Nut tree species have a great economic and cultural value for many countries across the world. Their highly nutritious content makes them one of the most favourite healthy foods of the global population, with strong evidence of their positive impact on human health. Nut tree crops are very diverse in terms of nut chemical content and shape, growing climates, and domestication history. However, all of them have a very long juvenile phase thatmakes classical breeding challenging and time consuming. Also, their cultivation is very resource demanding. The application of genomic-assisted breeding can accelerate the development of adaptive cultivars of nut tree species. In this article, we summarize recent successes in genomics for nut tree crops, specifically almond, chestnut, hazelnut, pecan, pistachio, and walnut. We report the recent discoveries on the genetic control of target traits for the genetic improvement of these species, with suggestions for future directions and breeding applications.

Simple sequence repeat (SSR) markers were used to authenticate ramets of 11 Persian walnut ( Juglans regia L.) varieties. All varieties and 28 of their ramets (n = 39) were genotyped with 17 SSR markers. The genetic profiles revealed two off-types: the ramets Serr 4 (S4) and Vina 1 (V1). SSR fingerprints individuating 11 walnut varieties were possible using 13 polymorphic SSRs that could be used in the future to identify clones of these varieties. Except for ‘Chandler’, each cultivar could be distinguished using a combination of two SSR loci. This result emphasizes the efficacy of the SSR markers in true-to-type validation of walnut orchards.

Juglans regia (L.) is cultivated worldwide for its nutrient-rich nuts. In Italy, despite the growing demand, walnut cultivation has gone through a strong decline in recent decades, which led to Italy being among the top five net importing countries. To promote the development of local high-quality Italian walnut production, we devised a multidisciplinary project to highlight the distinctive traits of three varieties grown in the mountainous region Trentino (northeast of Italy): the heirloom 'Bleggiana', a second local accession called local Franquette and the French cultivar 'Lara', recently introduced in the local production to increase yield. The genetic characterization confirmed the uniqueness of 'Bleggiana' and revealed local Franquette as a newly described autochthonous variety, thus named 'Blegette'. The metabolic profiles highlighted a valuable nutritional composition of the local varieties, richer in polyphenols and with a lower ω-6/ω-3 ratio than the commercial 'Lara'. 'Blegette' obtained the highest preference scores from consumers for both the visual aspect and tasting; however, the volatile organic compound profiles did not discriminate among the characterized cultivars. The described local varieties represent an interesting reservoir of walnut genetic diversity and quality properties, which deserve future investigation on agronomically useful traits (e.g., local adaptation and water usage) for a high-quality and sustainable production.

Background: The release of the first reference genome of walnut (Juglans regia L.) enabled many achievements in the characterization of walnut genetic and functional variation. However, it is highly fragmented, preventing the integration of genetic, transcriptomic, and proteomic information to fully elucidate walnut biological processes. Findings: Here, we report the new chromosome-scale assembly of the walnut reference genome (Chandler v2.0) obtained by combining Oxford Nanopore long-read sequencing with chromosome conformation capture (Hi-C) technology. Relative to the previous reference genome, the new assembly features an 84.4-fold increase in N50 size, with the 16 chromosomal pseudomolecules assembled and representing 95% of its total length. Using full-length transcripts from single-molecule real-time sequencing, we predicted 37,554 gene models, with a mean gene length higher than the previous gene annotations. Most of the new protein-coding genes (90%) present both start and stop codons, which represents a significant improvement compared with Chandler v1.0 (only 48%). We then tested the potential impact of the new chromosome-level genome on different areas of walnut research. By studying the proteome changes occurring during male flower development, we observed that the virtual proteome obtained from Chandler v2.0 presents fewer artifacts than the previous reference genome, enabling the identification of a new potential pollen allergen in walnut. Also, the new chromosome-scale genome facilitates in-depth studies of intraspecies genetic diversity by revealing previously undetected autozygous regions in Chandler, likely resulting from inbreeding, and 195 genomic regions highly differentiated between Western and Eastern walnut cultivars. Conclusion: Overall, Chandler v2.0 will serve as a valuable resource to better understand and explore walnut biology.

SummaryOver the last 20 years, global production of Persian walnut (Juglans regia L.) has grown enormously, likely reflecting increased consumption due to its numerous benefits to human health. However, advances in genome‐wide association (GWA) studies and genomic selection (GS) for agronomically important traits in walnut remain limited due to the lack of powerful genomic tools. Here, we present the development and validation of a high‐density 700K single nucleotide polymorphism (SNP) array in Persian walnut. Over 609K high‐quality SNPs have been thoroughly selected from a set of 9.6 m genome‐wide variants, previously identified from the high‐depth re‐sequencing of 27 founders of the Walnut Improvement Program (WIP) of University of California, Davis. To validate the effectiveness of the array, we genotyped a collection of 1284 walnut trees, including 1167 progeny of 48 WIP families and 26 walnut cultivars. More than half of the SNPs (55.7%) fell in the highest quality class of ‘Poly High Resolution’ (PHR) polymorphisms, which were used to assess the WIP pedigree integrity. We identified 151 new parent‐offspring relationships, all confirmed with the Mendelian inheritance test. In addition, we explored the genetic variability among cultivars of different origin, revealing how the varieties from Europe and California were differentiated from Asian accessions. Both the reconstruction of the WIP pedigree and population structure analysis confirmed the effectiveness of the Applied Biosystems™ Axiom™ J. regia 700K SNP array, which initiates a novel genomic and advanced phase in walnut genetics and breeding.

Genetic responses to environmental changes take place at different spatial scales. While the effect of environment on the distribution of species' genetic diversity at large geographical scales has been the focus of several recent studies, its potential effects on genetic structure at local scales are understudied. Environmental effects on fine‐scale spatial genetic structure (FSGS) were investigated in four Alpine conifer species (five to eight populations per species) from the eastern Italian Alps. Significant FSGS was found for 11 of 25 populations. Interestingly, we found no significant differences in FSGS across species but great variation among populations within species, highlighting the importance of local environmental factors. Interannual variability in spring temperature had a small but significant effect on FSGS of Larix decidua, probably related to species‐specific life history traits. For Abies alba, Picea abies and Pinus cembra, linear models identified spring precipitation as a potentially relevant climate factor associated with differences in FSGS across populations; however, models had low explanatory power and were strongly influenced by a P. cembra outlier population from a very dry site. Overall, the direction of the identified effects is according to expectations, with drier and more variable environments increasing FSGS. Underlying mechanisms may include climate‐related changes in the variance of reproductive success and/or environmental selection of specific families. This study provides new insights on potential changes in local genetic structure of four Alpine conifers in the face of environmental changes, suggesting that new climates, through altering FSGS, may also have relevant impacts on plant microevolution.

Using the latest sequencing and optical mapping technologies, we have produced a high-quality de novo assembly of the apple (Malus domestica Borkh.) genome. Repeat sequences, which represented over half of the assembly, provided an unprecedented opportunity to investigate the uncharacterized regions of a tree genome; we identified a new hyper-repetitive retrotransposon sequence that was over-represented in heterochromatic regions and estimated that a major burst of different transposable elements (TEs) occurred 21 million years ago. Notably, the timing of this TE burst coincided with the uplift of the Tian Shan mountains, which is thought to be the center of the location where the apple originated, suggesting that TEs and associated processes may have contributed to the diversification of the apple ancestor and possibly to its divergence from pear. Finally, genome-wide DNA methylation data suggest that epigenetic marks may contribute to agronomically relevant aspects, such as apple fruit development.

Conifers are key species of many boreal and temperate forests, providing crucial resources to human activities. Adaptation to local climate and long life span make tree species highly susceptible to both short- and long-term environmental changes. Thus, a substantial effect of ongoing climate change on forest viability and production potential is expected. A deeper understanding of the genetic mechanisms that control the adaptive ability of forests can be used for management strategies to assist forests through climate change. Indeed, a reliable and systematic genetic assessment of Norway spruce provenances for their response to extreme climatic conditions could be a prerequisite for a successful assisted migration of this species in a changing environment. The purpose of this study is to investigate, by mean of Single Nucleotide Polymorphisms (SNPs), the genetic bases of local adaptation along altitudinal gradients in 18 natural Alpine populations of Norway spruce (Picea abies [L.] Karst), sampled on a regional scale in Trentino-South Tyrol (Eastern Italian Alps). To account for patterns of gene flow and spatial genetic structure due to alpine landscape features, sampled stands were subdivided into three geographical groups, each including at least one sampled altitudinal gradient. As expected, hierarchical analyses of molecular variance revealed that most of the genetic variability was found within stands, though a little but significant variation was found among geographical groups. To detect potentially adaptive markers, two distinct approaches were used. First, classical outlier loci detection was applied along the altitudinal gradients using both a Bayesian analysis and a Hierarchical Island Model (HIM), which showed contrasting results. Subsequently, Moran's Eigenvector Maps (MEM) variables, which may account for spatial variation and un-accounted environmental factors, were applied to an allele distribution model, and 19 loci significantly associated to environmental variation were identified. Four of these loci were also detected as outliers by the HIM. The combined approach of selection scan and spatial analysis method allowed for a parallel investigation of Norway spruce adaptive potential in alpine stands, providing evidence for selective forces acting on adaptive loci that could be relevant in forest management.

Quantitative trait loci (QTL) mapping approaches rely on the correct ordering of molecular markers along the chromosomes, which can be obtained from genetic linkage maps or a reference genome sequence. For apple (Malus domestica Borkh), the genome sequence v1 and v2 could not meet this need; therefore, a novel approach was devised to develop a dense genetic linkage map, providing the most reliable marker-loci order for the highest possible number of markers. The approach was based on four strategies: (i) the use of multiple full-sib families, (ii) the reduction of missing information through the use of HaploBlocks and alternative calling procedures for single-nucleotide polymorphism (SNP) markers, (iii) the construction of a single backcross-type data set including all families, and (iv) a two-step map generation procedure based on the sequential inclusion of markers. The map comprises 15 417 SNP markers, clustered in 3 K HaploBlock markers spanning 1 267 cM, with an average distance between adjacent markers of 0.37 cM and a maximum distance of 3.29 cM. Moreover, chromosome 5 was oriented according to its homoeologous chromosome 10. This map was useful to improve the apple genome sequence, design the Axiom Apple 480 K SNP array and perform multifamily-based QTL studies. Its collinearity with the genome sequences v1 and v3 are reported. To our knowledge, this is the shortest published SNP map in apple, while including the largest number of markers, families and individuals. This result validates our methodology, proving its value for the construction of integrated linkage maps for any outbreeding species.

In temperate trees, growth resumption in spring time results from chilling and heat requirements, and is an adaptive trait under global warming. Here, the genetic determinism of budbreak and flowering time was deciphered using five related full-sib apple families. Both traits were observed over 3 years and two sites and expressed in calendar and degree-days. Best linear unbiased predictors of genotypic effect or interaction with climatic year were extracted from mixed linear models and used for quantitative trait locus (QTL) mapping, performed with an integrated genetic map containing 6849 single nucleotide polymorphisms (SNPs), grouped into haplotypes, and with a Bayesian pedigree-based analysis. Four major regions, on linkage group (LG) 7, LG10, LG12, and LG9, the latter being the most stable across families, sites, and years, explained 5.6-21.3% of trait variance. Co-localizations for traits in calendar days or growing degree hours (GDH) suggested common genetic determinism for chilling and heating requirements. Homologs of two major flowering genes, AGL24 and FT, were predicted close to LG9 and LG12 QTLs, respectively, whereas Dormancy Associated MADs-box (DAM) genes were near additional QTLs on LG8 and LG15. This suggests that chilling perception mechanisms could be common among perennial and annual plants. Progenitors with favorable alleles depending on trait and LG were identified and could benefit new breeding strategies for apple adaptation to temperature increase.

Cultivated apple (Malus × domestica Borkh.) is one of the most important fruit crops in temperate regions, and has great economic and cultural value. The apple genome is highly heterozygous and has undergone a recent duplication which, combined with a rapid linkage disequilibrium decay, makes it difficult to perform genome-wide association (GWA) studies. Single nucleotide polymorphism arrays offer highly multiplexed assays at a relatively low cost per data point and can be a valid tool for the identification of the markers associated with traits of interest. Here, we describe the development and validation of a 487K SNP Affymetrix Axiom(®) genotyping array for apple and discuss its potential applications. The array has been built from the high-depth resequencing of 63 different cultivars covering most of the genetic diversity in cultivated apple. The SNPs were chosen by applying a focal points approach to enrich genic regions, but also to reach a uniform coverage of non-genic regions. A total of 1324 apple accessions, including the 92 progenies of two mapping populations, have been genotyped with the Axiom(®) Apple480K to assess the effectiveness of the array. A large majority of SNPs (359 994 or 74%) fell in the stringent class of poly high resolution polymorphisms. We also devised a filtering procedure to identify a subset of 275K very robust markers that can be safely used for germplasm surveys in apple. The Axiom(®) Apple480K has now been commercially released both for public and proprietary use and will likely be a reference tool for GWA studies in apple.

Forest trees dominate many Alpine landscapes that are currently exposed to changing climate. Norway spruce is one of the most important conifer species of the Italian Alps, and natural populations are found across steep environmental gradients with large differences in temperature and moisture availability. This study seeks to determine and quantify patterns of genetic diversity in natural populations toward understanding adaptive responses to changing climate. Across the Italian species range, 24 natural stands were sampled with a major focus on the Eastern Italian Alps. Sampled trees were genotyped for 384 selected single nucleotide polymorphisms (SNPs) from 285 genes. A wide array of potential candidate genes was tested for correlation with climatic parameters. To minimize false-positive association between genotype and climate, population structure was investigated. Pairwise F ST estimates between sampled populations ranged between 0.000 and 0.075, with the highest values involving the two disjoint populations, Valdieri, on the western Italian Alps, and Campolino, the most southern population on the Apennines. Despite considerable genetic admixture among populations, both Bayesian and multivariate approach identified four genetic clusters. Selection scans revealed five F ST outliers, and the environmental association analysis detected ten SNPs associated to one or more climatic variables. Overall, 13 potentially adaptive loci were identified, three of which have been reported in a previous study on the same species conducted on a broader geographical scale. In our study, precipitation, more than temperature, was often associated with genotype; therefore, it appears as the most important environmental variable associated with the high sensitivity of Norway spruce to soil water supply. These findings provide relevant information for understanding and quantifying climate change effects on this species and its ability to genetically adapt.

Forest tree species of temperate and boreal regions have undergone a long history of demographic changes and evolutionary adaptations. The main objective of this study was to detect signals of selection in Norway spruce (Picea abies [L.] Karst), at different sampling-scales and to investigate, accounting for population structure, the effect of environment on species genetic diversity. A total of 384 single nucleotide polymorphisms (SNPs) representing 290 genes were genotyped at two geographic scales: across 12 populations distributed along two altitudinal-transects in the Alps (micro-geographic scale), and across 27 populations belonging to the range of Norway spruce in central and south-east Europe (macro-geographic scale). At the macrogeographic scale, principal component analysis combined with Bayesian clustering revealed three major clusters, corresponding to the main areas of southern spruce occurrence, i.e. the Alps, Carpathians, and Hercynia. The populations along the altitudinal transects were not differentiated. To assess the role of selection in structuring genetic variation, we applied a Bayesian and coalescent-based F-ST-outlier method and tested for correlations between allele frequencies and climatic variables using regression analyses. At the macrogeographic scale, the F-ST-outlier methods detected together 11 F-ST-outliers. Six outliers were detected when the same analyses were carried out taking into account the genetic structure. Regression analyses with population structure correction resulted in the identification of two (micro-geographic scale) and 38 SNPs (macro-geographic scale) significantly correlated with temperature and/or precipitation. Six of these loci overlapped with F-ST-outliers, among them two loci encoding an enzyme involved in riboflavin biosynthesis and a sucrose synthase. The results of this study indicate a strong relationship between genetic and environmental variation at both geographic scales. It also suggests that an integrative approach combining different outlier detection methods and population sampling at different geographic scales is useful to identify loci potentially involved in adaptation.

The Alpine Forest Genomics Network was formed in 2011 and held its first annual meeting on June 24–26, 2012, in the Natural Park Adamello Brenta in Trentino Region, Italy. The meeting was attended by 30 researchers from the alpine region of Europe and had two primary goals: (1) for researchers to introduce each other to current and planned research activities in forest landscape genomics and (2) to develop a strategic vision for the network. A steering committee was elected and will develop a white paper over the next year. The next annual meeting will be held in Austria in June 2013.

Climate is one of the most important drivers of local adaptation in forest tree species. Standing levels of genetic diversity and structure within and among natural populations of forest trees are determined by the interplay between climatic heterogeneity and the balance between selection and gene flow. To investigate this interplay, single nucleotide polymorphisms (SNPs) were genotyped in 24 to 37 populations from four subalpine conifers, Abies alba Mill., Larix decidua Mill., Pinus cembra L. and Pinus mugo Turra, across their natural ranges in the Italian Alps and Apennines. Patterns of population structure were apparent using a Bayesian clustering program, STRUCTURE, which identified three to five genetic groups per species. Geographical correlates with these patterns, however, were only apparent for P. cembra. Multivariate environmental variables [i.e. principal components (PCs)] were subsequently tested for association with SNPs using a Bayesian generalized linear mixed model. The majority of the SNPs, ranging from six in L. decidua to 18 in P. mugo, were associated with PC1, corresponding to winter precipitation and seasonal minimum temperature. In A. alba, four SNPs were associated with PC2, corresponding to the seasonal minimum temperature. Functional annotation of those genes with the orthologs in Arabidopsis revealed several genes involved in abiotic stress response. This study provides a detailed assessment of population structure and its association with environment and geography in four coniferous species in the Italian mountains.

Gliomas are very invasive brain tumors with poor prognosis and therefore any attempt to limit tumor cell dissemination in the brain is expected to improve glioma treatment. The recent deorphanization of CXCR7 as additional receptor for CXCL12 and CXCL11 has raised key issues on its interaction with the CXCL12/CXCR4 axis as a mechanism to modulate glioma cell migration. In this work we investigated protein and mRNA expression of the two chemokines CXCL12 and CXCL11, together with their receptors CXCR4 and CXCR7 in human glioma specimens and cell lines by immunohistochemistry, flow cytometry and quantitative real-time PCR. The main purpose of this study was to find out whether and at what extent CXCR4 and CXCR7 are differentially expressed in glioma cells. In human glioma specimens the levels of CXCL11 and CXCR4 mRNA were significantly higher in glioblastomas compared to non-tumor controls or low grade gliomas, whilst no difference was found for CXCL12 and CXCR7 mRNA expression. In cell lines, flow cytometry and immunocytochemical experiments showed CXCR4 was mainly expressed irrespective of its membrane or intracellular localization. In contrast, a predominant intracellular localization together with a negligible membrane expression of CXCR7 was found in all cells examined. In in vitro experiments CXCR4 and CXCR7 antagonists and the silencing of CXCR4 showed complete inhibition of glioma proliferation.Our findings, in agreement with previous data, suggest that in human glioma cells the prevalent intracellular localization of CXCR7 might modulate the functionality of CXCL11/12 either acting as a scavenger for these chemokines or interfering with the signaling pathways activated by the stimulation of CXCR4.