Simple sequence repeats (SSRs), consisting of tandemly repeated multiple copies of mono-, di-, tri-, or tetranucleotide motifs, are ubiquitous in eukaryotic genomes and are frequently used as genetic markers, taking advantage of their length polymorphism. We have examined the polymorphism of such sequences in the chloroplast genomes of plants, by using a PCR-based assay. GenBank searches identified the presence of several (dA).-(dT). mononucleotide stretches in chloroplast genomes. A chloroplast (cp) SSR was identified in three pine species (Pinus contorta, Pinus sylvestris, and Pinus thunbergii) 312 bp upstream of the psbA gene. DNA amplification of this repeated region from 11 pine species identified nine length variants. The polymorphic amplified fragments were isolated and the DNA sequence was determined, confirming that the length polymorphism was caused by variation in,the length of the repeated region. In the pines, the chloroplast genome is transmitted through pollen and this PCR assay may be used to monitor gene flow in this genus. (5,6). In plants, chloroplast and mitochondrial genomes exhibit different patterns of genetic differentiation compared to nuclear alleles (7) due to their generally uniparental mode of transmission. Thus, for a comprehensive understanding of plant population differentiation and evolution, three interrelated genomes must be considered. The analysis of the chloroplast organelle provides information on the population dynamics of plants that is complementary to that obtained from the nuclear genome (8). The chloroplast genome is highly conserved and has a much lower mutation rate than plant nuclear genomes (9, 10). Restriction site analysis of chloroplast (cp) DNA has been widely used for interspecific studies and in some cases the magnitude of intraspecific variation has been sufficient to allow populationbased studies (11). It has been observed, at least in Angiosperms, that the majority of the intraspecific variation is found between populations with little or no intrapopulational variation possibly due to the maternal mode of inheritance. The availability of a high-resolution chloroplast-specific polymorphic assay should facilitate the analysis of population differentiation and gene flow. Furthermore, in gymnosperms where chloroplasts are primarily paternally transmitted (12), such an assay would allow pollen movement to be monitored.Although the presence of repeated sequence motifs that evolve faster than the surrounding DNA has been reported in chloroplasts (13-15), the exploitation of PCR-based SSR length assays has been restricted to the nuclear genome. In this report we describe the occurrence of SSRs in the chloroplast genome and demonstrate that chloroplast mononucleotide repeat motifs are highly polymorphic in Pinus species. The SSR identified from database sequences of three pine species was amplified by using primers homologous to conserved flanking sequences. These primers were tested on a wide array of pine species. The level and pattern of chloroplast in...

We used chloroplast polymerase chain reaction-restriction-fragment length polymorphism (PCR-RFLP) and chloroplast microsatellites to assess the structure of genetic variation and postglacial history across the entire natural range of the common ash (Fraxinus excelsior L.), a broad-leaved wind-pollinated and wind-dispersed European forest tree. A low level of polymorphism was observed, with only 12 haplotypes at four polymorphic microsatellites in 201 populations, and two PCR-RFLP haplotypes in a subset of 62 populations. The clear geographical pattern displayed by the five most common haplotypes was in agreement with glacial refugia for ash being located in Iberia, Italy, the eastern Alps and the Balkan Peninsula, as had been suggested from fossil pollen data. A low chloroplast DNA mutation rate, a low effective population size in glacial refugia related to ash's life history traits, as well as features of postglacial expansion were put forward to explain the low level of polymorphism. Differentiation among populations was high (GST= 0.89), reflecting poor mixing among recolonizing lineages. Therefore, the responsible factor for the highly homogeneous genetic pattern previously identified at nuclear microsatellites throughout western and central Europe (Heuertz et al. 2004) must have been efficient postglacial pollen flow. Further comparison of variation patterns at both marker systems revealed that nuclear microsatellites identified complex differentiation patterns in south-eastern Europe which remained undetected with chloroplast microsatellites. The results suggest that data from different markers should be combined in order to capture the most important genetic patterns in a species.

Combining molecular analyses with geological and palaeontological data may reveal timing and modes for the divergence of lineages within species. The Mediterranean Basin is particularly appropriate for this kind of multidisciplinary studies, because of its complex geological history and biological diversity. Here, we investigated chloroplast DNA of Quercus suber populations in order to detect possible relationships between their geographical distribution and the palaeogeographical history of the western Mediterranean domain. We analysed 110 cork oak populations, covering the whole distribution range of the species, by 14 chloroplast microsatellite markers, among which eight displayed variation among populations. We identified five haplotypes whose distribution is clearly geographically structured. Results demonstrated that cork oak populations have undergone a genetic drift geographically consistent with the Oligocene and Miocene break-up events of the European-Iberian continental margin and suggested that they have persisted in a number of separate microplates, currently found in Tunisia, Sardinia, Corsica, and Provence, without detectable chloroplast DNA modifications for a time span of over 15 million years. A similar distribution pattern of mitochondrial DNA of Pinus pinaster supports the hypothesis of such long-term persistence, in spite of Quaternary climate oscillations and of isolation due to insularity, and suggests that part of the modern geographical structure of Mediterranean populations may be traced back to the Tertiary history of taxa.

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We investigated range-wide phylogeographic variation in three European ash species (Fraxinus sp., Oleaceae). Chloroplast DNA (cpDNA) microsatellites were typed in the thermophilous Fraxinus angustifolia and Fraxinus ornus and the observed haplotypes and the geographic distribution of diversity were compared to cpDNA data previously obtained in the more cold-tolerant Fraxinus excelsior. We found wide-ranging haplotype sharing between the phylogenetically close F. angustifolia and F. excelsior, suggesting hybridization (i) in common glacial refuges in the Iberian Peninsula, northern Italy, the eastern and/or Dinaric Alps and the Balkan Peninsula, and/or (ii) during postglacial recolonization. The data allowed us to propose additional glacial refuges for F. angustifolia in southern Italy and in Turkey, and populations from the latter region were particularly polymorphic. There was evidence for refuge areas in Italy, the Balkan Peninsula and Turkey for F. ornus, which did not share any single chloroplast haplotype with the other species. In both F. angustifolia and F. ornus, cpDNA diversity (h(S) = 0.027 and h(S) = 0.009, respectively) was lower and fixation levels (G(ST) = 0.964 and G(ST) = 0.983, respectively) higher than in sympatric F. excelsior (h(S) = 0.096, G(ST) = 0.870). These diversity patterns could be due to temperature tolerance or the demographic history.

Three chloroplast microsatellites (cpSSRs), previously sequence characterized and for which paternal inheritance was tested and confirmed, were used to assess their usefulness as informative markers for phylogeographic studies in Norway spruce (Picea abies K.) and to detect spatial genetic differentiation related to the possible recolonization processes in the postglacial period. Ninety-seven populations were included in the survey. Some 8, 7, and 6 different size variants for the three cpSSRs, respectively, were scored by analysing 1105 individuals. The above 21 variants combined into 41 different haplotypes. The distribution of some haplotypes showed a clear geographic structure and seems to be related to the existence of different refugia during the last glacial period. The analysis of chloroplast SSR variation detected the presence of two main gene pools (Sarmathic-Baltic and Alpine--Centre European) and a relatively low degree of differentiation (RST of about 10%), characteristic of tree species with large distribution and probably influenced by an intensive human impact on this species. Based on our data, we were not able to detect any evidence concerning the existence of additional gene pools (e.g., from Balkan and Carpathian glacial refugia), though we cannot exclude the existence of genetic discontinuity within the species' European range. A large proportion of population-specific haplotypes were scored in this species, thus indicating a possible usefulness of these markers for the identification of provenances, seed-lots, and autochthonous stands.

The olive is an important fruit species cultivated for oil and table olives in Italy and the Mediterranean basin. The conservation of cultivated plants in ex situ collections is essential for the optimal management and use of their genetic resources. The largest ex situ olive germplasm collection consists of approximately 500 Italian olive varieties and corresponding to 85% of the total Italian olive germplasm is maintained at the Consiglio per la Ricerca e sperimentazione per l'Agricoltura, Centro di Ricerca per l'Olivicoltura e l'Industria Olearia (CRA-OLI), in Italy. In this work, eleven preselected nuclear microsatellite markers were used to assess genetic diversity, population structure, and gene flows with the aim of assembling a core collection. The dendrogram obtained utilizing the unweighted pair group method highlights the presence of homonymy and synonymy in olive tree datasets analyzed in this study. 439 different unique genotype profiles were obtained with this combination of 11 loci nSSR, representing 89.8% of the varieties analyzed. The remaining 10.2% comprises different variety pairs in which both accessions are genetically indistinguishable. Clustering analysis performed using BAPS software detected seven groups in Italian olive germplasm and gene flows were determined among identified clusters. We proposed an Italian core collection of 23 olive varieties capturing all detected alleles at microsatellites. The information collected in this study regarding the CRA-OLI ex situ collection can be used for breeding programs, for germplasm conservation, and for optimizing a strategy for the management of olive gene pools.