Silvia Caporali

Olive phenolic compounds: metabolic and transcriptional profiling during fruit development

BackgroundOlive (Olea europaea L.) fruits contain numerous secondary metabolites, primarily phenolics, terpenes and sterols, some of which are particularly interesting for their nutraceutical properties. This study will attempt to provide further insight into the profile of olive phenolic compounds during fruit development and to identify the major genetic determinants of phenolic metabolism.ResultsThe concentration of the major phenolic compounds, such as oleuropein, demethyloleuropein, 3–4 DHPEA-EDA, ligstroside, tyrosol, hydroxytyrosol, verbascoside and lignans, were measured in the developing fruits of 12 olive cultivars. The content of these compounds varied significantly among the cultivars and decreased during fruit development and maturation, with some compounds showing specificity for certain cultivars. Thirty-five olive transcripts homologous to genes involved in the pathways of the main secondary metabolites were identified from the massive sequencing data of the olive fruit transcriptome or from cDNA-AFLP analysis. Their mRNA levels were determined using RT-qPCR analysis on fruits of high- and low-phenolic varieties (Coratina and Dolce d’Andria, respectively) during three different fruit developmental stages. A strong correlation was observed between phenolic compound concentrations and transcripts putatively involved in their biosynthesis, suggesting a transcriptional regulation of the corresponding pathways. OeDXS, OeGES, OeGE10H and OeADH, encoding putative 1-deoxy-D-xylulose-5-P synthase, geraniol synthase, geraniol 10-hydroxylase and arogenate dehydrogenase, respectively, were almost exclusively present at 45 days after flowering (DAF), suggesting that these compounds might play a key role in regulating secoiridoid accumulation during fruit development.ConclusionsMetabolic and transcriptional profiling led to the identification of some major players putatively involved in biosynthesis of secondary compounds in the olive tree. Our data represent the first step towards the functional characterisation of important genes for the determination of olive fruit quality.

A comparison of conservative DNA extraction methods from fins and scales of freshwater fish: A useful tool for conservation genetics

Key words: brown trout, conservative DNA extraction, fish fin clip, fish scales, northern pikeNon-destructive protocols for DNA isolationfrom fresh or preserved specimens are fundamen-tal to study endangered or elusive species, breedersor samples sibship and specimens derived frompublic or private collections (Nielsen et al. 1999;Wasko et al. 2003; Hansen and Jensen 2005;Lucentini et al. 2006). Because of the low yieldand poor quality DNA, particular laboratory careand standardizations were needed to allow repro-ducible results. We compared six DNA extractionprocedures from conservative samples: three arecommercial kits [Wizard Genomic DNA Purifi-cation Kit (WGDPK) (Promega); Wizard Mag-netic DNA Purification System for Food(WMDPF) (Promega); NucleoSpin Food (NSF)(Macheray–Nagel)] and three are largelyemployed methodologies [Trizol (Life Technolo-gies); Chelex (Sigma–Aldrich); C-TAB]. For eachmethod, the standard protocol (reported on datasheets or the first published one) and severalvariations were tested varying sample storage andpre-lysis conditions, homogenization procedures,buffer solutions and concentrations, incubationsand resuspension times and temperatures. DNAwas extracted from 200 northern pikes (Esoxlucius L.) and 100 brown trout (Salmo trutta farioL.) (Table 1) from small (£10 mg) fin pieces and5–10 scales stored dried or in absolute alcoholboth at )20 C and at room temperature. DNAextraction was carried out on fresh materials andrepeated within a period of three years (Table 1),and from 34 years old dried scales culled from amuseum collection. Furthermore, DNA wasextracted from liver and muscle of both species,from individuals that had naturally died and usedas controls. Spectrophotometric readings (260 and280 nm) and densitometric evaluations (Image J)of DNAs obtained through different methodolo-gies and protocols (conservative versus controlDNA) were analyzed by means of ANOVA andt-test. DNA suitability was assayed through PCR–RFLP and microsatellite analyses (Lucentini et al.(2006) (Table 2). Microsatellites were run onABI377 DNA sequencer whereas PCR–RFLPswere assayed on 2.5% agarose electrophoresis fortwo mtDNA NADH coding regions (ND-1 ND5/6) (Cronin et al. 1993). DNA stability andamplificability was controlled every three months,for three years, in parallel with control DNA. Toevaluate genotyping errors, null alleles presenceand allelic dropout, all the experiments were rep-licated and statistically treated as suggested(Hoffman and Amos 2005; Roon et al. 2005)through MICRO-CHECKER 2.2.3 (Vanoosterhout et al. 2004). Deviations from theHardy–Weinberg equilibrium were tested byArlequin 2000 and Genepop.Our results indicate that the quality and quan-tity of DNA extracted from fin clips and scalesvaried according to extraction methods (Table 2)and storage conditions. Alcohol preservation at)20 C is fundamental for fin specimens, althoughdried scales conservation at room temperatureallowed good DNA extraction. One percent

Tissue size and cell number in the olive (Olea europaea) ovary determine tissue growth and partitioning in the fruit

The relationship between tissue size and cell number in the ovary and tissue size in the fruit, was studied in eight olive (Olea europaea L.) cultivars with different fruit and ovary size. All tissues in the ovary increased in size with increasing ovary size. Tissue size in the fruits correlated with tissue size in the ovary for both mesocarp and endocarp, but with different correlations: the mesocarp grew about twice as much per unit of initial volume in the ovary. Tissue size in the fruit also correlated with tissue cell number in the ovary. In this case, a single regression fitted all data pooled for both endocarp and mesocarp, implying that a similar tissue mass was obtained in the fruit per initial cell in the ovary, independent of tissues and cultivars. Tissue relative growth from bloom to harvest (i.e. the ratio between final and initial tissue size) differed among cultivars and tissues, but correlated with tissue cell size at bloom, across cultivars and tissues. These results suggest that in olive, tissue growth and partitioning in the fruit is largely determined by the characteristics of the ovary tissues at bloom, providing important information for plant breeding and crop management.

Floral Biology: Implications for Fruit Characteristics and Yield

Floral biology has important practical implications, in addition to its scientific relevance, given that flower characteristics and bloom affect fruit characteristics and yield. Yield derives from fruit quality (e.g. weight) and quantity (i.e. number), which, in turns, depend on flower quantity and quality: flowers must be suitable to become fruits, and then must be pollinated and fertilized, and must set fruits, which must then grow. Not all flowers can do all of this: some flowers, for instance, have aborted ovaries which are partially developed or absent at bloom, depending on when the abortion occurred. Even when still present, these aborted ovaries are not capable of becoming fruits. Normal pistils, may not be pollinated or fertilized, but also fertilized ovaries may drop after some growth, resulting in fruit drop. From 100 flowers, in olive, all the above phenomena result in one to few fruits (Hartmann, 1950). Because of this low fruit set, it is often believed that cultural practices aimed at improving pollination, increasing fruit set or reducing ovary abortion or fruit drop, may lead to increased olive yields.