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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.

• Premise of the study: Large-scale studies on the genetic diversity of forest trees are relevant for the inventory, conservation, and management of genetic resources and provide an insight into the geographical origins of the species. This approach is appropriate to use with Castanea sativa, a tree of great economic importance and the only species from the genus Castanea in Europe. The history of C. sativa was deduced from fossil pollen data, but the large-scale genetic structure of this species needs to be elucidated. We evaluated the genetic diversity of C. sativa to define previously unclarified genetic relationships among the populations from Turkey and those from Greece and western Europe. The influence of natural events such as glaciations and human impact in terms of species distribution are discussed.• Methods: Wild chestnut trees (779) were sampled in 31 European sites. Six polymorphic microsatellites were used for the analysis. A set of measures of intra- and interpopulation genetic statistics were calculated. The population structure was inferred by using a Bayesian approach.• Key results: The population structure showed a genetic divergence between the eastern (Greek and Turkish) and western (Italian and Spanish) populations. Two gene pools and a zone of gene introgression in Turkey were revealed.• Conclusions: The inferred population structure shows a strong geographical correspondence with the hypothesized glacial refugia and rules out the migration of the chestnut from Turkey and Greece to Italy. The homogeneous gene pool observed in Italy and Spain could have been originated from common refugia along with human-mediated colonization.

Genetic erosion, mainly caused by the replacement of local landraces with high yielding or exotic varieties is causing loss of agrobiodiversity. Landraces of horticultural species, grown by small producers, represent today an important value in the preservation of agrobiodiversity. Umbria, a region of central Italy, is characterized by agricultural systems linked to tradition and cultivation of local landraces. In this study, the genetic profile of some traditional Umbrian tomato landraces was characterized for the first time, and the landraces uniqueness was evaluated by comparison with commercial varieties. One-hundred and twenty-one plants provided by local farmers and seed companies, represented by local and commercial varieties were analyzed using 19 SSRs markers. A total of 60 alleles were found with moderate levels of diversity. The mean number of alleles per locus was 3.158 and the average polymorphism information content was 0.38. Unweighted UPGMA clustered the accessions into four groups. The gene pool of Umbrian landraces seems to be highly differentiated compared to commercial varieties, with landraces showing a genetic distinctiveness. Furthermore, an identifying fingerprinting code of each tomato landrace was generated and an innovative method for varietal identification based on the ‘QR code’ was proposed. The results obtained in this study will be useful for a better management, conservation and propagation of tomato genetic resources in Umbria region.

Alnus cordata is an endemic tree species native to the Southern Italian Apennines and north‐eastern Corsica, renowned for its ecological significance. Climate change projections for the Mediterranean basin indicate range shifts and increased fragmentation for many forest trees, including A. cordata. Hybridization with the sympatric A. glutinosa in the central part of its Italian native range may also influence the genetic structure and conservation priorities for A. cordata. A comprehensive conservation strategy is needed to preserve its genetic resources in Italy. We analyzed the genetic diversity, population structure, and extent of hybridization with A. glutinosa in 23 A. cordata forest stands across its native Italian range using nuclear microsatellites. Habitat suitability was modeled under current and future climate scenarios using an ensemble forecasting approach. Conservation prioritization was guided by a genetically informed Reserve Selection analysis in DIVA‐GIS to identify areas of high conservation value and address gaps in genetic resource representation. Italian alder populations exhibit low genetic diversity, which decreases towards the southern latitudinal margins of the peninsula. Hybridization and introgression with Alnus glutinosa have a geographically localized impact on the genetic variation within A. cordata populations. Local increases in private allelic richness do not alter the spatial genetic structure of A. cordata, but they help mitigate the risk of severe genetic erosion. A significant proportion of the species' genetic diversity is effectively preserved through in situ conservation. Model projections under future climate scenarios indicate a substantial decline in habitat suitability for A. cordata stands with high priority for in situ conservation. This highlights the need for complementary strategies, including ex situ conservation measures. Our study highlights the importance of integrating genetic analyses, habitat suitability modeling, and spatial prioritization techniques for effective conservation planning of A. cordata in the face of climate change across the Mediterranean. Alnus cordata is an endemic tree species native to the Southern Italian Apennines and north‐eastern Corsica, renowned for its ecological significance. We analyzed the genetic diversity, population structure, and extent of hybridization with A. glutinosa in 23 A. cordata forest stands across its native Italian range. Conservation prioritization was guided by a genetically informed Reserve Selection analysis in DIVA‐GIS to identify areas of high conservation value and address gaps in genetic resource representation.

Biogenic fraction of airborne PM10 which includes bacteria, viruses, fungi and pollens, has been proposed as one of the potential causes of the PM10 toxicity. The present study aimed to provide a comprehensive understanding of the microbial community variations associated to PM10, and their main local sources in the surrounding environment in three urban sites of Rome, characterized by differential pollution rate: green area, residential area and polluted area close to the traffic roads. We combined high-throughput amplicon sequencing of the bacterial 16S rRNA gene and the fungal internal transcribed spacer (ITS) region, with detailed chemical analysis of particulate matter sampled from air, paved road surfaces and leaf surfaces of Quercus ilex. Our results demonstrated that bacterial and fungal airborne communities were characterized by the highest alpha-diversity and grouped separately from epiphytic and road dust communities. The reconstruction of source-sink relationships revealed that the resuspension/deposition of road dust from traffic might contribute to the maximum magnitude of microbial exchanges. The relative abundance of extremotolerant microbes was found to be enhanced in epiphytic communities and was associated to a progressively increase of pollution levels as well as opportunistic human pathogenicity in fungal communities.

Biotechnological tools have emerged as key alternatives for the protection, improvement, and sustainable use of forest species. This paper analyzes the main biotechnological strategies applied to the European chestnut, a species of significant ecological, economic, and cultural importance in many temperate regions. However, in recent decades, it has been seriously threatened by various factors, including devastating diseases such as chestnut blight and ink disease, as well as the impacts of climate change. First, classical and assisted breeding techniques are discussed, including controlled hybridization and the use of molecular markers to accelerate the selection of genotypes of interest. In the field of molecular biotechnology, studies related to the identification of key genes, the development of genetic markers (e.g., SSRs and SNPs), and the omics characterization of chestnut are reviewed. The use of micropropagation techniques for the clonal multiplication of elite individuals is also included. Furthermore, advances in genetic modifications are explored, highlighting the introduction of resistance genes through transgenic and cisgenic approaches, as well as emerging technologies such as CRISPR/Cas9. In the future, the integration of classical breeding with advanced genomics will enable the precise selection and accelerated development of European chestnut varieties, combining traditional trait improvement with genomic tools such as marker-assisted selection, genomic prediction, and gene editing to enhance disease resistance and climate resilience.

Castanea sativa is classified as non-indigenous in Britain and Ireland. It was long held that it was first introduced into Britain by the Romans, until a recent study found no corroborative evidence of its growing here before c. AD 650. This paper presents new data on the genetic diversity of C. sativa in Britain and Ireland and potential ancestral sources in continental Europe. Microsatellite markers and analytical methods tested in previous European studies were used to genotype over 600 C. sativa trees and coppice stools, sampled from ancient semi-natural woodlands, secondary woodlands and historic cultural sites across Britain and Ireland. A single overall genepool with a diverse admixture of genotypes was found, containing two sub groups differentiating Wales from Ireland, with discrete geographical and typological clusters. C. sativa genotypes in Britain and Ireland were found to relate predominantly to some sites in Portugal, Spain, France, Italy and Romania, but not to Greece, Turkey or eastern parts of Europe. C. sativa has come to Britain and Ireland from these western European areas, which had acted as refugia in the Last Glacial Maximum; we compare its introduction with the colonization/translocation of oak, ash, beech and hazel into Britain and Ireland. Clones of C. sativa were identified in Britain, defining for the first time the antiquity of some ancient trees and coppice stools, evincing both natural regeneration and anthropogenic propagation over many centuries and informing the chronology of the species' arrival in Britain. This new evidence on the origins and antiquity of British and Irish C. sativa trees enhances their conservation and economic significance, important in the context of increasing threats from environmental change, pests and pathogens.

The Tuscan Archipelago is one of the most ancient and ecologically heterogeneous island systems in the Mediterranean. The biodiversity of these islands was strongly shaped by the Pliocene and Pleistocene sea regressions and transgression, resulting in different waves of colonization and isolation of species coming from the mainland. The Italian wall lizard, Podarcis siculus, is present on the following islands of the Tuscan Archipelago: Elba, Giglio, Giannutri, Capraia, Montecristo and Cerboli. The species in the area displays a relatively high morphological variability that in the past led to the description of several subspecies. In this study, both the genetic and morphological diversity of P. siculus of the Tuscan Archipelago were investigated. Specifically, the meristic characters and the dorsal pattern were analyzed, while the genetic relationships among these populations were explored with mtDNA and microsatellite nuclear markers to reconstruct the colonization history of the Archipelago. Our results converge in the identification of at least two different waves of colonization in the Archipelago: Elba, and the populations of Cerboli and Montecristo probably originate from historical introductions from mainland Tuscany, while those of Giglio and Capraia are surviving populations of an ancient lineage which colonized the Tuscan Archipelago during the Pliocene and which shares a common ancestry with the P. siculus populations of south-eastern Italy. Giannutri perhaps represents an interesting case of hybridization between the populations from mainland Tuscany and the Giglio-Capraia clade. Based on the high phenotypic and molecular distinctiveness of this ancient clade, these populations should be treated as distinct units deserving conservation and management efforts as well as further investigation to assess their taxonomic status.

Sweet chestnut is a tree of great economic (fruit and wood production), ecological, and cultural importance in Europe. A large-scale landscape genetic analysis of natural populations of sweet chestnut across Europe is applied to (1) evaluate the geographic patterns of genetic diversity, (2) identify spatial coincidences between genetic discontinuities and geographic barriers, and (3) propose certain chestnut populations as reservoirs of genetic diversity for conservation and breeding programs. Six polymorphic microsatellite markers were used for genotyping 1608 wild trees sampled in 73 European sites. The Geostatistical IDW technique (ArcGIS 9.3) was used to produce maps of genetic diversity parameters (He, Ar, PAr) and a synthetic map of the population membership (Q value) to the different gene pools. Genetic barriers were investigated using BARRIER 2.2 software and their locations were overlaid on a Digital Elevation Model (GTOPO30). The DIVA-GIS software was used to propose priority areas for conservation. High values of genetic diversity (He) and allelic richness (Ar) were observed in the central area of C. sativa ’s European distribution range. The highest values of private allelic richness (PAr) were found in the eastern area. Three main gene pools and a significant genetic barrier separating the eastern from the central and western populations were identified. Areas with high priority for genetic conservation were indicated in Georgia, eastern Turkey, and Italy. Our results increase knowledge of the biogeographic history of C. sativa in Europe, indicate the geographic location of different gene pools, and identify potential priority reservoirs of genetic diversity.

The Asian chestnut gall wasp (ACGW) (Hymenoptera Dryocosmus kuriphilus Yasumatsu) is a severe pest of sweet chestnut (Castanea sativa Mill.) with a strong impact on growth and nut production. A comparative field trial in Central Italy, including provenances from Spain, Italy, and Greece, was screened for ACGW infestation over consecutive years. The Greek provenance Hortiatis expressed a high proportion of immune plants and was used to perform a genome-wide association study based on DNA pool sequencing (Pool-GWAS) by comparing two DNA pools from 25 susceptible and 25 resistant plants. DNA pools were sequenced with 50X coverage depth. Sequence reads were aligned to a C. mollissima reference genome and the pools were compared to identify SNPs associated with resistance. Twenty-one significant SNPs were identified and highlighted a small genomic region on pseudochromosome 3 (Chr 3), containing 12 candidate genes of three gene families: Cytochrome P450, UDP-glycosyltransferase, and Rac-like GTP-binding protein. Functional analyses revealed a putative metabolic gene cluster related to saccharide biosynthesis in the genomic regions associated with resistance that could be involved in the production of a toxic metabolite against parasites. The comparison with previous genetic studies confirmed the involvement of Chr 3 in the control of resistance to ACGW.