The identification and characterization of transcripts involved in flower organ development, plant reproduction and metabolism represent key steps in plant phenotypic and physiological pathways, and may generate high-quality transcript variants useful for the development of functional markers. This study was aimed at obtaining an extensive characterization of the olive flower transcripts, by providing sound information on the candidate MADS-box genes related to the ABC model of flower development and on the putative genetic and molecular determinants of ovary abortion and pollen-pistil interaction. The overall sequence data, obtained by pyrosequencing of four cDNA libraries from flowers at different developmental stages of three olive varieties with distinct reproductive features (Leccino, Frantoio and Dolce Agogia), included approximately 465,000 ESTs, which gave rise to more than 14,600 contigs and approximately 92,000 singletons. As many as 56,700 unigenes were successfully annotated and provided gene ontology insights into the structural organization and putative molecular function of sequenced transcripts and deduced proteins in the context of their corresponding biological processes. Differentially expressed genes with potential regulatory roles in biosynthetic pathways and metabolic networks during flower development were identified. The gene expression studies allowed us to select the candidate genes that play well-known molecular functions in a number of biosynthetic pathways and specific biological processes that affect olive reproduction. A sound understanding of gene functions and regulatory networks that characterize the olive flower is provided.

Circadian clocks are internal molecular time-keeping mechanisms that provide living organisms with the ability to adjust their growth and physiology and to anticipate diurnal environmental changes. Circadian clocks, without exception, respond to light and, in plants, light is the most potent and best characterized entraining stimulus. The capacity of plants to respond to light is achieved through a number of photo-perceptive proteins including cryptochromes and phytochromes. There is considerable experimental evidence demonstrating the roles of photoreceptors in providing light input to the clock. In order to identify genes regulated by diurnal and circadian rhythms, and to establish possible functional relations between photoreceptors and the circadian clock in tomato, we monitored the temporal transcription pattern in plants entrained to long-day conditions, either by large scale comparative profiling, or using a focused approach over a number of photosensory and clock-related genes by QRT-PCR. In parallel, focused transcription analyses were performed in cry1a- and in CRY2-OX tomato genotypes. We report a large series of transcript oscillations that shed light on the complex network of interactions among tomato photoreceptors and clock-related genes. Alteration of cryptochrome gene expression induced major changes in the rhythmic oscillations of several other gene transcripts. In particular, over-expression of CRY2 had an impact not only on day/night fluctuations but also on rhythmicity under constant light conditions. Evidence was found for widespread diurnal oscillations of transcripts encoding specific enzyme classes (e.g. carotenoid biosynthesis enzymes) as well as for post-transcriptional diurnal and circadian regulation of the CRY2 transcript.

Rapid and effective genotyping is an important goal to discriminate among the numerous olive cultivars and their wild related forms. The largely used di-nucleotide simple sequence repeat (SSR) markers show a high level of polymorphism and have strongly contributed to solve many inconsistencies in varietal identity, but many problems related to difficult discrimination of neighboring alleles and low comparability of data among different labs severely reduce their applicability for large-scale screening. The availability of numerous transcriptome libraries, which were developed from different tissues of several olive varieties, has allowed their intensive screening to search for polynucleotide microsatellite regions with long core repeats, potentially polymorphic among varieties. An accurate screening of all these polymorphisms has allowed to select a set of 25 trinucleotide and one tetranucleotide SSRs, showing a good level of discrimination power with a high allele pattern resolution and repeatability. They were preliminarily tested on a group of cultivated varieties then validated on a wider group of cultivated and wild plants, and related species and subspecies, demonstrating a good transferability within the entire Olea taxon. Furthermore, an in silico functional prediction has allowed to assign each transcribed sequence to their gene functions and biological process categories, highlighting their potential application of these new EST-SSRs as functional markers.