The importance of natural selection for shaping adaptive trait differentiation among natural populations of allogamous tree species has long been recognized. Determining the molecular basis of local adaptation remains largely unresolved, and the respective roles of selection and demography in shaping population structure are actively debated. Using a multilocus scan that aims to detect outliers from simulated neutral expectations, we analyzed patterns of nucleotide diversity and genetic differentiation at 11 polymorphic candidate genes for drought stress tolerance in phenotypically contrasted Pinus pinaster Ait. populations across its geographical range. We compared 3 coalescent-based methods: 2 frequentist-like, including 1 approach specifically developed for biallelic single nucleotide polymorphisms (SNPs) here and 1 Bayesian. Five genes showed outlier patterns that were robust across methods at the haplotype level for 2 of them. Two genes presented higher F(ST) values than expected (PR-AGP4 and erd3), suggesting that they could have been affected by the action of diversifying selection among populations. In contrast, 3 genes presented lower F(ST) values than expected (dhn-1, dhn2, and lp3-1), which could represent signatures of homogenizing selection among populations. A smaller proportion of outliers were detected at the SNP level suggesting the potential functional significance of particular combinations of sites in drought-response candidate genes. The Bayesian method appeared robust to low sample sizes, flexible to assumptions regarding migration rates, and powerful for detecting selection at the haplotype level, but the frequentist-like method adapted to SNPs was more efficient for the identification of outlier SNPs showing low differentiation. Population-specific effects estimated in the Bayesian method also revealed populations with lower immigration rates, which could have led to favorable situations for local adaptation. Outlier patterns are discussed in relation to the different genes' putative involvement in drought tolerance responses, from published results in transcriptomics and association mapping in P. pinaster and other related species. These genes clearly constitute relevant candidates for future association studies in P. pinaster.

Circadian clock performance during winter dormancy has been investigated in chestnut by using as marker genes CsTOC1 and CsLHY, which are homologous to essential components of the central circadian oscillator in Arabidopsis. During vegetative growth, mRNA levels of these two genes in chestnut seedlings and adult plants cycled daily, as expected. However, during winter dormancy, CsTOC1 and CsLHY mRNA levels were high and did not oscillate, indicating that the circadian clock was altered. A similar disruption was induced by chilling chestnut seedlings (to 4°C). Normal cycling resumed when endodormant or cold-treated plants were returned to 22°C. The behavior of CsTOC1 and CsLHY during a cold response reveals a relevant aspect of clock regulation not yet encountered in Arabidopsis.chilling ͉ winter dormancy ͉ ecodormancy ͉ cold response W inter dormancy is an important adaptive strategy that enables plants to persist during periods of stressful environmental conditions (1). Dormancy parameters are key determinants in woody plants in agriculture and forestry. Dormancy determines to what degree fruit crops will survive winter and early spring without shoot and flower bud damage, and, in long-lived forest species, the length of rest limits the growing season and thus affects wood production and quality. The onset of winter deep dormancy (endodormancy) is preceded by a stage of ecodormancy. Endodormancy is caused by plant endogenous factors, and, once established,

Two unigene datasets of Pinus taeda and Pinus pinaster were screened to detect di-, tri- and tetranucleotide repeated motifs using the SSRIT script. A total of 419 simple sequence repeats (SSRs) were identified, from which only 12.8% overlapped between the two sets. The position of the SSRs within their coding sequences were predicted using FrameD. Trinucleotides appeared to be the most abundant repeated motif (63 and 51% in P. taeda and P. pinaster, respectively) and tended to be found within translated regions (76% in both species), whereas dinucleotide repeats were preferentially found within the 5'- and 3'-untranslated regions (75 and 65%, respectively). Fifty-three primer pairs amplifying a single PCR fragment in the source species (mainly P. taeda), were tested for amplification in six other pine species. The amplification rate with other pine species was high and corresponded with the phylogenetic distance between species, varying from 64.6% in P. canariensis to 94.2% in P. radiata. Genomic SSRs were found to be less transferable; 58 of the 107 primer pairs (i.e. 54%) derived from P. radiata amplified a single fragment in P. pinaster. Nine cDNA-SSRs were located to their chromosomes in two P. pinaster linkage maps. The level of polymorphism of these cDNA-SSRs was compared to that of previously and newly developed genomic-SSRs. Overall, genomic SSRs tend to perform better in terms of heterozygosity and number of alleles. This study suggests that useful SSR markers can be developed from pine ESTs.

Oaks are long-standing models for the study of gene flow and hybridisation. Temperate (Quercus petraea) and subMediterranean (Quercus pyrenaica) oaks coexist in central Spain, showing remarkable differences in population size and structure. Q. petraea has a scattered distribution in central Spain, where it is at one of the southernmost limits of its range, and forms low-density stands; in contrast, Q. pyrenaica is widespread in the region. We selected a mixed population of the two species (B13 ha, 176 adults and 96 saplings) to compare the patterns of gene flow within each species and the extent of introgression between them. Using five nuclear microsatellite markers, we performed a parentage analysis and found considerable immigration from outside the stand (B38% for Q. petraea and B34% for Q. pyrenaica), and estimated average seed-dispersal distances of 42 and 14 m for Q. petraea and Q. pyrenaica, respectively. Introgression between species was also estimated using our microsatellite battery. First, we developed a multivariate discriminant approach and, second, we compared our results with a widely used clustering method (STRUCTURE). Both analyses were consistent with a low level of introgression between Q. petraea and Q. pyrenaica. Indeed, only 15 adult trees, B8.5%, were identified as putative hybrids when both methods of analysis were combined. Hybrids may be most common in contact zones due merely to physical proximity. Heredity (2005) 95, 457-465.

A small heat-shock protein (sHSP) that shows molecular chaperone activity in vitro was recently purified from mature chestnut (Castanea sativa) cotyledons. This protein, renamed here as CsHSP17.5, belongs to cytosolic class I, as revealed by cDNA sequencing and immunoelectron microscopy. Recombinant CsHSP17.5 was overexpressed in Escherichia coli to study its possible function under stress conditions. Upon transfer from 37°C to 50°C, a temperature known to cause cell autolysis, those cells that accumulated CsHSP17.5 showed improved viability compared with control cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cell lysates suggested that such a protective effect in vivo is due to the ability of recombinant sHSP to maintain soluble cytosolic proteins in their native conformation, with little substrate specificity. To test the recent hypothesis that sHSPs may be involved in protection against cold stress, we also studied the viability of recombinant cells at 4°C. Unlike the major heat-induced chaperone, GroEL/ES, the chestnut sHSP significantly enhanced cell survivability at this temperature. CsHSP17.5 thus represents an example of a HSP capable of protecting cells against both thermal extremes. Consistent with these findings, high-level induction of homologous transcripts was observed in vegetative tissues of chestnut plantlets exposed to either type of thermal stress but not salt stress.