Search

A multi-technique approach, combining circular dichroism spectroscopy, ultraviolet resonance Raman spectroscopy and small angle scattering techniques, has been deployed to elucidate how the structural features of the human telomeric G-quadruplex d[A(GGGTTA)3GGG] (Tel22) change upon thermal unfolding. The system is studied both in the free form and when it is bound to Actinomycin D (ActD), an anticancer ligand with remarkable conformational flexibility. We find that at room temperature binding of Tel22 with ActD involves end-stacking upon the terminal G-tetrad. Structural evidence for drug-driven dimerization of a significant fraction of the G-quadruplexes is provided. When the temperature is raised, both free and bound Tel22 undergo melting through a multi-state process. We show that in the intermediate states of Tel22 the conformational equilibrium is shifted toward the (3+1) hybrid-type, while a parallel structure is promoted in the complex. The unfolded state of the free Tel22 is consistent with a self-avoiding random-coil conformation, whereas the high-temperature state of the complex is observed to assume a quite compact form. Such an unprecedented high-temperature arrangement is caused by the persistent interaction between Tel22 and ActD, which stabilizes compact conformations even in the presence of large thermal structural fluctuations.

There is evidence that the ABA signaling pathway has greatly contributed to increase the complexity of land plants, thereby sustaining their ability to adapt in an ever-changing environment. The regulatory functions of the ABA signaling pathway go well beyond the movements of stomata and the dormancy of seeds. For instance, the ABA signaling regulates the flavonoid biosynthesis, consistent with the high integration of ABA and light signaling pathways, which occurs at the level of key signaling components, such as the bZIP transcription factors HY5 and ABI5. Here we focus on the regulation of 'colorless' (UV-absorbing) flavonol biosynthesis by the ABA signaling and, about how flavonols may regulate, in turn, the ABA signaling network. We discuss very recent findings that quercetin regulates the ABA signaling pathway, and hypothesize this might occur at the level of second messenger and perhaps of primary signaling components as well. We critically review old and recent suggestions of the primary roles played by flavonols, the ancient class of flavonoids already present in bryophytes, in the evolution of terrestrial plants. Our reasoning strongly supports the view that the ABA-flavonol relationship may represent a robust trait of land plants, and might have contributed to their adaptation on land.

Sweet basil (Ocimum basilicum), one of the most popular cultivated herbs worldwide, displays a number of varieties differing in several characteristics, such as the color of the leaves. The development of a reference transcriptome for sweet basil, and the analysis of differentially expressed genes in acyanic and cyanic cultivars exposed to natural sunlight irradiance, has interest from horticultural and biological point of views. There is still great uncertainty about the significance of anthocyanins in photoprotection, and how green and red morphs may perform when exposed to photo-inhibitory light, a condition plants face on daily and seasonal basis. We sequenced the leaf transcriptome of the green-leaved Tigullio (TIG) and the purple-leaved Red Rubin (RR) exposed to full sunlight over a four-week experimental period. We assembled and annotated 111,007 transcripts. A total of 5,468 and 5,969 potential SSRs were identified in TIG and RR, respectively, out of which 66 were polymorphic in silico. Comparative analysis of the two transcriptomes showed 2,372 differentially expressed genes (DEGs) clustered in 222 enriched Gene ontology terms. Green and red basil mostly differed for transcripts abundance of genes involved in secondary metabolism. While the biosynthesis of waxes was up-regulated in red basil, the biosynthesis of flavonols and carotenoids was up-regulated in green basil. Data from our study provides a comprehensive transcriptome survey, gene sequence resources and microsatellites that can be used for further investigations in sweet basil. The analysis of DEGs and their functional classification also offers new insights on the functional role of anthocyanins in photoprotection.

International audience; Genomic resources have recently been developed for a number of species of Fagaceae, with the purpose of identifying the genetic factors underlying the adaptation of these long-lived, biologically predominant, commercially and ecologically important species to their environment. The sequencing of genomes of the size of the oak genome (740 Mb/C) is now becoming both possible and affordable due to breakthroughs in sequencing technology. However, an understanding of the composition and structure of the oak genome is required before launching a sequencing initiative. We constructed random (Rd) and hypomethylated (Hp) genomic libraries for pedunculate oak (Quercus robur) and carried out a sample sequencing of 2.33 and 2.36 Mb of shotgun DNA from the Rd and Hp libraries, respectively, to provide a first insight into the repetitive element and gene content of the oak genome. We found striking similarities between Rd sequences and previously analyzed BAC end sequences of pedunculate oak, with a similar percentage of known repeat elements (5.56%), an almost identical simple sequence repeat density (i.e., 29 SSRs per 100 kb), an identical profile of SSR motifs (in descending order of frequency—dinucleotide, pentanucleotide, trinucleotide, tetranucleotide, and hexanucleotide motifs). Conversely, the Hp fraction was, as expected, enriched in nuclear genes (2.44-fold enrichment). This enrichment was associated with a lower frequency of retrotransposons than for Rd sequences. We also identified twice as many SSR motifs in the Rd library as in the Hp library. This work provides useful information before opening a new chapter in oak genome sequencing.

Boreal and cool temperate forests are the major land cover of northern Eurasia and information about continental-scale genetic structure and past demographic history of forest species is important from an evolutionary perspective and has conservation implications. However, although many population genetics studies of forest tree species have been conducted in Europe or Eastern Asia, continental-scale genetic structure and past demographic history remain poorly known. Here, we focus on the birch genus Betula, which is commonly distributed in boreal and cool temperate forests, and examine 129 populations of 2 tetraploid and 4 diploid species collected from Iceland to Japan. All individuals were genotyped at 7 to 18 nuclear simple sequence repeats (nSSRs). Pairwise F’ST among the six species ranged from 0.285 to 0.903, and genetic differentiation among them was clear. STRUCTURE analysis suggested that Betula pubescens is an allotetraploid and one of the parental species was B. pendula. In both species pairs of B. pendula and B. plathyphylla, and B. pubescens and B. ermanii, genetic diversity was highest in central Siberia. A hybrid zone was detected around Lake Baikal for eastern and western species pairs regardless of ploidy level. Approximate Bayesian Computation (ABC) suggested that the divergence of B. pendula and B. platyphylla occurred around the beginning of the last ice age (36,300 years BP, 95%CI: 15,330 – 92,700) and hybridization between them was inferred to have occurred after the last glacial maximum (1,614 years BP, 95%CI: 561 – 4,710), with B. pendula providing a higher contribution to hybrids. This article is protected by copyright. All rights reserved.

Twenty populations of the species aggregate Rubus fruticosus were collected throughout European natural forests and analyzed by chloroplast microsatellites (SSR). Results showed high genetic diversity (hT = 0.73) and haplotipic richness (17 haplotypes were detected), and the presence of several unique alleles. The value of genetic differentiation between populations was low for unordered alleles (GST = 0.29) and for ordered alleles (NST = 0.30), revealing the absence of phylogeographic structure of the haplotypic diversity. This can be mainly ascribed to the mechanisms of seed dispersal, mostly mediated by animal ingestion, which are responsible for a efficient gene flow through seeds. Rubus L. species are characterized by the ability to colonizing disturbed, but also intact forest communities, rapidly propagating though suckering and hybridizing with native species. Our results suggest that efficient seed dispersal can counterbalance the effects of vegetative propagation, maintaining a high genetic diversity.

Random amplified polymorphic DNA (RAPDs) from plants contains numerous fragments of mitochondrial origin. In the present study, the association between RAPD bands and previously recognized mitochondrial polymorphism in a Larix population was used to identify fragments of mitochondrial origin and to develop PCR-based mitochondrial DNA markers useful to study phylogeny in larches, Larix sp. (Pinaceae).

Analyses of 55 individual and 31 concatenated protein data sets encoded in Reclinomonas americana and Marchantia polymorpha mitochondrial genomes revealed that current methods for constructing phylogenetic trees are insufficiently sensitive (or artifact-insensitive) to ascertain the sister of mitochondria among the current sample of eight alpha-proteobacterial genomes using mitochondrially-encoded proteins. However, Rhodospirillum rubrum came as close to mitochondria as any alpha-proteobacterium investigated. This prompted a search for methods to directly compare eukaryotic genomes to their prokaryotic counterparts to investigate the origin of the mitochondrion and its host from the standpoint of nuclear genes. We examined pairwise amino acid sequence identity in comparisons of 6,214 nuclear protein-coding genes from Saccharomyces cerevisiae to 177,117 proteins encoded in sequenced genomes from 45 eubacteria and 15 archaebacteria. The results reveal that approximately 75% of yeast genes having homologues among the present prokaryotic sample share greater amino acid sequence identity to eubacterial than to archaebacterial homologues. At high stringency comparisons, only the eubacterial component of the yeast genome is detectable. Our findings indicate that at the levels of overall amino acid sequence identity and gene content, yeast shares a sister-group relationship with eubacteria, not with archaebacteria, in contrast to the current phylogenetic paradigm based on ribosomal RNA. Among eubacteria and archaebacteria, proteobacterial and methanogen genomes, respectively, shared more similarity with the yeast genome than other prokaryotic genomes surveyed.

In the present work, we report the development of 11 microstallite markers (SSR) for Punica granatum. Evaluated on a set of 27 pomegranate accessions sampled in Tunisia, they displayed 25 alleles, with number of alleles per locus ranging between 1 and 4, and an observed heterozygosity from 0.037 and 0.592. This set of SSR markers can be very useful for studies dealing with genetic diversity assessment of germplasm, with cultivars/varieties fingerprinting and pedigree analysis of this economically important fruit species.

An enriched genomic library was constructed and 9 novel polymorphic tetranucleotide microsatellite markers developed for Austrocedrus chilensis, the most economically important native conifer in the Andean Patagonian region. Polymorphism was investigated for these markers in 48 individuals from two populations. Numbers of alleles ranged from 3 to 19 and levels of observed heterozygosity among the 9 loci ranged from 0.32 to 0.95. No locus combinations exhibited linkage disequilibrium. These polymorphic markers will be useful tools for the study of demography and gene flow and more in general for population and conservation genetics of this species.

We have developed a set of eight polymorphic nuclear microsatellite markers for the Mediterranean shrub Pistacia lentiscus by means of an enriched library method. Characterization for the eight loci was carried out on 42 individuals from two populations sampled in southern Spain. The overall number of alleles detected was 59, ranging from three to 13 per locus. Expected heterozygosity per locus and population ranged from 0.139 to 0.895. Two loci albeit only in one population (Seville) departed significantly from Hardy–Weinberg equilibrium expectations and no linkage disequilibrium between pairs of loci was detected. These markers will be used in studies of gene flow across a fragmented landscape.

• Premise of the study Pinus pinea is one of the few widespread plant species that are also genetically depauperate. It is also an important commercial species with high market value seeds. A deeper knowledge of the existing population genetic variation was needed. • Methods and Results Twelve nuclear microsatellites were isolated from genomic and cDNA sequences and screened for variability in 729 individuals from 33 natural populations. Low level of genetic variability was confirmed with average expected heterozygosity of 0.11. Hardy-Weinberg equilibrium expectations were not met in only ~10% of the possible locus/population combinations. All loci were in linkage equilibrium, and the frequency of null alleles was very low (≤ 1% in 332 out of 396 locus/ population combinations). Nine out of the 12 microsatellites were successfully transferred to P. halepensis. • Conclusions Despite low polymorphism, these new markers will be useful to resolve population structure and hold potential for seed origin identification and traceability. © 2012 Botanical Society of America.

Pomegranate (Punica granatum L.) is one of the oldest known edible fruits and more and more it arouse interest of scientific community given its numerous biological activities. However, information about its genetic resources and characterization using reliable molecular markers are still scarce. In the present study, we report the development of 4 new polymorphic SSR markers. They have been used in addition to 11 SSRs previously published to investigate molecular diversity of 33 P. granatum ecotypes. Based on the multi-locus profiles, twenty-two distinctive genotypes were identified. Globally, quite low genetic diversity has been revealed, as measured by allele richness (2.83 per locus) and heterozygosity (He = 0.245; Ho = 0.243), reflecting the narrow genetic background of the plant material. Four synonymous groups could be detected involving 15 accessions. Results of ordination and cluster analysis suggested that almost all the Tunisian cultivars share similar genetic background, and are likely derived from a small number of introductions in ancient times. Results issued from this study provide essential information to project a pomegranate core-collection without plant material duplication and for sustainable management of pomegranate landraces at national and international level. Furthermore, these SSR markers are powerful tool for marker assisted selection (MAS) program and for QTL studies.

We developed eight polymorphic nuclear microsatellite markers for the Swiss stone pine (Pinus cembra L.), of which seven may be amplified in a multiplex polymerase chain reaction. Allelic polymorphism across all loci and 40 individuals representing two populations in the Swiss Alps was high (mean = 7.6 alleles). No significant linkage disequlibrium was displayed between pairs of loci. Significant deviation from Hardy-Weinberg equilibrium was revealed at three loci in one population. Cross-amplification was achieved in two related species within the genus (P. sibirica and P. pumila). Thus, the markers may be useful for population genetic studies in these three pine species. They will be applied in ongoing projects on genetic diversity and patterns of gene flow in P. cembra.

Scots pine (Pinus sylvestris L.) is one of the most widespread forest trees in the world, ranging from southern Mediterranean mountains to eastern Siberia. Ten polymorphic microsatellite loci were isolated from Scots pine cDNA sequences and were screened for variability in three natural populations. High levels of genetic variability were observed with effective number of alleles per locus ranging from 1.0 to 4.6 and average expected heterozygosity per population of 0.79. With only two exceptions, Hardy-Weinberg expectations were confirmed. All loci were in linkage equilibrium and there was little evidence for confounding null alleles. These new markers will be used to resolve population structure and gene flow patterns in this major Eurasian forest tree. © 2011 Springer Science+Business Media B.V.

Premise of the study To characterize the level of genetic diversity and gene flow, as well as to identify unambiguously two African tropical tree species, Erythrophleum ivorense and E. suaveolens, we have developed a set of nuclear SSR (Simple Sequence Repeats) markers. Methods and Results Nine SSRs that display polymorphism in both species were identified. The nine newly developed SSR markers can be amplified in only two multiplexed reactions. Levels of polymorphism were assessed in two populations per species, yielding two to fifteen alleles per locus in E. ivorense and three to sixteen alleles per locus in E. suaveolens. Conclusions The SSR markers developed here are promising to study the spatial distribution of genetic diversity and the genetic delimitation of two Erythrophleum species from central Africa. © 2011 Botanical Society of America.

International audience; This study combines neutrality tests and environmental correlations to identify non-neutral patterns of evolution in candidate genes related to drought stress in two closely-related Mediterranean conifers, Pinus pinaster Ait. and Pinus halepensis Mill. Based on previous studies, we selected 12 amplicons covering six candidate genes that were sequenced in a large sample spanning the full range of these two species. Neutrality tests relatively robust to demography (DHEW compound test and ML-HKA test) were used to detect selection events at different temporal scales. Environmental associations between variation at candidate genes and climatic variables were also examined. These combined approaches detected distinct genes that may be targeted by selection, most of them specific to only one of the two conifers, despite their recent divergence (< 10 Ma). An exception was 4-coumarate: CoA ligase (4cl), a gene involved in the production of various important secondary products that appeared to play a role in local adaptation processes of both pines. Another remarkable result was that all significant environmental correlations involved temperature indices, highlighting the importance of this climatic factor as a selective driver on Mediterranean pines. The ability to detect natural selection at the DNA sequence level depends on the nature and the strength of the selection events, on the timescale at which they occurred and on the sensitivity of the methods to other evolutionary forces that can mimic selection (e.g. demography, population structure). Using complementary approaches can help to capture different aspects of the evolutionary processes that govern molecular variation at both intra- and interspecific levels.

5 pages; International audience; This article documents the addition of 411 microsatellite marker loci and 15 pairs of Single Nucleotide Polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Acanthopagrus schlegeli, Anopheles lesteri, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus oryzae, Aspergillus terreus, Branchiostoma japonicum, Branchiostoma belcheri, Colias behrii, Coryphopterus personatus, Cynogolssus semilaevis, Cynoglossus semilaevis, Dendrobium officinale, Dendrobium officinale, Dysoxylum malabaricum, Metrioptera roeselii, Myrmeciza exsul, Ochotona thibetana, Neosartorya fischeri, Nothofagus pumilio, Onychodactylus fischeri, Phoenicopterus roseus, Salvia officinalis L., Scylla paramamosain, Silene latifo, Sula sula, and Vulpes vulpes. These loci were cross-tested on the following species: Aspergillus giganteus, Colias pelidne, Colias interior, Colias meadii, Colias eurytheme, Coryphopterus lipernes, Coryphopterus glaucofrenum, Coryphopterus eidolon, Gnatholepis thompsoni, Elacatinus evelynae, Dendrobium loddigesii Dendrobium devonianum, Dysoxylum binectariferum, Nothofagus antarctica, Nothofagus dombeyii, Nothofagus nervosa, Nothofagus obliqua, Sula nebouxii, and Sula variegata. This article also documents the addition of 39 sequencing primer pairs and 15 allele specific primers or probes for Paralithodes camtschaticus.

For the isolation of the microsatellite markers, we directly sequenced a large number of clones from a non-enriched random genomic library. Briefl y, we extracted ca. 2 μ g of total genomic DNA from a single myrtle individual with the Invisorb Spin Plant Mini Kit (Invitek, Berlin, Germany) following the manufacturer ’ s protocol. Genomic DNA was sheared with a Nebulizer Kit (Invitrogen Corporation, Carlsbad, CA) applying high-pressure nitrogen to obtain DNA fragments ranging between 500 and 1000bp. Sheared DNA was repaired with the End-It DNA End-Repair Kit (Epicentre Biotechnologies, Madison, WI), and then the fragments were routinely cloned using the Zero Background Cloning Kit (Invitrogen Corporation, Carlsbad, CA). Four hundred twenty-fi ve randomly chosen clones were sequenced in the forward direction with the M13 universal primer and using the Big Dye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and run on an ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, CA) automatic sequencer. Sequences were checked for the occurrence of di-, tri, and tetranucleotide repeats with the on-line software Sputnik (available at http://www.cbib.ubordeaux2.fr/pise/sputnik.html). Microsatellite motifs were detected in 56 (13%) of the sequences, among which half were discarded because microsatellite motifs were too short, nucleotide repeats were too close to the vector for primer design, or because the clones showed high sequence homology. We sequenced the reverse direction of the selected clones and designed 28 primer 1

International audience; Tree species are thought to be relatively resistant to habitat fragmentation because of their longevity and their aptitude for extensive gene flow, although recent empirical studies have reported negative genetic consequences, in particular after long-term habitat fragmentation in European temperate regions. Yet the response of each species to habitat loss may differ greatly depending on their biological attributes, in particular seed dispersal ability. In this study, we used demographic and molecular data to investigate the genetic consequences of chronic habitat fragmentation in remnant populations of Taxus baccata in the Montseny Mountains, northeast Spain. The age structure of populations revealed demographic bottlenecks and recruitment events associated with exploitation and management practices. We found a strong genetic structure, both at the landscape and within-population levels. We also detected high levels of inbreeding for a strictly outcrossing species. Chronic forest fragmentation resulting from long-term exploitation in the Montseny Mountains seems the most plausible explanation for the strong genetic structure observed. Our results support the view that, contrary to some predictions, tree species are not buffered from the adverse effects of habitat fragmentation, even in the case of species with a high dispersal potential.

Background Expressed Sequence Tags (ESTs) are a source of simple sequence repeats (SSRs) that can be used to develop molecular markers for genetic studies. The availability of ESTs for Quercus robur and Quercus petraea provided a unique opportunity to develop microsatellite markers to accelerate research aimed at studying adaptation of these long-lived species to their environment. As a first step toward the construction of a SSR-based linkage map of oak for quantitative trait locus (QTL) mapping, we describe the mining and survey of EST-SSRs as well as a fast and cost-effective approach (bin mapping) to assign these markers to an approximate map position. We also compared the level of polymorphism between genomic and EST-derived SSRs and address the transferability of EST-SSRs in Castanea sativa (chestnut). Results A catalogue of 103,000 Sanger ESTs was assembled into 28,024 unigenes from which 18.6% presented one or more SSR motifs. More than 42% of these SSRs corresponded to trinucleotides. Primer pairs were designed for 748 putative unigenes. Overall 37.7% (283) were found to amplify a single polymorphic locus in a reference full-sib pedigree of Quercus robur. The usefulness of these loci for establishing a genetic map was assessed using a bin mapping approach. Bin maps were constructed for the male and female parental tree for which framework linkage maps based on AFLP markers were available. The bin set consisting of 14 highly informative offspring selected based on the number and position of crossover sites. The female and male maps comprised 44 and 37 bins, with an average bin length of 16.5 cM and 20.99 cM, respectively. A total of 256 EST-SSRs were assigned to bins and their map position was further validated by linkage mapping. EST-SSRs were found to be less polymorphic than genomic SSRs, but their transferability rate to chestnut, a phylogenetically related species to oak, was higher. Conclusion We have generated a bin map for oak comprising 256 EST-SSRs. This resource constitutes a first step toward the establishment of a gene-based map for this genus that will facilitate the dissection of QTLs affecting complex traits of ecological importance.

Knowledge about current patterns of genetic structure of populations together with the evolutionary history of a species helps to understand and predict the adaptation of populations to future climate change. We assayed variation at nuclear microsatellite markers among peripheral vs. continuous populations of the temperate South American species Austrocedrus chilensis, to investigate the role of historical vs. demographical forces in shaping population genetic structure. This species occurs in continuous populations in the west and central distribution range, but becomes highly fragmented at the eastern limit, which comprised ice-free areas during Quaternary glaciations and has extreme climatic conditions at present times. Bayesian analysis methods identified two contrasting patterns of genetic structure; (I) populations from humid, mesic and peri-glacial regions formed a single deme with relatively low genetic differentiation and high admixture levels whereas (II) a highly heterogeneous genetic structure with low level of admixture was found in the steppe, towards the east and northeast limit of the distribution range. In the steppe, population fragmentation, restricted gene flow and isolation-by-distance were also inferred. In addition, several small steppe populations showed high genetic diversity and divergent gene pools, suggesting that they constitute ancient refuges from pre-Holocene glaciations with just a subgroup of them contributing significantly to post-glacial spread. These results are discussed in relation to patterns of genetic variation found for other temperate species and the contribution of the particular southern Andes topography and climate to post-glacial spread.

This study represents the first large-scale analysis using nuclear molecular markers to assess genetic diversity and structure of Cupressus sempervirens L. Genetic and fossil data were combined to infer the possible role of human activity and evolutionary history in shaping the diversity of cypress populations. We analysed 30 populations with six polymorphic nuclear microsatellite markers. Dramatic reductions in heterozygosity and allelic richness were observed from east to west across the species range. Structure analysis assigned individuals to two main groups separating central Mediterranean and eastern populations. The two main groups could be further divided into five subgroups which showed the following geographical distributions Turkey with the Greek islands Rhodes and Samos, Greece (Crete), Southern Italy, Northern Italy, Tunisia with Central Italy. This pattern of genetic structure is also supported by samova and Barrier analyses. Palaeobotanical data indicated that Cupressus was present in Italy in the Pliocene, Pleistocene and Holocene. Furthermore, our molecular survey showed that Italian cypress populations experienced bottlenecks that resulted in reduced genetic diversity and allelic richness and greater genetic differentiation. Recent colonization or introduction may also have influenced levels of diversity detected in the Italian populations, as most individuals found in this range today have multilocus genotypes that are also present in the eastern range of the species. The data reveal a new interpretation of the history of cypress distribution characterized by ancient eastern populations (Turkey and Greek islands) and a mosaic of recently introduced trees and remnants of ancient, depauperate populations in the central Mediterranean range. © 2009 Blackwell Publishing Ltd.

English yew (Taxus baccata L.;Taxaceae), a Tertiary relict, provides a seminal example of a widespread albeit locally endangered (often close to extinction) tree species. In order to gain detailed insights into the evolutionary dynamics of the species on a broad geographical scale, over 1000 trees from 91 populations of English yew in the western Mediterranean were analyzed using seven nuclear microsatellite markers. Our results revealed contrasting patterns of genetic structure at different spatial scales genetic variation was highly structured at the local scale, while only a low proportion of the observed variation was attributed to regional differences. We also found a geographic gradient of decreasing diversity and increasing population divergence from northwest (central Europe and northern Iberian Peninsula) to southeast (Mediterranean Iberia and North Africa). The patterns revealed in this study probably reflect the combined effects of Quaternary climatic changes and recent impact of human activities, and potentially also more ancient events dating back to the Tertiary. Both climatic and anthropogenic factors seem to have conducted to a long history of population isolation, which may have contributed significantly to enhance population divergence through restricted gene flow and genetic drift. © 2010 Elsevier Inc. All rights reserved.

Seven polymorphic nuclear microsatellite markers for Taxus baccata L. (English yew) were developed using an enriched-library method. An additional polymorphic SSR was obtained by testing eight primer pairs from the congeneric species Taxus sumatrana. Mendelian inheritance for the seven Taxus baccata SSRs was proved by genotyping 17 individuals and 124 megagametophytes (conifer seed haploid tissue). A total of 96 individuals from 5 different populations (10-26 samples per population) were used to estimate genetic diversity parameters. High levels of genetic diversity, with values ranging from 0.533 to 0.929 (6-28 alleles per SSR) were found. No linkage disequilibrium between pairs of loci was detected. All loci but one showed significant departures from Hardy-Weinberg equilibrium. Excess of homozygosity was probably due to high inbreeding in English yew populations, an outcome of low effective population size and/or fragmented distribution. Highly polymorphic SSRs will be used to conduct population genetic studies at different geographical scales and to monitor gene flow. © 2008 Springer Science+Business Media B.V.

Genetic variation is generally considered a prerequisite for adaptation to new environmental conditions. Thus the discovery of genetically depauperate but geographically widespread species is unexpected. We used 12 paternally inherited chloroplast microsatellites to estimate population genetic variation across the full range of an emblematic circum-Mediterranean conifer, stone pine (Pinus pinea L.). The same chloroplast DNA haplotype is fixed in nearly all of the 34 investigated populations. Such a low level of variation is consistent with a previous report of very low levels of diversity at nuclear loci in this species. Stone pine appears to have passed through a severe and prolonged demographic bottleneck, followed by subsequent natural- and human-mediated dispersal across the Mediterranean Basin. No other abundant and widespread plant species has as little genetic diversity as P. pinea at both chloroplast and nuclear markers. However, the species harbors a nonnegligible amount of variation at adaptive traits. Thus a causal relationship between genetic diversity, as measured by marker loci, and the evolutionary precariousness of a species, cannot be taken for granted. © 2007 The Author(s).

Some 1339 trees from 48 Pinus pinaster stands were characterized by five chloroplast microsatellites, detecting a total of 103 distinct haplotypes. Frequencies for the 16 most abundant haplotypes (pk > 0.01) were spatially interpolated over a lattice made by 430 grid points. Fitting of spatially interpolated values on raw haplotype frequencies at the same geographical location was tested by regression analysis. A range-wide 'diversity map' based on interpolated haplotype frequencies allowed the identification of one 'hotspot' of diversity in central and southeastern Spain, and two areas of low haplotypic diversity located in the western Iberian peninsula and Morocco. Principal component analysis (PCA) carried out on haplotypes frequency surfaces allowed the construction of a colour-based 'synthetic' map of the first three PC components, enabling the detection of the main range-scale genetic trends and the identification of three main 'gene pools' for the species (i) a 'southeastern' gene pool, including southeastern France, Italy, Corsica, Sardinia, Pantelleria and northern Africa; (ii) an 'Atlantic' gene pool, including all the western areas of the Iberian peninsula; and (iii) a 'central' gene pool, located in southeastern Spain. Multivariate and amova analyses carried out on interpolated grid point frequency values revealed the existence of eight major clusters ('gene zones'), whose genetic relationships were related with the history of the species. In addition, demographic models showed more ancient expansions in the eastern and southern ranges of maritime pine probably associated to early postglacial recolonization. The delineation of the gene zones provides a baseline for designing conservation areas in this key Mediterranean pine. © 2007 The Authors.

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.

International audience; 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.

We developed five nuclear microsatellite markers in Juniperus communis L. using an enriched library method. Samples from 28 juniper individuals were collected in Spain, Germany and Slovakia and were analysed at the five loci. A high level of allelic diversity with values ranging from nine to 23 alleles was found. These highly polymorphic markers will be used in ongoing population genetic studies to evaluate the genetic resources and to contribute to the maintenance of genetic diversity of juniper in Middle Europe.

Crossings between strains of Saccharomyces cerevisiae and Saccharomyces bayanus were carried out. Genetic, molecular and electrophoretic karyotyping data indicated that interspecific hybrids were obtained. The hybrid cells segregated "grande" and "petite" colonies, and the latter ranged between 20 and 50%; unlike "grande" colonies, "petite" colonies did not sporulate and did not ferment maltose. In the hybrids, the extent of sporulation varied between 10 and 20%; only very rare asci (around 10(-4)) held viable ascospores. Clones from the viable ascospores sporulated and produced asci with viable ascospores able to give mating with spores from both hybrid derivatives and parental species. Fertile asci could derive from allotetraploid cells generated by endomitotic events in allodiploid cells, a mechanism that enables overcoming the species barrier between S. cerevisiae and S. bayanus.