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The genus Brachypodium is generally considered taxonomically difficult because of the high levels of phenotypic variation and frequent hybridization between its species. In this study, the patterns of genetic variation of 180 lines of B. distachyon (B. distachyon, B Stacei and B. hybridum) collected from diverse regions in Tunisia were investigated using 18 morpho-phenological traits and 15 SSR markers. Our results showed that the polyploid species B. hybridum is the most abundant in Tunisia. Moreover, considerable morphological variation was revealed for the majority of analyzed traits. The variation was found to be more pronounced within B. hybridum, than its two progenitors. Importantly, we found that B. distachyon was characterized by early maturity, B. stacei differed from the two other species by plant height, whereas the allotetraploid species was mainly characterized by traits related to biomass production. At the molecular level, the fifteen SSR marker were found to be very informative in terms of polymorphism and allelic diversity (P = 100%, Na = 20.3 and Ne = 10.8). B. hybridum was found to be much variable than B. stacei and B. distachyon (He = 0.86, He = 0.78 and He = 0.58, respectively). FCA and Bayesian clustering showed a clear separation between the three species, which suggests the high usefulness of SSR markers used in our study in the delimitation between B. distachyon, B. stacei and B. hybridum.

Oxidations in grape berries are gaining major interest as they affect grape characteristics and quality. Considering berries, Reactive Oxygen Species are involved in the responses to both ripening process and stresses, including photooxidative sunburn. Redox metabolism involves a multitude of chemical and enzymatic reactions. In this study, four white grape cultivars were examined for natural ripening and photooxidative sunburn effects (obtained in artificial conditions) on berry pigmentation, chemical composition and enzymatic activity. The measured parameters included reflectance spectra, pigmentation (including berry browning), content of photosynthetic pigments, organic acid profiles, antioxidant activity, concentrations of antioxidants (total phenolics, ascorbic acid and reduced glutathione), enzymatic activities (guaiacol peroxidases, ascorbate peroxidase and catalase). The effects of the treatment (natural ripening and artificial photooxidative sunburn) on each considered parameter are described in the paper. Photooxidative sunburn strongly affected the contents of antioxidants and chlorophylls, increased the browning index and modulated the enzymatic activities investigated. Samples clearly clustered depending on the oxidation status. Furthermore, the PCA highlighted the similarities and differences in the responses to oxidative stress during ripening and photooxidative sunburn. PCA produced five functions with eigenvalues higher than 1, representing 87.03% of the total variability. In particular, the scores of the function 1 discriminated the samples based on the oxidation status, while the function 2 separated the samples based on the sampling date, representing the physiological responses characteristic of ripening. Our work sheds light on this topic, and will allow a more conscious vineyard management, thus supporting the agricultural adaptation to climate changes.

Sharka, a virosis that affects the most important fruit species belonging to the Prunus genus, is a major disease whose consequences heavily impact stone fruit production. Despite many decades have passed since the disease was first reported and its etiological agent, the potyvirus-type Plum Pox Virus, was recognized as the causative agent, the mechanism of its virulence still remains unclear. The virus is propagated by both its natural insect vectors, mainly aphids and by agronomical practices like the propagation through infected material. The diffusion of the disease can only be attained through removal of infected plants in the orchards, provided that appropriate early diagnostics measures are adopted. A second measure of intervention is provided by the control of aphid populations in the orchards although this seems to be a time-limited strategy. A third strategy is represented by the cultivation of plant material featuring inborn resistance to the virus. Although this seems the most appropriate way to address the problem, it is of limited applicability. In fact, while in some Prunus species, like apricot, there is evidence of some type of resistance, no clear sources of resistance are known for others like peach or plum. A fourth strategy is represented by transgenesis, provided that solid sources (genes) of resistance are identified and transformation and regeneration protocols are available. While this seems to be the most promising way, in the medium term, its applicability is still limited by public and political acceptance of the transgenic approach. Various programs are underway to investigate the type of resistance and the factors involved. In this review we address the various aspects of the investigation on PPV and its control which encompass the genetics and the physiology of plant-virus interaction, the identification of candidate genes to be used to limit its infection and spread, the most modern ways to early diagnose the infection and the cutting-edge approaches of gene silencing.

Sharka, a common disease among most stone fruit crops, is caused by the Plum Pox Virus (PPV). Resistant genotypes have been found in apricot (Prunus armeniaca L.), one of which--the cultivar 'Lito' heterozygous for the resistance--has been used to map a major quantitative trait locus (QTL) on linkage group 1, following a pseudo-test-cross mating design with 231 individuals. In addition, 19 SNP markers were selected from among the hundreds previously developed, which allowed the region to be limited to 236 kb on chromosome 1. A 'Lito' bacterial artificial chromosome (BAC) library was produced, screened with markers of the region, and positive BAC clones were sequenced. Resistant (R) and susceptible (S) haplotypes were assembled independently. To refine the assembly, the whole genome of 'Lito' was sequenced to high coverage (98×) using PacBio technology, enabling the development of a detailed assembly of the region that was able to predict and annotate the genes in the QTL region. The selected cultivar 'Lito' allowed not only to discriminate structural variants between the two haplotypic regions but also to distinguish specific allele expression, contributing towards mining the PPVres locus. In light of these findings, genes previously indicated (i.e., MATHd genes) to have a possible role in PPV resistance were further analyzed, and new candidates were discussed. Although the results are not conclusive, the accurate and independent assembly of R and S haplotypes of 'Lito' is a valuable resource to predict and test alternative transcription and regulation mechanisms underpinning PPV resistance.

For a long time ‘Chinotto di Savona’, a Citrus tree cultivated in Liguria region (northern Italy), has been considered a variety of Citrus myrtifolia (known with the Italian name ‘chinotto’). Nevertheless, molecular analysis to address its true genetic origin has been not performed yet. Fruits of ‘Chinotto di Savona’ are highly appreciated and often preferred to common chinotto for its superior organoleptic characteristics by food and beverage industries. This work reports a first morphological and genetic characterization of ‘Chinotto di Savona’ aiming at inferring its origin and main pomological traits. The main phenotypic characteristics of this Citrus accession were also evaluated and compared with those of the ‘common’ chinotto, considering leaves, shoots, fruits and seed morphology and juice composition. In addition, the colour evolution of fruit flavedo was monitored by reflectance spectroscopy. Molecular analyses were performed using SSR, AFLP and RNA-seq-derived markers, providing tools to discriminate ‘Chinotto di Savona’ from C. myrtifolia. A cytogenetic analysis was performed to assess ploidy of the Chinotto accessions. Findings confirm the genetic similarity of ‘Chinotto di Savona’ and C. myrtifolia, supporting the running hypothesis of their origin from somatic mutations of sour orange. Further analysis will be needed in order to fully understand whether they are independent somatic mutations of sour orange or if they originated from each other.

Applying next-generation sequencing (NGS) technologies to species of agricultural interest has the potential to accelerate the understanding and exploration of genetic resources. The storage, availability and maintenance of huge quantities of NGS-generated data remains a major challenge. The PeachVar-DB portal, available at http://hpc-bioinformatics.cineca.it/peach, is an open-source catalog of genetic variants present in peach (Prunus persica L. Batsch) and wild-related species of Prunus genera, annotated from 146 samples publicly released on the Sequence Read Archive (SRA). We designed a user-friendly web-based interface of the database, providing search tools to retrieve single nucleotide polymorphism (SNP) and InDel variants, along with useful statistics and information. PeachVar-DB results are linked to the Genome Database for Rosaceae (GDR) and the Phytozome database to allow easy access to other external useful plant-oriented resources. In order to extend the genetic diversity covered by the PeachVar-DB further, and to allow increasingly powerful comparative analysis, we will progressively integrate newly released data.

Brachypodium hybridum has recently been recognized as an allotetraploid species deriving from natural hybridization between B. distachyon and B. stacei . The identification of this species which emerged as a new model for polyploid species has spawned considerable scientific interest. A lot of investigations using highly variable markers and a broad geographical sampling are underway to understand the patterns of their genetic diversity and the contribution of polyploidy to adaptation. As a step towards understanding the genetic basis for natural variation of this species in Tunisia, 15 microsatellite markers and 145 genotypes representing 9 natural populations were used. Results exhibit a high level of polymorphism (159 polymorphic alleles with an average of 8.6 alleles per locus) and high level of genetic diversity in all the studied populations with expected and observed heterozygosities averaging 0.79 and 0.31, respectively. AMOVA resulted in a high variation within population (97%) and altitude as compared to the variation among populations (3%, Φ PT = 0.023), altitude and eco-region. Results also showed that the 9 populations grouped into 3 clusters without geographical or altitudinal clustering. This grouping was accompanied by a high rate of gene flow between populations (Nm = 2.31). Interestingly, Mantel test identified that the extent of genetic differentiation was not correlated to geographic distances ( r = 0.046; p = 0.78) nor to the altitudinal differences ( r = −0.29; p = 0.75). In the present study we report a high rate of successful cross-amplification of B. distachyon SSR markers in B. hybridum , which implies the great transferability between these two species. Moreover, genetic characterization of the Tunisian populations of B. hybridum by SSR markers suggests that the spatial genetic structure of this species may be affected by a multidirectional gene flow via long-distance seed dispersal, leading to a weak genetic structure.

Background Plum pox virus (PPV), agent of Sharka disease, is the most important quarantine pathogen of peach (P. persica L. Batsch). Extensive evaluation of peach germplasm has highlighted the lack of resistant sources, while suggesting the presence of a quantitative disease resistance, expressed as reduction in the intensity of symptoms. Unravelling the genetic architecture of peach response to PPV infection is essential for pyramiding resistant genes and for developing more tolerant varieties. For this purpose, a genome-wide association (GWA) approach was applied in a panel of accessions phenotyped for virus susceptibility and genotyped with the IPSC peach 9 K SNP Array, and coupled with an high-coverage resequencing of the tolerant accession ‘Kamarat’. Results Genome-wide association identified three highly significant associated loci on chromosome 2 and 3, accounting for most of the reduction in PPV-M susceptibility within the analysed peach population. The exploration of associated intervals through whole-genome comparison of the tolerant accession ‘Kamarat’ and other susceptible accessions, including the PPV-resistant wild-related species P. davidiana, allow the identification of allelic variants in promising candidate genes, including an RTM2-like gene already characterized in A. thaliana. Conclusions The present study is the first effort to identify genetic factors involved in Sharka disease in peach germplasm through a GWA approach. We provide evidence of the presence of quantitative resistant loci in a collection of peach accessions, identifying major loci and highly informative SNPs that could be useful for marker assisted selection. These results could serve as reference bases for future research aimed at the comprehension of genetic mechanism regulating the complex peach-PPV interaction. Electronic supplementary material The online version of this article (10.1186/s12870-017-1117-0) contains supplementary material, which is available to authorized users.

Sharka, caused by plum pox virus, is the most important viral disease of stone fruits. Important progresses have been recently achieved in apricot (Prunus armeniaca), identifying a major locus on chromosome 1 which explains most of the variability for plum pox virus (PPV) resistance trait. A set of molecular markers associated with the resistance has been developed and validated in different genetic backgrounds, endorsing their application for breeding purposes. Particularly for complex traits as the PPV resistance, requiring long and expensive phenotyping procedures, marker-assisted selection (MAS) bears a great potential to improve the efficiency of conventional breeding. In this work, novel HRM (high-resolution melting) assays were designed for the genotyping of resistant/susceptible alleles at PPV resistance (PPVres) locus. The assays were tested on 51 apricot cultivars and breeding selections already phenotyped for PPV resistance and cross-validated with standard short simple repeat marker data. We demonstrated that three HRM assays, PGS1.21_SNP, PGS1.24_SNP, and ZP002_DEL, represent a reliable, quick, and cost-effective genotyping approach, particularly suitable as high-throughput screening method for large-scale breeding programs.

Sharka is a devastating disease in stone fruit species caused by the Plum pox virus (PPV). Unlike other plant pathogens, PPV cannot be directly controlled by chemical sprays. Alternative approaches include the control of insect vectors and the use of pathogen-resistant cultivars. This work is aimed at developing new tools to understand the genetic control of resistance. A BAC library, covering about 10× the 'Lito' haploid genome, was used in order to identify candidate genes for Sharka resistance in apricot. SSRs markers covering the linkage group 1 of 'Lito', where resistance is located, were used for a preliminary screening of the BAC library. In a second step the screening was focused in a region between 6 and 8.5 Mbp (on the reference peach genome), that was covered by more than 50 new markers developed on both 'Lito' re-sequenced genome and sequenced BAC ends. Several BAC clones from both the resistant and susceptible chromosomes were identified (17 and 18 clones, respectively) in order to obtain the minimum tiling path covering the whole resistant and susceptible chromosome regions. The BAC clones sequencing by Illumina and assembly is in progress. The new markers developed in this work could be used to speed up the selection process in apricot breeding programs.

SUMMARYBrachypodium hybridumbelongs to thePoaceaegrass subfamily. It has a close genetic relationship with temperate cereal crops, which means that it can be used as a model for temperate cereal and grass crops. In order to improve knowledge on the genetic diversity of this species, 145 lines ofB. hybridumrepresentative of nine populations and all the ecoregions of Tunisia were characterized on the basis of 18 morpho-phenologic features. The results show a considerable variation between populations and ecoregions in all traits studied. Variation was relatively higher for reproductive than vegetative traits. The majority of traits showed very low to high heritability with low border value for average length of spikelet (ALS) and an average value of 0·64. It is noticeable that high values of heritability were observed for most vegetative descriptors, with low values for reproductive ones. Differentiation between populations (QST) varied from 0·02 for ALS to 0·78 for average length of leaves with a mean value across traits of 0·4, which confirms the wide intra-population variation in Tunisian natural population ofB. hybridum. PairwiseQSTshowed that the greatest differentiation among populations was registered between Ain Drahem and Jbel Zaghouan and the smallest between Haouria and Raoued. Overall, the Ain Draham population showed the largest differentiation from the rest of the populations. To infer the effect of geographic distribution of the species, a Mantel test was applied between observed pairwise differentiation and geographic distance between populations and between ecoregions: the results show a positive, but not significant, relationship. In addition a significant negative relationship was found between phenotypic diversity and altitude, indicating that genetic diversity decreased with increasing altitude. Taken together, the high levels of intra-population variation and the lack of correlation between genetic differentiation and geographic distribution suggest a potentially important rate of long-distance seed dispersal and confirm the role played by natural selection in the population structure of Tunisian natural populations ofB. hybridum.

Sharka disease, caused by the Plum Pox Virus (PPV), is endangering apricot industry in many countries worldwide. Both PPV-D (Dideron) and PPV-M (Marcus) strains are able to determine severe crop losses, with the latter strain being the most dangerous. We selected 'Lito', among the cultivars described in literature as resistant, and crossed to the susceptible accession 'B081604311' to produce a double pseudo-test cross progeny. A population of 118 individuals was phenotyped and genotyped to identify molecular markers linked to the genetic determinant/s of Sharka resistance. Both PPV D and M strains were inoculated in seedling replicates and symptoms visually scored for three years. ELISA and PCR assays were also done. A detailed SSR-based molecular map was developed and used for QTL analysis. A major QTL for both PPV-M and PPV-D strains was found at the top of the linkage group 1 of 'Lito', in the same region where a QTL of resistance to Sharka was found in 'Stark Early Orange', the resistant parent of 'Lito', by Lambert and coworkers (2007). To pave the way for the positional cloning of the DNA sequence, the 'Lito' × 'BO81604311' progeny has been enlarged and further 241 seedlings were genotyped with old and new SSR primers that allowed saturating the QTL region in the LG1. Phenotyping with both PPV strains is under way. A random shearing 10X BAC library prepared with inserts >100 kb is being screened with markers surrounding the QTL. Positive BACs will be physically mapped to produce a contig of the genomic region including the main QTL. Finally, as a first step towards genetic transformation of apricot for resistance to PPV, constructs for either CP overexpression or post-transcriptional silencing were used, and transgenic callus lines were recovered from Agrobacterium-inoculated mature tissues, highly recalcitrant to shoot regeneration.

We report here the first integrated investigation of both ancient DNA and proteins in archaeobotanical samples: medieval grape (Vitis vinifera L.) seeds, preserved by anoxic waterlogging, from an early medieval (seventh–eighth century A.D.) Byzantine rural settlement in the Salento area (Lecce, Italy) and a late (fourteenth–fifteenth century A.D.) medieval site in York (England). Pyrolysis gas chromatography mass spectrometry documented good carbohydrate preservation, whilst amino acid analysis revealed approximately 90% loss of the original protein content. In the York sample, mass spectrometry-based sequencing identified several degraded ancient peptides. Nuclear microsatellite locus (VVS2, VVMD5, VVMD7, ZAG62 and ZAG79) analysis permitted a tentative comparison of the genetic profiles of both the ancient samples with the modern varieties. The ability to recover microsatellite DNA has potential to improve biomolecular analysis on ancient grape seeds from archaeological contexts. Although the investigation of five microsatellite loci cannot assign the ancient samples to any geographic region or modern cultivar, the results allow speculation that the material from York was not grown locally, whilst the remains from Supersano could represent a trace of contacts with the eastern Mediterranean. Electronic supplementary material The online version of this article (doi:10.1007/s00114-009-0629-3) contains supplementary material, which is available to authorized users.

Amplified fragment length polymorphism (AFLP) molecular markers were employed for the fingerprinting of 118 accessions of apricot, including cultivated varieties and related apricot species. Five primer combinations were tested and 165 polymorphic bands produced which could uniquely differentiate all accessions under investigation. Primer combinations were rated according to the multiplex ratio, the polymorphic index content and the discrimination power parameters and superior combinations were identified. AFLP markers were used in cluster analysis carried out with the UPGMA and the neighbour-joining methods and parsimony analysis. Four groups of similar accessions were identified: (i) those from the Mediterranean basin, (ii) from China, (iii) from continental Europe and (iv) mixed Europe-North America accessions. In the principal component analysis the first three components accounted for 17% of the total variability existing among accessions. Because the most important regions where apricot is cultivated in the world were represented in the analysis, with a large number of varieties, it was possible to discuss the data in the light of current phylogenetic hypotheses on the origin and evolution of the species.

International audience; Sharka is one of the most serious viral diseases affecting stone fruit species and, in apricot, resistance to its viral agent, the Plum Pox Virus (PPV), is conferred by one major quantitative trait locus (QTL), named PPVres for PPV resistance. Previous studies indicated that PPV-resistant cultivars and breeding progenies can be selected by using a set of SSR markers (named PGS) targeting the PPVres locus. However, before these markers can be employed for marker-assisted selection, they were validated in a wide range of genetic backgrounds and environments. We used a total of 11 mapping populations issued from three distinct environments to confirm that this marker set located within the QTL adequately predicted PPV resistance. In this study, we show that selection of PPV-resistant material based only on markers co-localizing with the PPVres major locus is not fully reliable. Indeed, genotype-phenotype discrepancies were observed depending on the progeny and the PPV-resistant/susceptible parents. While most of the PPV-resistant individuals displayed the resistant alleles, a significant number of PPV-susceptible individuals showed the same resistant haplotype. An effect of the PPV strain used for phenotyping was also demonstrated. We thus hypothesize that the presence of other factors or genes involved in the mechanism of resistance to sharka in apricot could explain these unexpected results. Our work indicates that the current PGS marker set is not broadly applicable for MAS and that marker-assisted breeding based on the sole PPVres locus is not sufficient to unambiguously select PPV-resistant apricot cultivars.

A quick, inexpensive, and reliable protocol for the extraction of RNA from grapevine berry skins containing large quantities of polyphenols, procyanidins, and polysaccharides is described. The method involves an extraction step in the presence of ribonuclease inhibitors and compounds that compete with vacuolar contaminants for binding to RNA. After extraction with organic solvents, RNA is bound to a fibrous cellulose matrix and processed to eliminate the remaining contaminants and ribonucleases. Following this method, highly stable RNA, sufficiently pure for northern hybridizations and enzymatic processing, may be obtained from as little as 200 mg of starting amounts of fresh material and without multiple, time consuming precipitations or ultracentrifugation steps. This procedure may also prove useful for extracting RNA from recalcitrant tissues of other plant species. Abbreviations: ATA, aurintricarboxylic acid; CF11, cellulose fibrous medium (type 11); PVPP, polyvinylpolypyrrolidone; RT room temperature; VRC, vanadyl ribonucleoside complex.

The degree of polymorphism present in 5′ untranslated regions of stilbene synthase (StSy)-like loci was assessed. A ligation-mediated polymerase chain reaction (LM-PCR) cloning strategy was adopted to isolate sequences located immediately upstream of StSy coding regions. Among several clones, 13 randomly chosen fragments were analyzed at the sequence level. Four of the analyzed fragments appeared of particular interest. Two carried sequences reminiscent of micro-satellites, while the remaining fragments contained direct repeats. Oligonucleotides constructed against the specific DNA sequence of these clones disclosed a complex banding pattern when used in polymerase chain reaction (PCR)-analysis of 22 ancient varieties of grapevine. A total of 40 polymorphic bands could be identified and used to calculate coefficients of genetic similarity (GS) between varieties. GS values were used in cluster analysis to differentiate the 22 varieties. The data obtained are in good agreement with available information concerning the relationships between the varieties considered. This suggests the use of the method we have developed in fingerprinting studies of Vitis vinifera germ plasma.

Sharka is a severe apricot viral disease caused by the plum pox virus (PPV) and is responsible for large crop losses in many countries. Among the known PPV strains, both PPV-D (Dideron) and PPV-M (Marcus) are virulent in apricot, the latter being the most threatening. An F1 apricot progeny derived from Lito, described in the literature as resistant, crossed to the susceptible selection BO81604311 (San Castrese × Reale di Imola) was used to study the genetic control of resistance to PPV. A population of 118 individuals was phenotyped by inoculating both PPV-D and PPV-M strains in replicated seedlings and scored for 3 years. An additional set of 231 seedlings from the same cross was also phenotyped for 2 years. SSR-based linkage maps were used for quantitative trait locus (QTL) analysis. A major QTL of resistance to both PPV-M and PPV-D strains was found in the top half of the Lito linkage group 1, where a QTL was previously described in Stark Earli-Orange, the donor of Lito resistance. The LOD score was considerably enhanced when the recovery of plants from infection was taken into account. The results obtained in Lito were compared with those observed in a second apricot cross progeny (Harcot × Reale di Imola) in which QTL of resistance to sharka were also mapped in the same linkage group 1 for both PPV strains. Several models of resistance to sharka disease are discussed considering the segregation frequencies, the QTL alignment in the two maps and the information gathered from the literature.

Considering the high degree of sequence conservation within the Rosaceae family and, in particular, among the Prunus species, we employed the first available peach oligonucleotide microarray (μPEACH 1.0) for studying the transcriptomic profile during apricot (cv. ‘Goldrich’) fruit development. Apricot fruits were harvested at three distinct developmental stages, corresponding to immature green (6 weeks before fully ripe stage), mature firm ripe (change of peel colour, 1 week before fully ripe stage) and fully ripe, namely T1, T2 and T3, respectively. When applied to μPEACH1.0, apricot target cDNAs showed significant hybridization with an average of 43% of spotted probes, validating the use of μPEACH1.0 to profile the transcriptome of apricot fruit. Microarray analyses carried out separately on peach (cv. ‘Fantasia’) and apricot fruit to profile transcriptome changes during fruit development showed that 70% of genes had the same expression pattern in both species. Such data indicate that the transcriptome is quite similar in apricot and peach fruit and also highlight the presence of species-specific transcript changes. In apricot, 400 and 74 differentially expressed genes were found during the transition from T1 to T2 and from T2 to T3, respectively. Among these, a considerable number of genes encoding IAA protein in action regulators (Aux/IAA) and heat shock proteins (HSPs) were highly up-regulated at early and late ripening, respectively. Intriguingly, the expression profiles of all considered HSPs and some of IAA protein /IAA genes showed different patterns between apricot and peach during the last stages of on-tree fruit development, suggesting the presence of diverse mechanism regulating ripening in these two close phylogenetically related species.

There is a high degree of sequence conservation within the Family Rosaceae and, in particular, among the Prunus species. The first available peach microarray (µPEACH1.0) was employed for the investigation of the transcription profile during apricot (Prunus armeniaca) fruit growth and development. Fruits (cv. ‘Goldrich’) were harvested at two growth stages, corresponding to immature (6 weeks before harvest) and ripe (at harvest) stage. Overall, this preliminary transcriptomic approach suggests that peach microarray can be employed in a heterologous fashion. Amongst the 746 genes which could be statistically analysed, 287 were induced, while 260 were down-regulated. In addition to the well documented role of genes implicated in ethylene biosynthesis perception and transduction as well as those involved in cell wall metabolism, this study brings into light a putative role for genes encoding auxin-regulated proteins, and those responsive to stress conditions and to other biotic or abiotic stimuli. The data of the present study can also be used for comparative purposes within Prunus species, since for 203 and 189 genes that were up- and down-regulated respectively during apricot fruit development, data exist for their regulation during the last stages of peach fruit ripening. Future experiments should include analysis of more fruit stages and data validation for the genes of interest.

In this study, a total of 14 agronomic traits, five AFLP primer combinations and ten SSR loci were used to describe and to classify a group of Tunisian olive genotypes into groups based on molecular profiles and agronomic traits. The analysis of variance of the agronomical data revealed significant differences among accessions for all measured traits. The mean phenotypic dissimilarity (0.34 with a range of 0.08-0.6) was low in comparison to dissimilarity calculated using AFLP (0.50 with a range of 0.16-0.70) and SSR markers (0.76 with a range 0.35-0.94). The correlation between the agronomical dissimilarity matrix and the matrices of genetic dissimilarity based on SSR and AFLP markers was very weak: 0.156 (p = 0.05) and 0.185 (p = 0.05), respectively. The SSR-AFLP dendrogram based on unweighted pair-group cluster analysis using Jaccard's index revealed that the genetic diversity was predominantly structured according to fruit size. A trend of clustering together of accessions originating from the same or adjacent regions was also observed. The data obtained can be used for the varietal survey and construction of a database of all olive varieties grown in Tunisia and providing also additional information that could form the basis for the rational design of breeding programs.

Cultivated tomato (Solanum lycopersicum L.) germplasm shows limited genetic variation. Many DNA marker systems have been used for genetic diversity studies in wild and cultivated tomatoes, but their usefulness for characterizing phenotypic differences among very closely related cultivars remains uncertain. We have used 19 selected simple sequence repeat (SSR) markers and 7 amplified fragment length polymorphism (AFLP) primer combinations to characterize 48 cultivars of tomato, mainly traditional cultivars from the south-east of Spain. The main types were Solanum lycopersicum L. 'Muchamiel', 'De la pera', and 'Moruno'. The robustness of the dendrograms and the discrimination power reached with each marker type were similar. Unique fingerprinting even of the most closely related tomato cultivars could be obtained using a combination of some SSR and AFLP markers. A better grouping of the 'Muchamiel' cultivars was observed with SSR markers, whereas the grouping of cultivars of 'De la pera' type was best achieved with AFLPs. However, both types of markers adequately grouped cultivars of the main types, confirming the utility of SSR and AFLP markers for the identification of traditional cultivars of tomato.Key words: genetic variability, molecular markers, Solanum lycopersicum.

The application of the complementary DNA (cDNA)–amplified fragment length polymorphism (AFLP) technique to clone transcripts differentially expressed during fruit development and ripening is reported. Using 34 different primer combinations, 265 cDNA–AFLP bands were found differentially expressed in six fruit developmental stages, with leaf and developing seed as controls. One hundred twenty-five bands were cloned and sequenced and database search allowed to identify genes involved in cell wall, sugar, lipid, organic acids, and protein metabolism, as well as genes participating in hormonal signaling and in signal transduction (51 sequences, 41% of total). Genomic and expression analyses for a group of genes as well as phylogenetic investigations were carried out. cDNA–AFLP profiles have been also compared to the corresponding Northern hybridization data. Several genes are described for the first time in apricot or in other Prunus species. They establish the basis for further investigation of the ripening process in stone fruits.

The R gene family determines the timing, distribution and amount of anthocyanin pigmentation in maize. This family comprises a set of regulatory genes, consisting of a cluster of several elements at the R locus, on chromosome 10, the Lc and Sn gene lying about two units R distal and B on chromosome 2. Each gene determines a tissue-specific pigmentation of different parts of the seed and plant. The proposed duplicated function of R, Sn, Lc and B loci is reflected in cDNA sequence similarity. In this paper an extensive analysis of the predicted proteins of the R, Sn, Lc and B genes together with a search for putative sites of post-translational modification is reported. A comparison with the prosite database discloses several N-glycosylation and phosphorylation sites, as well as the basic Helix-Loop-Helix (HLH) domain of transcriptional activators. Sn, Lc, and R-S show a high conservation of these sites, while B is more divergent. Analysis of the 5' leader of mRNA sequences discloses the presence of five ATG triplets with two upstream open reading frames (uORFs) of 38 and 15 amino acids and a loop structure indicating a possible mechanism of control at the translational level. It is conceivable that possible mechanisms acting at the translational and post-translational level could modulate the expression and the activation of these transcription factors. Northern analysis of various tissues of different R alleles highlights a strict correlation between pigment accumulation in different tissues and the expression of the regulatory and structural genes suggesting that the pattern of pigmentation relies on a mechanism of differential expression of the members of the R family. Analysis of the Sn promoter discloses the presence of several sequences resembling binding sites of known transcription factors (as GAGA and GT) that might be responsible for the spatial and light-induced expression of this gene. Two regions include a short sequence homologous to the consensus binding site of the B-HLH domain suggesting a self-regulatory control of the Sn gene.

Isolating nucleic acids from sources rich in contaminants is particularly cumbersome when treating a large number of samples. Several protocols have been published that address the problem of nucleic acid extraction and purification, but few address sample number. We describe a method for extracting DNA from recalcitrant tree species by using a commercial grinding apparatus. This alleviates the hard work of sample preparation prior to lysis and purification. Our method has been tested extensively on different fruit tree species and in projects that require the simultaneous processing of hundreds of samples. Moreover, it does not require the availability of robotic workstations.

SummaryTwenty-eight accessions of the Tunisian olive (Olea europaea L.) cultivars ‘Chemlali’ and ‘Chetoui’ were screened using 11 microsatellite (SSR) primer pairs to evaluate within-cultivar variability, and to eliminate mis-labelling and redundancies before their introduction into the ‘Borj Cedria’ olive collection. Variations in microsatellite patterns were observed among accessions under the same denomination. Accessions designated ‘Chemlali’ showed three different microsatellite profiles, which differed at all loci analysed and should therefore correspond to three different varieties. However, real clonal or intra-cultivar variability was observed in one of the three varieties, as well as in the cultivar ‘Chetoui’, with differences between accessions at only one of the 11 loci screened. These results confirm the efficacy of microsatellite (SSR) markers for the classification and selection of olive accessions for germplasm collections.