The first genetic linkage map for Fraxinus pennsylvanica and syntenic relationships with four related species.

KEY MESSAGE: The genetic linkage map for green ash (Fraxinus pennsylvanica) contains 1201 DNA markers in 23 linkage groups spanning 2008.87cM. The green ash map shows stronger synteny with coffee than tomato. Green ash (Fraxinus pennsylvanica) is an outcrossing, diploid (2n = 46) hardwood tree species, native to North America. Native ash species in North America are being threatened by the rapid spread of the emerald ash borer (EAB, Agrilus planipennis), an invasive pest from Asia. Green ash, the most widely distributed ash species, is severely affected by EAB infestation, yet few genomic resources for genetic studies and improvement of green ash are available. In this study, a total of 5712 high quality single nucleotide polymorphisms (SNPs) were discovered using a minimum allele frequency of 1% across the entire genome through genotyping-by-sequencing. We also screened hundreds of genomic- and EST-based microsatellite markers (SSRs) from previous de novo assemblies (Staton et al., PLoS ONE 10:e0145031, 2015; Lane et al., BMC Genom 17:702, 2016). A first genetic linkage map of green ash was constructed from 90 individuals in a full-sib family, combining 2719 SNP and 84 SSR segregating markers among the parental maps. The consensus SNP and SSR map contains a total of 1201 markers in 23 linkage groups spanning 2008.87 cM, at an average inter-marker distance of 1.67 cM with a minimum logarithm of odds of 6 and maximum recombination fraction of 0.40. Comparisons of the organization the green ash map with the genomes of asterid species coffee and tomato, and genomes of the rosid species poplar and peach, showed areas of conserved gene order, with overall synteny strongest with coffee.

High-quality genetic mapping with ddRADseq in the non-model tree Quercus rubra.

BACKGROUND: Restriction site associated DNA sequencing (RADseq) has the potential to be a broadly applicable, low-cost approach for high-quality genetic linkage mapping in forest trees lacking a reference genome. The statistical inference of linear order must be as accurate as possible for the correct ordering of sequence scaffolds and contigs to chromosomal locations. Accurate maps also facilitate the discovery of chromosome segments containing allelic variants conferring resistance to the biotic and abiotic stresses that threaten forest trees worldwide. We used ddRADseq for genetic mapping in the tree Quercus rubra, with an approach optimized to produce a high-quality map. Our study design also enabled us to model the results we would have obtained with less depth of coverage. RESULTS: Our sequencing design produced a high sequencing depth in the parents (248×) and a moderate sequencing depth (15×) in the progeny. The digital normalization method of generating a de novo reference and the SAMtools SNP variant caller yielded the most SNP calls (78,725). The major drivers of map inflation were multiple SNPs located within the same sequence (77% of SNPs called). The highest quality map was generated with a low level of missing data (5%) and a genome-wide threshold of 0.025 for deviation from Mendelian expectation. The final map included 849 SNP markers (1.8% of the 78,725 SNPs called). Downsampling the individual FASTQ files to model lower depth of coverage revealed that sequencing the progeny using 96 samples per lane would have yielded too few SNP markers to generate a map, even if we had sequenced the parents at depth 248×. CONCLUSIONS: The ddRADseq technology produced enough high-quality SNP markers to make a moderately dense, high-quality map. The success of this project was due to high depth of coverage of the parents, moderate depth of coverage of the progeny, a good framework map, an optimized bioinformatics pipeline, and rigorous premapping filters. The ddRADseq approach is useful for the construction of high-quality genetic maps in organisms lacking a reference genome if the parents and progeny are sequenced at sufficient depth. Technical improvements in reduced representation sequencing (RRS) approaches are needed to reduce the amount of missing data.

The green ash transcriptome and identification of genes responding to abiotic and biotic stresses.

BACKGROUND: To develop a set of transcriptome sequences to support research on environmental stress responses in green ash (Fraxinus pennsylvanica), we undertook deep RNA sequencing of green ash tissues under various stress treatments. The treatments, including emerald ash borer (EAB) feeding, heat, drought, cold and ozone, were selected to mimic the increasing threats of climate change and invasive pests faced by green ash across its native habitat. RESULTS: We report the generation and assembly of RNA sequences from 55 green ash samples into 107,611 putative unique transcripts (PUTs). 52,899 open reading frames were identified. Functional annotation of the PUTs by comparison to the Uniprot protein database identified matches for 63 % of transcripts and for 98 % of transcripts with ORFs. Further functional annotation identified conserved protein domains and assigned gene ontology terms to the PUTs. Examination of transcript expression across different RNA libraries revealed that expression patterns clustered based on tissues regardless of stress treatment. The transcripts from stress treatments were further examined to identify differential expression. Tens to hundreds of differentially expressed PUTs were identified for each stress treatment. A set of 109 PUTs were found to be consistently up or down regulated across three or more different stress treatments, representing basal stress response candidate genes in green ash. In addition, 1956 simple sequence repeats were identified in the PUTs, of which we identified 465 high quality DNA markers and designed flanking PCR primers. CONCLUSIONS: North American native ash trees have suffered extensive mortality due to EAB infestation, creating a need to breed or select for resistant green ash genotypes. Stress from climate change is an additional concern for longevity of native ash populations. The use of genomics could accelerate management efforts. The green ash transcriptome we have developed provides important sequence information, genetic markers and stress-response candidate genes.

Susceptibility of Walnut and Hickory Species to Geosmithia morbida.

Thousand cankers disease (TCD) of walnut is a result of feeding in the phloem by the walnut twig beetle (WTB), Pityophthorus juglandis, and subsequent canker formation caused by Geosmithia morbida around galleries. TCD has caused extensive morbidity and mortality to Juglans nigra in the western United States and, in 2010, was discovered in the eastern United States, where the tree is a highly valuable timber resource. WTB and G. morbida also have been found in J. regia orchards throughout major production areas in California, and the numbers of damaged trees are increasing. We tested the susceptibility of walnut and hickory species to G. morbida in greenhouse and field studies. Carya illinoinensis, C. aquatica, and C. ovata were immune. All walnut species tested, including J. ailantifolia, J. californica, J. cinerea, J. hindsii, J. major, J. mandshurica, J. microcarpa, J. nigra, and J. regia, developed cankers following inoculation with G. morbida. J. nigra was the most susceptible, whereas J. major, a native host of the WTB and, presumably, G. morbida, had smaller and more superficial cankers. Canker formation differed among maternal half-sibling families of J. nigra and J. cinerea, indicating genetic variability in resistance to G. morbida. Our inoculation studies with G. morbida have corroborated many of the field observations on susceptibility of walnut and hickory species to TCD, although the ability of the WTB to successfully attack and breed in walnut is also an important component in TCD resistance.

Quantification and characterization of biological activities of glansreginin A in black walnuts (Juglans nigra).

Glansreginin A has been reported to be an indicator of the quality of walnuts (Juglans spp.). However, bioactive properties of glansreginin A have not been adequately explored. In the present study, we quantified concentrations of glansreginin A in black walnuts (Juglans nigra) using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and performed an array of in vitro bioassays to characterize biological activities (e.g., antibacterial, antioxidant, anticancer capacities) of this compound. Results from HPLC-MS/MS analysis indicated that glansreginin A was presented in all 12 black cultivars examined and its contents were variable among black walnut cultivars, ranged from 6.8 mg/kg (Jackson) to 47.0 mg/kg (Hay). Glansreginin A possessed moderate antibacterial activities against Gram-positive pathogens (Staphylococcus aureus and Bacillus anthracis). This compound exhibited no antioxidant activities, did not induce the activity of antioxidant response element signaling pathways, and exerted no antiproliferative effects on tumorigenic alveolar epithelial cells and non-tumorigenic lung fibroblast cells.

A haplotype-resolved chromosome-scale genome for Quercus rubra L. provides insights into the genetics of adaptive traits for red oak species.

Northern red oak (Quercus rubra L.) is an ecologically and economically important forest tree native to North America. We present a chromosome-scale genome of Q. rubra generated by the combination of PacBio sequences and chromatin conformation capture (Hi-C) scaffolding. This is the first reference genome from the red oak clade (section Lobatae). The Q. rubra assembly spans 739 Mb with 95.27% of the genome in 12 chromosomes and 33,333 protein-coding genes. Comparisons to the genomes of Quercus lobata and Quercus mongolica revealed high collinearity, with intrachromosomal structural variants present. Orthologous gene family analysis with other tree species revealed that gene families associated with defense response were expanding and contracting simultaneously across the Q. rubra genome. Quercus rubra had the most CC-NBS-LRR and TIR-NBS-LRR resistance genes out of the 9 species analyzed. Terpene synthase gene family comparisons further reveal tandem gene duplications in TPS-b subfamily, similar to Quercus robur. Phylogenetic analysis also identified 4 subfamilies of the IGT/LAZY gene family in Q. rubra important for plant structure. Single major QTL regions were identified for vegetative bud break and marcescence, which contain candidate genes for further research, including a putative ortholog of the circadian clock constituent cryptochrome (CRY2) and 8 tandemly duplicated genes for serine protease inhibitors, respectively. Genome-environment associations across natural populations identified candidate abiotic stress tolerance genes and predicted performance in a common garden. This high-quality red oak genome represents an essential resource to the oak genomic community, which will expedite comparative genomics and biological studies in Quercus species.

Microsatellite Borders and Micro-sequence Conservation in Juglans.

Walnuts (Juglans spp.) are economically important nut and timber species with a worldwide distribution. Using the published Persian walnut genome as a reference for the assembly of short reads from six Juglans species and several interspecific hybrids, we identified simple sequence repeats in 12 Juglans nuclear and organellar genomes. The genome-wide distribution and polymorphisms of nuclear and organellar microsatellites (SSRs) for most Juglans genomes have not been previously studied. We compared the frequency of nuclear SSR motifs and their lengths across Juglans, and identified section-specific chloroplast SSR motifs. Primer pairs were designed for more than 60,000 SSR-containing sequences based on alignment against assembled scaffold sequences. Of the >60,000 loci, 39,000 were validated by e-PCR using unique primer pairs. We identified primers containing 100% sequence identity in multiple species. Across species, sequence identity in the SSR-flanking regions was generally low. Although SSRs are common and highly dispersed in the genome, their flanking sequences are conserved at about 90 to 95% identity within Juglans and within species. In a few rare cases, flanking sequences are identical across species of Juglans. This comprehensive report of nuclear and organellar SSRs in Juglans and the generation of validated SSR primers will be a useful resource for future genetic analyses, walnut breeding programs, high-level taxonomic evaluations, and genomic studies in Juglandaceae.

Black Walnut (Juglans nigra) Extracts Inhibit Proinflammatory Cytokine Production From Lipopolysaccharide-Stimulated Human Promonocytic Cell Line U-937.

Black walnut (Juglans nigra L.) is an excellent source of health-promoting compounds. Consumption of black walnuts has been linked to many health benefits (e.g., anti-inflammatory) stemming from its phytochemical composition and medicinal properties, but these effects have not been systematically studied or characterized. In this study, potential anti-inflammatory compounds found in kernel extracts of 10 black walnut cultivars were putatively identified using a metabolomic profiling analysis, revealing differences in potential anti-inflammatory capacities among examined cultivars. Five cultivars were examined for activities in the human promonocytic cell line U-937 by evaluating the effects of the extracts on the expression of six human inflammatory cytokines/chemokines using a bead-based, flow cytometric multiplex assay. The methanolic extracts of these cultivars were added at four concentrations (0.1, 0.3, 1, and 10 mg/ml) either before and after the addition of lipopolysaccharide (LPS) to human U-937 cells to examine their effect on cytokine production. Results from cytotoxicity and viability assays revealed that the kernel extracts had no toxic effect on the U-937 cells. Of the 13 cytokines [interleukin (IL)-1ß, tumor necrosis factor alpha (TNF-α), monocyte chemoattractant protein (MCP)-1, IL-6, IL-8, IL-10, IL-12, IL-17, IL-18, IL-23, IL-33, interferon (IFN)-α, IFN-γ] measured, only six were detected under the culture conditions. The production of the six detected cytokines by phorbol 12-myristate 13-acetate (PMA)-differentiated, LPS-stimulated U-937 was significantly inhibited by the kernel extracts from two cultivars Surprise and Sparrow when the extracts were added before the addition of LPS. Other cultivars (Daniel, Mystry, and Sparks) showed weak or no significant effects on cytokine production. In contrast, no inhibitory effect was observed on the production of cytokines by PMA-differentiated, LPS-stimulated U-937 when the kernel extracts were added after the addition of LPS. The findings suggest that the extracts from certain black walnut cultivars, such as Sparrow and Surprise, are promising biological candidates for potentially decreasing the severity of inflammatory disease.

Identification and Characterization of Phenolic Compounds in Black Walnut Kernels.

Black walnuts ( Juglans nigra L.) are highly valued for producing phenolic-enriched nuts. The objectives of this study were to identify and characterize the phenolic contents of 11 different black walnut cultivars and compare the levels of these phenolics between black walnuts and English walnut ( Juglans regia L.). Totally, 16 phenolics including phenolic acids, flavonoids, and catechins were identified in the black walnut kernels, with ellagic acid predominating over the other phenolics. Significant differences were noted for the levels of quinic acid, gallic acid, 1,3,6-trigalloylglucose, catechin, and penta- O-galloyl-ß-d-glucose between the studied black walnuts and English walnut. Through principal component analysis, 51.54% of the variance in the phenolic data was explained. The hierarchical cluster analysis results showed three groups to which each walnut sample belongs. Most of the phenolics identified in this study have been reported to exert potential health-promoting activities. The findings of this study will provide critical information for consumers, nutritional therapy practitioners, researchers, and producers.

Pollen flow and paternity in an isolated and non-isolated black walnut (Juglans nigra L.) timber seed orchard.

Artificial pollination of black walnut (Juglans nigra L.) is not practical and timber breeders have historically utilized only open-pollinated half-sib families. An alternate approach called "breeding without breeding," consists of genotyping open-pollinated progeny using DNA markers to identify paternal parents and then constructing full-sib families. In 2014, we used 12 SSR markers to genotype 884 open-pollinated half-sib progeny harvested from two clonal orchards containing 206 trees, comprised of 52 elite timber selections. Seed was harvested in 2011 from each of two ramets of 23 clones, one upwind and one downwind, based on prevailing wind direction from the west-southwest. One orchard was isolated from wild black walnut and composed of forward selections while the other orchard was adjacent to a natural forest containing mature black walnut composed of backward selections. Isolation significantly increased within-orchard pollination (85%) of the progeny from the isolated orchard compared to 42% from the non-isolated orchard. Neither prevailing wind direction nor seed tree position in the orchard affected paternity patterns or wild pollen contamination. Genetic diversity indices revealed that progeny from both orchards were in Hardy-Weinberg equilibrium with very little inbreeding and no selfing. A significant level of inbreeding was present among the forward selected parents, but not the first generation (backward selected) parents. Some orchard clones failed to sire any progeny while other clones pollinated upwards of 20% of progeny.

A novel molecular toolkit for rapid detection of the pathogen and primary vector of thousand cankers disease.

Thousand Cankers Disease (TCD) of Juglans and Pterocarya (Juglandaceae) involves a fungal pathogen, Geosmithia morbida, and a primary insect vector, Pityophthorus juglandis. TCD was described originally from dying Juglans nigra trees in the western United States (USA), but it was reported subsequently from the eastern USA and northern Italy. The disease is often difficult to diagnose due to the absence of symptoms or signs on the bark surface of the host. Furthermore, disease symptoms can be confused with those caused by other biotic and abiotic agents. Thus, there is a critical need for a method for rapid detection of the pathogen and vector of TCD. Using species-specific microsatellite DNA markers, we developed a molecular protocol for the detection of G. morbida and P. juglandis. To demonstrate the utility of the method for delineating TCD quarantine zones, we tested whether geographical occurrence of symptoms and signs of TCD was correlated with molecular evidence for the presence of the cryptic TCD organisms. A total of 1600 drill cores were taken from branch sections collected from three regions (n = 40 trees for each location): California-J. hindsii (heavy disease incidence); Tennessee-J. nigra (mild disease incidence); and outside the known TCD zone (Missouri-J. nigra, no record of the disease). California samples had the highest incidence of the TCD organisms (85%, 34/40). Tennessee had intermediate incidence (42.5%, 17/40), whereas neither organism was detected in samples from Missouri. The low cost molecular protocol developed here has a high degree of sensitivity and specificity, and it significantly reduces sample-processing time, making the protocol a powerful tool for rapid detection of TCD.

Identifying Antibacterial Compounds in Black Walnuts (Juglans nigra) Using a Metabolomics Approach.

Black walnut (Juglans nigra L.) is one of the most economically valuable hardwood species and a high value tree for edible nut production in the United States. Although consumption of black walnut has been linked to multiple health-promoting effects (e.g., antioxidant, antimicrobial, anti-inflammatory), the bioactive compounds have not been systematically characterized. In addition, the associations between different black walnut cultivars and their health-promoting compounds have not been well established. In this study, the kernels of twenty-two black walnut cultivars selected for nut production by the University of Missouri Center for Agroforestry (Columbia, MO, USA) were evaluated for their antibacterial activities using agar-well diffusion assay. Among the selected cultivars, four black walnut cultivars (i.e., Mystry, Surprise, D.34, and A.36) exhibited antibacterial activity against a Gram-positive bacterium (Staphylococcus aureus), whereas other cultivars showed no effect on the inhibition of this bacterium. The antibacterial compounds showing the strongest activity were isolated with bioassay-guided purification and identified using a metabolomics approach. Six antibacterial bioactive compounds responsible for antimicrobial activity were successfully identified. Glansreginin A, azelaic acid, quercetin, and eriodictyol-7-O-glucoside are novel antibacterial compounds identified in the kernels of black walnuts. The metabolomics approach provides a simple and cost-effective tool for bioactive compound identification.

Genomic Variation Among and Within Six Juglans Species.

Genomic analysis in Juglans (walnuts) is expected to transform the breeding and agricultural production of both nuts and lumber. To that end, we report here the determination of reference sequences for six additional relatives of Juglans regia: Juglans sigillata (also from section Dioscaryon), Juglans nigra, Juglans microcarpa, Juglans hindsii (from section Rhysocaryon), Juglans cathayensis (from section Cardiocaryon), and the closely related Pterocarya stenoptera While these are 'draft' genomes, ranging in size between 640Mbp and 990Mbp, their contiguities and accuracies can support powerful annotations of genomic variation that are often the foundation of new avenues of research and breeding. We annotated nucleotide divergence and synteny by creating complete pairwise alignments of each reference genome to the remaining six. In addition, we have re-sequenced a sample of accessions from four Juglans species (including regia). The variation discovered in these surveys comprises a critical resource for experimentation and breeding, as well as a solid complementary annotation. To demonstrate the potential of these resources the structural and sequence variation in and around the polyphenol oxidase loci, PPO1 and PPO2 were investigated. As reported for other seed crops variation in this gene is implicated in the domestication of walnuts. The apparently Juglandaceae specific PPO1 duplicate shows accelerated divergence and an excess of amino acid replacement on the lineage leading to accessions of the domesticated nut crop species, Juglans regia and sigillata.

Development of novel genic microsatellite markers from transcriptome sequencing in sugar maple (Acer saccharum Marsh.).

BACKGROUND: Sugar maple (Acer saccharum Marsh.) is a hardwood tree species native to northeastern North America and economically valued for its wood and sap. Yet, few molecular genetic resources have been developed for this species to date. Microsatellite markers have been a useful tool in population genetics, e.g., to monitor genetic variation and to analyze gene flow patterns. The objective of this study is to develop a reference transcriptome and microsatellite markers in sugar maple. FINDINGS: A set of 117,861 putative unique transcripts were assembled using 29.2 Gb of RNA sequencing data derived from different tissues and stress treatments. From this set of sequences a total of 1068 microsatellite motifs were identified. Out of 58 genic microsatellite markers tested on a population of 47 sugar maple trees in upper Michigan, 22 amplified well, of which 16 were polymorphic and 6 were monomorphic. Values for expected heterozygosity varied from 0.224 to 0.726 for individual loci. Of the 16 polymorphic markers, 15 exhibited transferability to other Acer L. species. CONCLUSIONS: Genic microsatellite markers can be applied to analyze genetic variation in potentially adaptive genes relative to genomic reference markers as a basis for the management of sugar maple genetic resources in the face of climate change.

Preliminary Genomic Characterization of Ten Hardwood Tree Species from Multiplexed Low Coverage Whole Genome Sequencing.

Forest health issues are on the rise in the United States, resulting from introduction of alien pests and diseases, coupled with abiotic stresses related to climate change. Increasingly, forest scientists are finding genetic/genomic resources valuable in addressing forest health issues. For a set of ten ecologically and economically important native hardwood tree species representing a broad phylogenetic spectrum, we used low coverage whole genome sequencing from multiplex Illumina paired ends to economically profile their genomic content. For six species, the genome content was further analyzed by flow cytometry in order to determine the nuclear genome size. Sequencing yielded a depth of 0.8X to 7.5X, from which in silico analysis yielded preliminary estimates of gene and repetitive sequence content in the genome for each species. Thousands of genomic SSRs were identified, with a clear predisposition toward dinucleotide repeats and AT-rich repeat motifs. Flanking primers were designed for SSR loci for all ten species, ranging from 891 loci in sugar maple to 18,167 in redbay. In summary, we have demonstrated that useful preliminary genome information including repeat content, gene content and useful SSR markers can be obtained at low cost and time input from a single lane of Illumina multiplex sequence.

Development of genomic microsatellites in Gleditsia triacanthos (Fabaceae) using Illumina sequencing.

PREMISE OF THE STUDY: Fourteen genomic microsatellite markers were developed and characterized in honey locust, Gleditsia triacanthos, using Illumina sequencing. Due to their high variability, these markers can be applied in analyses of genetic diversity and structure, and in mating system and gene flow studies. • METHODS AND RESULTS: Thirty-six individuals from across the species range were included in a genetic diversity analysis and yielded three to 20 alleles per locus. Observed heterozygosity and expected heterozygosity ranged from 0.214 to 0.944 and from 0.400 to 0.934, respectively, with minimal occurrence of null alleles. Regular segregation of maternal alleles was observed at seven loci and moderate segregation distortion at four of 11 loci that were heterozygous in the seed parent. • CONCLUSIONS: Honey locust is an important agroforestry tree capable of very fast growth and tolerance of poor site conditions. This is the first report of genomic microsatellites for this species.