to enable improvement in current standards of care. The questionnaire included 5 sections about quality and 1 section for suggestions. Patients were asked to provide answers on a 5-item Likert scale. Areas assessed included quality of staff, organization, boarding, privacy, and transfer of care. Among 51 recipients, satisfaction was high (>50%) in all areas. Lower satisfaction scores were given for room services, diet, and background music. The most frequently reported area of dissatisfaction (12%) was lack of availability of in-hospital physical rehabilitation programs. Despite overall satisfaction with quality of care, recipients reported lack of appropriate physical rehabilitation programs in the early posttransplantation period.

The "You Are Not Alone" care program for liver transplantation.

Current clinical practice is based on the principles of efficacy, appropriateness, efficiency, quality, and safety. Compliance with these tenets requires experienced medical and nursing staff, and active participation of patients and their families in the planned therapeutic program. To match patients' expectations on quality and safety of care and spur active participation in the transplant care process, we set up an integrated, multiphase, multidisciplinary care program devoted to liver transplantation (LT) candidates, engrafted patients, and their families: the "Non Sei Solo" care program (You Are Not Alone). The basic principle of the care program was that, to provide efficient and effective education to their patients, health care professionals need to learn how to teach and what to teach, acquire successful communication skills, and monitor the process of education. The methodology encompassed 5 distinct phases: phase 1, exploration of patients' needs, by means of a questionnaire devoted to waitlisted and engrafted patients and their care givers; and phase 2, creation of 16 patient-oriented educational brochures directed to patients and their families. Once created, the educational brochures were presented, discussed, and amended during a consensus meeting involving all transplantation nurses and physicians (phase 3). To acquire the necessary skills and ease communication with patients, the transplantation nurses, physicians, surgeons, and anesthesiologists attended a 6-month counseling course under the tutorial of an expert counselor (phase 4). Finally, in June 2007 the program started officially with monthly meetings with patients and their families, guided hospital tours on patient request, and activation of a toll-free phone number to provide support to patients and answer their questions.

Microsatellite markers are powerful tools for discriminating among olive cultivars and assigning them to geographically defined populations.

Twelve simple sequence repeat (SSR) loci were used to differentiate among 118 cultivars sampled in several countries of the Mediterranean basin and to analyze the genetic structure of olive cultivar gene pools. The markers were found to have high discrimination power. On average, with a single assay it was possible to discriminate 96% of the pairwise comparisons and, with a combination of 3 loci, virtually all cultivars were distinguished. The SSR markers were also tested for their ability to assign cultivars to their geographic population of origin. A selection of 6 loci was found to maximize assignment accuracy, correctly reallocating up to 75.4% of cultivars to their population of origin. Because of the confusion surrounding the origin of most olive cultivars, their molecular identification and ascertainment of origin will be extremely useful for germplasm management and breeding.

Transferability of olive microsatellite loci across the genus Olea.

The transferability of microsatellite markers developed for olive cultivars ( Olea europaea L.) has been tested and confirmed in the Olea complex. Thirty two genotypes, belonging to different taxa of the genus Olea, have been analyzed with four olive SSRs. Positive amplifications at all loci were obtained in 13 taxa (at least one accession per species). Sixty seven different alleles have been detected at the four loci analyzed. Polymorphic products have been observed at the inter- and intra-species level. Some SSR loci have shown multiple amplification products in some species. The high number of unique alleles has allowed the unambiguous discrimination of most accessions. Similarity coefficients and relationships among the Olea taxa have been calculated based on SSR amplification results. The reliability of SSRs as markers for intra-species variability evaluation has been confirmed while their use to explore relationships at the inter-species level is discussed, being dependent on the locus analyzed.

Comparative study of the discriminating capacity of RAPD, AFLP and SSR markers and of their effectiveness in establishing genetic relationships in olive.

RAPDs, AFLPs and SSRs were compared in terms of their informativeness and efficiency in a study of genetic diversity and relationships among 32 olive cultivars cultivated in Italy and Spain. SSRs presented a higher level of polymorphism and a greater information content, as assessed by the expected heterozygosity, than AFLPs and RAPDs. The lowest values of expected heterozygosity were obtained for AFLPs, which, nevertheless were the most efficient marker system due to their capacity to reveal the highest number of bands per reaction and because of the high values achieved for a considerable number of indexes. All three techniques discriminated the genotypes very effectively, but only SSRs were able to discriminate the cultivars Frantoio and Cellina. The correlation coefficients of similarity were statistically significant for all three marker systems used but were lower for the SSR data than for RAPDs and AFLPs. For all markers a high similarity in dendrogram topologies was obtained although some differences were observed. All the dendrograms, including that obtained by the combined use of all the marker data, reflect some relationships for most of the cultivars according to their geographic diffusion. AMOVA analysis detected greater genetic differentiation among cultivars within each country than it did between the two countries.

A first linkage map of olive (Olea europaea L.) cultivars using RAPD, AFLP, RFLP and SSR markers.

The first linkage map of the olive (Olea europaea L.) genome has been constructed using random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphisms (AFLP) as dominant markers and a few restriction fragment length polymorphisms (RFLP) and simple-sequence repeats (SSR) as codominant markers. Ninety-five individuals of a cross progeny derived from two highly heterozygous olive cultivars, Leccino and Dolce Agogia, were used by applying the pseudo test-cross strategy. From 61 RAPD primers 279 markers were obtained - 158 were scored for Leccino and 121 for Dolce Agogia. Twenty-one AFLP primer combinations gave 304 useful markers - 160 heterozygous in Leccino and 144 heterozygous in Dolce Agogia. In the Leccino map 249 markers (110 RAPD, 127 AFLP, 8 RFLP and 3 SSR) were linked. This resulted in 22 major linkage groups and 17 minor groups with fewer than four markers. In the Dolce Agogia map, 236 markers (93 RAPD, 133 AFLP, 6 RFLP and 4 SSR) were linked; 27 major linkage groups and three minor groups were obtained. Codominant RFLPs and SSRs, as well as few RAPDs in heteroduplex configuration, were used to establish homologies between linkage groups of both parents. The total distance covered was 2,765 cM and 2,445 cM in the Leccino and Dolce Agogia maps, respectively. The mean map distance between adjacent markers was 13.2 cM in Leccino and 11.9 cM in Dolce Agogia, respectively. Both AFLP and RAPD markers were homogeneously distributed in all of the linkage groups reported. The stearoyl-ACP desaturase gene was mapped on linkage group 4 of cv. Leccino.

Diversity of Olea genotypes and the origin of cultivated olives.

Tandem repeats belonging to three DNA sequence families ( OeTaq80, OeTaq178, and OeGEM86) were isolated from the nuclear DNA of Olea europaea cv. Carolea and dot-hybridized to the genomic DNA of 14 hypothetically different Olea species, 78 olive cultivars, and 14 wild olives. The copy number per unreplicated haploid genome of OeTaq80- and OeTaq178-related sequences was in the 10(7)-10(6) range and that of OeGEM86-related sequences was in the 10(5) range in cultivars, wild olives and some Olea species. A large variation in the frequency of repeats belonging to each sequence family was observed within each group of plants. Positive correlations existed in each genome between the frequencies of repeats belonging to each family, and their overall frequency was positively correlated to the genome size. Duncan grouping showed that the frequency variation of tandem repeats within each group of plants was not continuous. Two main groups and several subgroups of genotypes could be separated within both the olive cultivars and the wild olives. Discrete areas in the Mediterranean Basin could be delimited by the geographic distribution of cultivated olives with different genotypes and the wild plants were associated with the cultivars in these areas according to genotypic similarity. The Olea species could be divided into four genotypic groups. Three of these, comprising accessions from Asia and North Africa, showed similarity with the genotypes of cultivars and wild olives. These results suggest a polyphyletic origin of cultivated olives from different wild Olea forms distributed throughout the Mediterranean Basin.

Amount and organization of the heterochromatin in Olea europaea and related species.

The amount and spatial organization of the heterochromatin in nuclei of the shoot meristem and the frequency in the nuclear DNA of sequences belonging to a family of tandem repeats were investigated in cultivars of Olea europaea and related species. Significant differences between Olea species and between cultivars of O. europaea were observed: (i) in the spatial organization of the heterochromatin in interphase nuclei as determined by the number and surface area of the chromocentres; (ii) in genome size; and (iii) in the amount of condensed chromatin as measured by cytophotometry carried out at different thresholds of optical density. DNA elements belonging to a family of tandem repeats about 80 bp in length (OeTaq80 repeats) were isolated from the genomic DNA of an olive cultivar. It was shown: (i) by nucleotide sequence comparisons, that these repeats display variability in structure even within the same array, where different elements may share no more than 74% homology; (ii) by in situ hybridization, that OeTaq80-related DNA sequences are mainly localized in the heterochromatin at the chromosome ends; (iii) by dot-blot hybridization experiments, that these sequences are highly represented in the genome of all the olive cultivars and the majority of Olea species studied, and that their frequency may differ significantly even between olive cultivars; and (iv) by calculating the copy number of OeTaq80-related sequences per haploid (1C) genome, that the redundancy of these DNA elements may differ significantly between the genomes tested. It is suggested that the inter- and intraspecific changes in the nuclear and genomic traits observed can contribute to the understanding of the phylogenetic relationships between Olea species and in defining parameters to be exploited in varietal identification within cultivated olives.