Identification of a Unique Genomic Region in Sweet Chestnut (Castanea sativa Mill.) That Controls Resistance to Asian Chestnut Gall Wasp Dryocosmus kuriphilus Yasumatsu.

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.

The Intriguing Biogeographic Pattern of the Italian Wall Lizard Podarcis siculus (Squamata: Lacertidae) in the Tuscan Archipelago Reveals the Existence of a New Ancient Insular Clade.

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.

Biocultural diversity of common walnut (Juglans regia L.) and sweet chestnut (Castanea sativa Mill.) across Eurasia.

A biocultural diversity approach integrates plant biology and germplasm dispersal processes with human cultural diversity. An increasing number of studies have identified cultural factors and ethnolinguistic barriers as the main drivers of the genetic diversity in crop plants. Little is known about how anthropogenic processes have affected the evolution of tree crops over the entire time scale of their interaction with humans. In Asia and the Mediterranean, common walnut (Juglans regia L.) and sweet chestnut (Castanea sativa Mill.) have been economically and culturally important crops for millennia; there, in ancient times, they were invested with symbolic and religious significance. In this study, we detected a partial geographic congruence between the ethno-linguistic repartition of human communities, the distribution of major cognitive sets of word-related terms, and the inferred genetic clusters of common walnut and sweet chestnut populations across Eurasia. Our data indicated that isolation by distance processes, landscape heterogeneity and cultural boundaries might have promoted simultaneously human language diversification and walnut/chestnut differentiation across the same geographic macro-regions. Hotspots of common walnut and sweet chestnut genetic diversity were associated with areas of linguistic enrichment in the Himalayas, Trans-Caucasus, and Pyrenees Mountains, where common walnuts and sweet chestnuts had sustained ties to human culture since the Early Bronze Age. Our multidisciplinary approach supported the indirect and direct role of humans in shaping walnut and chestnut diversity across Eurasia from the EBA (e.g., Persian Empire and Greek-Roman colonization) until the first evidence of active selection and clonal propagation by grafting of both species. Our findings highlighted the benefit of an efficient integration of the relevant cultural factors in the classical genome (G) × environmental (E) model and the urgency of a systematic application of the biocultural diversity concept in the reconstruction of the evolutionary history of tree species.

Genetic Analysis by nuSSR Markers of Silver Birch (Betula pendula Roth) Populations in Their Southern European Distribution Range.

In the main distribution area the genetic pattern of silver birch is dominated by two haplotypes: haplotype A located in the western and north-western Europe, and haplotype C in eastern and southeastern Europe, characterized by high levels of neutral genetic variability within populations, and low differentiation among populations. Information about the amount and structure of genetic variation in the southern marginal areas, representing rear populations left during the expansion of this species from southern glacial refugia, are lacking. The general aim of the study was to investigate the existence of the climatic characteristics typical of the environmental niche of the species, jointly to genetic organization, variation and gene flow, in marginal populations on the Italian Apennines and Greek Southern Rhodope and compare them with populations of the southern part of the main distribution range on the Alps and Balkans. Genetic analysis was performed using nuclear microsatellites loci on 311 trees sampled from 14 populations. Environmental analysis was performed on the multivariate analysis of derived climatic variables. The allelic pattern was analyzed to assess genetic diversity, population diversity and differentiation, population structure and gene flow. The geographic and environmental peripherality did not always match, with some Apennine sites at higher elevation enveloped in the environmental niche. In the peripheral populations on the Apennines, we observed a lower genetic diversity and higher differentiation, with evident genetic barriers detected around these sites. These characteristics were not shown in the marginal Greek populations. Unexpectedly, the southern Italian marginal populations showed genetic links with the Greek and central area of the distribution range. The Greek populations also showed evident gene flow with the Alpine and Balkan areas. The disparity of results in these two marginal areas show that it is not the geographic peripherality or even the ecological marginality that may shape the genetic diversity and structure of marginal populations, but primarily their position as part of the continuous range or as disjunct populations. This outcome suggests different considerations on how to manage their gene pools and the role that these rear populations can play in maintaining the biodiversity of this species.

DNA analysis of Castanea sativa (sweet chestnut) in Britain and Ireland: Elucidating European origins and genepool diversity.

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.

An Assessment of Genetic Diversity and Drought Tolerance in Argan Tree (Argania spinosa) Populations: Potential for the Development of Improved Drought Tolerance.

The argan tree (Argania spinosa) occurs in a restricted area of Southwestern Morocco characterized by low water availability and high evapotranspirative demand. Despite the adaptation of the argan tree to drought stress, the extent of the argan forest has declined markedly due to increased aridity, land use changes and the expansion of olive cultivation. The oil of the argan seed is used for cooking and as the basis for numerous cosmetics. The identification of argan tree varieties with enhanced drought tolerance may minimize the economic losses associated with the decline of the argan forest and constrain the spread of desertification. In this study we collected argan ecotypes from four contrasting habitats and grew them under identical controlled environment conditions to investigate their response to drought. Leaf gas exchange analysis indicated that the argan ecotypes showed a high degree of adaptation to drought stress, maintaining photosynthetic activity at low levels of foliar water content and co-ordinating photosynthesis, stomatal behavior and metabolism. The stomata of the argan trees were highly sensitive to increased leaf to air vapor pressure deficit, representing an adaptation to growth in an arid environment where potential evapotranspiration is high. However, despite originating in contrasting environments, the four argan ecotypes exhibited similar gas exchange characteristics under both fully irrigated and water deficit conditions. Population genetic analyses using microsatellite markers indicated a high degree of relatedness between the four ecotypes; indicative of both artificial selection and the transport of ecotypes between different provinces throughout centuries of management of the argan forest. The majority of genetic variation across the four populations (71%) was observed between individuals, suggesting that improvement of argan is possible. Phenotypic screening of physiological responses to drought may prove effective in identifying individuals and then developing varieties with enhanced drought tolerance to enable the maintenance of argan production as climate change results in more frequent and severe drought events in Northern Africa.

Microsatellite markers reveal a strong geographical structure in European populations of Castanea sativa (Fagaceae): evidence for multiple glacial refugia.

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.

Comparative mapping in the Fagaceae and beyond with EST-SSRs.

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.

Microsatellite development for the relictual conifer Araucaria araucana (Araucariaceae) using next-generation sequencing.

PREMISE OF THE STUDY: In this study, the 454 GS-FLX genome sequence system was used for the identification and characterization of microsatellites in Araucaria araucana, one of the most important and endangered species of Chilean and Argentinean native forests. METHODS AND RESULTS: A total of 35876 reads were identified, 96% of which were within the size range selected for further analysis. Of these, 1563 contained a microsatellite insert suitable for primer design. Ten simple sequence repeat (SSR) markers provided easily interpretable patterns and were used to evaluate the genetic diversity in four populations of the species. The 10 microsatellites showed high polymorphism levels, with a total of 99 alleles and 32 private alleles. The observed heterozygosity was high and ranged from 0.513 to 0.723. CONCLUSIONS: The primers presented in this study display high genetic diversity and may provide useful information for the design of conservation strategies in Araucaria araucana.

A fast and cost-effective approach to develop and map EST-SSR markers: oak as a case study.

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.