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Persian walnut (Juglans regia L.), also known as common walnut, is an economically and ecologically important species in the Kyrgyz Republic. Natural forests of J. regia, walnut forests, grow between western and southwestern slopes of the Fergana and Chatkal mountain ridges in the southern Kyrgyz Republic. We investigated 11 natural populations of J. regia across its geographic range in the Kyrgyz Republic using eight nuclear microsatellite (nSSR) and eight expressed sequence tag (EST)-derived SSR (EST-SSR) markers. The SSR markers revealed comparatively high levels of genetic diversity (HO = 0.538, HE = 0.524). The overall genetic differentiation among populations was low, but statistically significant (FST = 0.068). Significant correlation between genetic and geographic distances was observed, indicating an isolation-by-distance (IBD) model for J. regia. Spatial analysis of molecular variance (SAMOVA) and Bayesian analysis assigned individuals to two main genetic groups separating central region walnut populations (Jalal-Abad province) and southeastern region walnut populations (Osh province). Genetic diversity parameters such as gene diversity and allelic richness were significantly lower in the Osh province than in the Jalal-Abad province. Detection of significant genetic discontinuities suggested the existence of physical barriers to migration and gene flow among regions. We suggest that the Osh province and Jalal-Abad province populations should be considered as two evolutionary significant units (ESUs) for conservation efforts.

Juniperus excelsa is one of the most common tree species and has a wide geographical and altitudinal distribution in Turkey. It is also resistant to drought and frost damages and can cope with poor soils. In this study, we explore whether there are any differences among eight J. excelsa populations from a narrow geographic region grown in a common garden test site in terms of growth and contents of photosynthetic pigments, proline and nutrients of their 10-year-old saplings. Phenotypic trait differentiation (QST) at all traits, FST at neutral SSRs among six of the populations and associations of traits with environmental conditions at provenance regions were also analysed to test for patterns of local adaptation. Sapling traits of eight J. excelsa populations of from Lakes District in Turkey at the test site showed that populations significantly differed for growth, photosynthetic pigments, proline and nutrient contents. The mean height and diameter of 10-year-old saplings were found as 94.5 cm and 41.6 mm, respectively. Egirdir-Barla and Sütçüler-Tota populations showed the highest performance for the majority of traits at age 10 compared to all other populations. Nutrient contents in leaves were generally in the sufficiency range reported for plant growth. Considering photosynthetic pigments and proline, it could be concluded that the populations were not exposed to severe stress. Among the environmental variables, the best predictors of growth were annual mean minimum temperature and soil texture at the populations’ origin, accounting for 49% of the variation in height and diameter, respectively. Also, higher phenotypic trait differentiation for most traits than genetic differentiation at neutral genetic markers suggests local adaptation at a small geographic scale. The present study revealed adaptive divergence between populations at a small geographic scale. However, environmental similarity between region of origin and test site was not a good indicator of growth-related traits. The results can be used in the early selection of provenances for J. excelsa for plantation establishment.

Background Genetic markers and linkage mapping are basic prerequisites for comparative genetic analyses, QTL detection and map-based cloning. A large number of mapping populations have been developed for oak, but few gene-based markers are available for constructing integrated genetic linkage maps and comparing gene order and QTL location across related species. Results We developed a set of 573 expressed sequence tag-derived simple sequence repeats (EST-SSRs) and located 397 markers (EST-SSRs and genomic SSRs) on the 12 oak chromosomes (2n = 2x = 24) on the basis of Mendelian segregation patterns in 5 full-sib mapping pedigrees of two species: Quercus robur (pedunculate oak) and Quercus petraea (sessile oak). Consensus maps for the two species were constructed and aligned. They showed a high degree of macrosynteny between these two sympatric European oaks. We assessed the transferability of EST-SSRs to other Fagaceae genera and a subset of these markers was mapped in Castanea sativa , the European chestnut. Reasonably high levels of macrosynteny were observed between oak and chestnut. We also obtained diversity statistics for a subset of EST-SSRs, to support further population genetic analyses with gene-based markers. Finally, based on the orthologous relationships between the oak, Arabidopsis , grape, poplar, Medicago , and soybean genomes and the paralogous relationships between the 12 oak chromosomes, we propose an evolutionary scenario of the 12 oak chromosomes from the eudicot ancestral karyotype. Conclusions This study provides map locations for a large set of EST-SSRs in two oak species of recognized biological importance in natural ecosystems. This first step toward the construction of a gene-based linkage map will facilitate the assignment of future genome scaffolds to pseudo-chromosomes. This study also provides an indication of the potential utility of new gene-based markers for population genetics and comparative mapping within and beyond the Fagaceae.

In order to construct a molecular phylogeny of Indonesian Dipterocarpoideae (Dipterocarpaceae), PCR-RFLP of the chloroplast regions rbcL, petB, psbA, psaA, and trnL-F was performed with seven restriction enzymes in 129 samples including 58 species from nine genera. In the strict consensus tree with Monotes kerstingii as outgroup Indonesian Dipterocarpaceae were divided into two major clades. One clade (bootstrap value = 71) consisted of Upuna, Cotylelobium, Anisoptera, Vatica, Dipterocarpus (tribe Dipterocarpeae, bootstrap value = 83) and Dryobalanops (tribe Shoreae, bootstrap value = 99) in a basal position. The second clade consisted of Hopea, Parashorea, and Shorea (tribe Shoreae) with 95% bootstrap support. Tribe Dipterocarpeae is monophyletic, tribe Shoreae is polyphyletic since Dryobalanops is sister to tribe Dipterocarpeae. In the neighbour-joining tree the sister group position of Dryobalanops to tribe Dipterocarpeae is not supported by the bootstrap analysis. Alternatively, we used Upuna borneensis as outgroup. The effect of outgroup selection on tree topology, taxonomic classification and the interpretation of character evolution is discussed.

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.

The reduction of inner (adaxial) pollen sacs (microsporangia, MS) as a diagnostic character for the three asteracean species, Microseris bigelovii, Microseris elegans and Microseris pygmaea, was analysed in an interspecific cross between Microseris douglasii and Microseris bigelovii with 4 MS and 2 MS, respectively, using the average number of MS per plant as a quantitative character. A previous QTL (Quantitative Trait Locus) analysis had revealed one major QTL (3B) and three modifier QTLs (3A, 4A, 7A) with epistatic effects only on the homozygous recessive 2 MS genotype of QTL 3B. Here we performed a bulked segregant analysis on four 2 MS and four 4 MS DNA-bulks with 407 EcoRI/ MseI AFLP-primer combinations each. In this way additional AFLP markers were mapped close to QTL 3B and QTL 3A. Three of them were converted to SCAR (Sequence Characterized Amplified region) markers. All markers were tested in natural populations of the disporangiate (2 MS) species M. bigelovii, M. elegans and M. pygmaea, and in different populations of tetrasporangiate (4 MS) M. douglasii. The marker distribution suggests that locus 3B mutated in a progenitor of the disporangiate species. QTL 3A has evolved in the 2 MS background of the major gene in the disporangiate species. Since M. pygmaea and M. bigelovii are the sister group to M. elegans, the 4 MS genotype for (markers of) QTL 3A in M. pygmaea populations is most likely due to a back mutation to the 4 MS state and could explain the slight instability of the 2 MS phenotype in this species.

The development of molecular markers unambiguously distinguishing groups at different taxonomic levels has numerous forensic applications. The identification of tropical timber is of particular interest in this context. We describe the development of SCAR (Sequence Characterized Amplified Region) markers for forensic applications taking the example of two closely related species of the tropical tree family Shorea (Dipterocarpaceae). Two AFLP (Amplified Fragment Length Polymorphism) fragments have been described earlier showing strong differentiation between S. leprosula and S. parvifolia. The AFLP markers were isolated from a gel, re-amplified, cloned and sequenced. Primer sets were designed from these sequences and AFLP fragments were converted into SCAR markers. The SCAR markers and PCR-RFLP markers of the chloroplast region trnLF digested with HinfI were used to screen in total 557 samples of S. parvifolia and S. leprosula from nineteen widely separated populations in Indonesia. Complete genetic differentiation between species was observed based on the putatively nuclear SCAR marker and the PCR-RFLP of the cpDNA region. We found a good agreement between leaf morphological variation and species identification based on both marker types and no indication for interspecific hybridization.

Oak species show a wide variation in morphological and physiological characters, and species boundaries between closely related species are often not clear-cut. Still, despite frequent interspecific gene flow, oaks maintain distinct morphological and physiological adaptations. In sympatric stands, spatial distribution of species with different ecological requirements is not random but constrained by soil and other microenvironmental factors. Pre-zygotic isolation (e.g. cross incompatibilities, asynchrony in flowering, pollen competition) and post-zygotic isolation (divergent selection) contribute to the maintenance of species integrity in sympatric oak stands. The antagonistic effects of interspecific gene flow and divergent selection are reflected in the low genetic differentiation between hybridizing oak species at most genomic regions interspersed by regions with signatures of divergent selection (outlier regions). In the near future, the availability of high-density genetic linkage maps anchored to scaffolds of a sequenced Q. robur genome will allow to characterize the underlying genes in these outlier regions and their putative role in reproductive isolation between species. Reciprocal transplant experiments of seedlings between parental environments can be used to characterize selection on outlier genes. High transferability of gene-based markers will enable comparative outlier screens in different oak species.

Highly polymorphic AFLP markers were applied to analyse the phylogenetic relationships of seven species from three sections within genus Araucaria (Araucariaceae) with cladistic and phenetic approaches. The objectives of the study were to compare the intrageneric relationships within Araucaria assessed by AFLP markers with the classification according to chloroplast DNA sequences and morphological characters. The AMOVA revealed 48% of the variation among species. The results of the principal coordinate analysis revealed three distinct groups: (1) A. angustifolia and A. araucana (= section Araucaria), (2) A. bidwillii (= section Bunya) and (3) A. cunninghamii, A. heterophylla, A. rulei and A. scopulorum (= section Eutacta). In the cladistic and phenetic analyses, phylogenetic trees were subdivided into two sister clades, one comprising the samples from section Eutacta, the other one was divided again into two sister clades corresponding to sections Araucaria and Bunya. These results are congruent with a previous phylogenetic study of the family Araucariaceae based on rbcL sequences and with the classification of genus Araucaria based on morphological characters. Both rbcL sequence data and AFLP analyses do not support section Bunya as one of the oldest sections within genus Araucaria, as suggested by the fossil record. The utility of AFLP markers for phylogenetic analyses is discussed.

Species boundaries in oaks are often not clear-cut, which is potentially a result of interspecific hybridization with trait introgression and phenotypic plasticity. Quercus rubra L. and Quercus ellipsoidalis E.J. Hill are two interfertile partially sympatric red oak species (section Lobatae) with different adaptations to drought. Quercus ellipsoidalis is the most drought tolerant of the North American red oak species and is characterized by deep tap roots, a shrubby growth and by deeply dissected leaves. Genetic differentiation between species is low for most molecular markers. However, one genic microsatellite in a CONSTANS-like (COL) gene, FIR013, was previously identified as outlier locus under strong divergent selection between species. In this study, we analyzed leaf morphometric traits in neighboring (parapatric) Q. rubra/Q. ellipsoidalis populations and in one sympatric population from the same region along an environmental gradient. Using multivariate statistics of leaf traits both species showed distinct bimodal frequency distributions for the first canonical discriminant function with some overlap in the phenotypic extremes, especially in the sympatric population. Leaf dissection traits showed strong and consistent differentiation between species in sympatric and parapatric populations, while differentiation for leaf size was lower in the sympatric population under more similar environmental conditions. Leaf phenotypes in F1 hybrids and introgressive forms suggested maternal effects and introgression of leaf traits between species. The association of outlier gene copy number at FIR013 with species-discriminating leaf traits in Quercus rubra can be a reflection of population differences since outlier gene copy number and population membership show significant collinearity. Similar environmental selection pressures on outlier alleles and leaf shape could also have resulted in this association. In future studies, segregating full-sib families could be used to test whether outlier alleles and associated genomic regions are indeed associated with leaf traits or other species-discriminating characters.