Abstract Juglans regia cv. Zijing, known as ‘Five-purple walnut’ exhibits distinct purple phenotypes in leaves, husks, flowers, branches, and seed coats. Despite its high horticultural value and stress resistance, the genetic basis of its unusual anthocyanin accumulation and the regulatory mechanisms underlying its purple phenotypes remain poorly understood. Here, we report chromosome-level genomes for ZJ and wild-type ‘Lvling’ (LL) walnuts, enabling comparative insights into genes regulating anthocyanin accumulation in plants. We identified genetic variations of JrATs, JrGHs, JrGH1s, and JrNLRs as unique to ZJ. By integrating transcriptomic and metabolomic analyses, we identified 590 differentially expressed genes (DEGs) and 118 differentially accumulated metabolites (DAMs) between purple and wild-type tissues. Consistent with leaf and husk data, enzymes JrANS54, JrANS102, JrC4H122, JrUFGT46, and transcription factors (TFs), particularly the MYB-bHLH-WD40 were expressed highly in ZJ leaves and husks. Multi-omics integration converged on JrMYB216 and JrMYB85 as important TFs regulating purple colorations in ZJ. Functional validation through stable Arabidopsis transformation and transient walnut husk assays confirmed that JrMYB216 and JrMYB85 form MYB-bHLH-WD40 complex with JrbHLH42 and JrWD40–168. These complexes synergistically activated structural genes downstream of the anthocyanin biosynthetic pathway, promoting proanthocyanidins and delphinidin derivative accumulation, thereby driving the purple phenotype. Notably, JrMYB216 contributed more significantly to anthocyanin accumulation than JrMYB85, with its enhanced regulatory activity potentially linked to a 23 bp deletion in the second intron. This study systematically elucidates the regulatory network governing anthocyanin mediated purple phenotyping in walnut, providing novel theoretical insights into the molecular mechanisms underlying purple coloration traits in perennial woody plants.

Members of the genus Juglans (walnuts, family Juglandaceae) are typical Tertiary relict tree species. They are widely distributed in China and are well known for their edible nuts and high-quality wood. The genetic structure and historical gene flow patterns of Chinese Juglans still need to be resolved. Here, we collected samples from a total of 2242 trees from 142 populations of three walnut species (Juglans regia L., Juglans sigillata Dode, and Juglans mandshurica Maxim.) and a hybrid taxon Juglans x hopeiensis. These samples were distributed throughout China. We analyzed population genetic structure, interspecific relationships, lineage differentiation, and speciation using 21 EST-SSR genetic markers. All populations of J. regia and J. sigillata clustered into one lineage, corresponding to the Juglans sect. Dioscaryon, and all J. mandshurica and Juglans x hopeiensis populations (section Cardiocaryon) clustered into a second lineage. For J. mandshurica, there was an obvious north-south genetic cline. Interestingly, except for seven populations of the hybrid Juglans x hopeiensis, there was almost no gene flow between the populations of section Juglans/Dioscaryon and section Cardiocaryon. A DIYABC analysis showed that J. regia and J. mandshurica differentiated during the Oligocene. Juglans sigillata originated during the Pliocene to the Pleistocene. Subsequently, during the Middle Pleistocene, J. regia and J. mandshurica hybridized within a narrow zone to produce Juglans x hopeiensis. These results demonstrate the impact of repeated turbulent climate changes in the Quaternary on the evolutionary history of Tertiary relict plants.

Intraspecific genetic variance and gene flow can support the adaptive evolution of species challenged by climate shifts or novel environmental conditions. Less well understood is how genome organization and gene flow interact in closely related species during evolutionary divergence and differentiation. Here we conducted genomic footprint analyses to determine how three species of Pterocarya (P . stenoptera, P. hupehensis, and P. macroptera), which are sympatric but occupy different elevational niches, adapted to the heterogeneous environment of the Qinling-Daba Mountains, China. We identified candidate genes for environmental adaptation (i.e., PIEZO1, WRKY39, VDAC3, CBL1, and RAF), and also identified regions of gene introgression between P. hupehensis and P. macroptera that show lower genetic load and higher genetic diversity than the rest of their genomes. The same introgressed regions are notably situated in areas of minimal genetic divergence yet they are characterized by elevated recombination rates. We also identified candidate genes within these introgressed regions related to environmental adaptation (TPLC2, CYCH;1, LUH, bHLH112, GLX1, TLP-3, and ABC1). Our findings have thus clarified the important role of gene flow in ecological adaptation and revealed genomic signatures of past introgression. Together, these findings provide a stronger theoretical basis for understanding the ecological adaptation and conservation of Quaternary relict woody plants in East Asia.

Elucidating the impacts of demographic history and genomic selection on species evolution is a central topic in phylogeography and evolutionary biology. Black walnuts (Juglans section Rhysocaryon) are native nut trees of the NEW WORLD, with a broad distribution ranging from southern Canada to northern Argentina. The demographic history and genomic dynamics of Rhysocaryon species remain poorly understood. Here, we employed population genomics and chloroplast data to construct a high-density map of genomic variation across 108 Rhysocaryon accessions. Despite gene introgression, these accessions were clearly delimited into four groups. Evolutionary scenarios analysis showed that the diversification of black walnuts might have occurred approximately 28.74 million years ago during the late Oligocene, with the clade comprising Juglans hindsii and Juglans californica diverging earliest. The gene introgression and hybridization analysis indicated that Juglans microcarpa might be a hybrid descendant of Juglans nigra and J. hindsii. As the climate oscillated, these ancestral populations kept diverging, laying the basis for their colonization of South America. Quaternary climatic oscillations also exerted a profound influence on black walnut population size, which exhibited sensitive fluctuations in response to alternation of glacial and interglacial periods. The selection sweeps analysis unveiled highly divergent genomic regions in the economic species J. nigra, which were associated with development, reproduction, disease resistance, and stress tolerance. The genes WRKY41 and ERF012 were identified as potential drivers of J. nigra's adaptation. Our findings illuminated the demographic history and selective signatures of black walnuts, thereby providing a genetic foundation for future breeding, conservation, and genomic studies.

With the advent of affordable and more accurate third generation sequencing technologies and the associated bioinformatic tools, it is now possible to sequence, assemble, and annotate more species of conservation concern than ever before. Juglans cinerea , commonly known as butternut or white walnut, is a member of the walnut family, native to the Eastern United States and Southeastern Canada. The species is currently listed as Endangered on the IUCN Red List due to decline from an invasive fungus known as Ophiognomonia clavigignenti-juglandacearum (Oc-j) that causes butternut canker. Oc-j creates visible sores on the trunks of the tree which essentially starves and slowly kills the tree. Natural resistance to this pathogen is rare. Conserving butternut is of utmost priority due to its critical ecosystem role and cultural significance. As part of an integrated undergraduate and graduate student training program in biodiversity and conservation genomics, the first reference genome for Juglans cinerea is described here. This chromosome-scale 539 Mb assembly was generated from over 100X coverage of Oxford Nanopore long reads and scaffolded with the Juglans mandshurica genome. Scaffolding with a closely related species oriented and ordered the sequences in a manner more representative of the structure of the genome without altering the sequence. Comparisons with sequenced Juglandaceae revealed high levels of synteny and further supported J. cinerea’s recent phylogenetic placement. Comparative assessment of gene family evolution revealed a significant number of contracting families, including several associated with biotic stress response.

Current techniques of forest inventory rely on manual measurements and are slow and labor intensive. Recent developments in computer vision and depth sensing can produce accurate measurement data at significantly reduced time and labor costs. We developed the ForSense system to measure the diameters of trees at various points along the stem as well as stem straightness. Time use, mean absolute error (MAE), and root mean squared error (RMSE) metrics were used to compare the system against manual methods, and to compare the system against itself (reproducibility). Depth-derived diameter measurements of the stems at the heights of 0.3, 1.4, and 2.7 m achieved RMSE of 1.7, 1.5, and 2.7 cm, respectively. The ForSense system produced straightness measurement data that was highly correlated with straightness ratings by trained foresters. The ForSense system was also consistent, achieving sub-centimeter diameter difference with subsequent measures and less than 4% difference in straightness value between runs. This method of forest inventory, which is based on depth-image computer vision, is time efficient compared to manual methods and less computationally and technologically intensive compared to Structure-from-Motion (SFM) photogrammetry and ground-based LiDAR or terrestrial laser scanning (TLS).

Juglans regia is an important perennial crop cultivated for its high-quality nuts and wood. It is generally believed that J. regia survived and expanded in almost completely isolated stands in Asia after the last glaciation. Humans subsequently dispersed J. regia through cultural expansion and trade. We evaluated the spatial genetic structure and genetic diversity of 2,929 J . regia samples from 150 populations using 14 Simple Sequence Repeats (SSRs) markers. Our study revealed that regions with the highest genetic diversity included Southern Asia, Western Asia, Western Europe, and China, as illustrated using a Geostatistical Inverse Distance Weighting (IDW) interpolation of observed heterozygosity (H O ), expected (HE) heterozygosity (H E ), percentage of polymorphic loci (PPL), the total number of alleles (N A ) , and Allelic richness (R S ) in Arc Geographic Information System (ArcGIS). The ecological Niche Model (ENM) showed J. regia had a high probability of association with Central Asian and Eastern Asian habitats. Population genetic structure, phylogeny, and Principal Coordinate Analysis (PCoA) identified three genetic groups corresponding to three geographic sources. Turkish and Georgian populations served as a bridge between Asian populations and Europe populations. We suggest that J. regia evolved in central Asian mountain ranges ~ 65 million years ago (Mya) and dispersed across Eurasia during climate shifts (~ 65 to 3Mya). The population contracted into multiple refugia during the Last Glacial Maximum. The current distribution of J. regia across Eurasia was shaped by the cumulative effects of contraction or expansion of different refugia and human exploitation after LGM.

Walnut (Juglans) species are used as nut crops worldwide. Eastern black walnut (EBW, Juglans nigra), a diploid, horticultural important woody species is native to much of eastern North America. Although it is highly valued for its wood and nut, there are few resources for understanding EBW genetics. Here, we present a high-quality genome assembly of J. nigra based on Illumina, Pacbio, and Hi-C technologies. The genome size was 540.8 Mb, with a scaffold N50 size of 35.1 Mb, and 99.0% of the assembly was anchored to 16 chromosomes. Using this genome as a reference, the resequencing of 74 accessions revealed the effective population size of J. nigra declined during the glacial maximum. A single whole-genome duplication event was identified in the J. nigra genome. Large syntenic blocks among J. nigra, Juglans regia, and Juglans microcarpa predominated, but inversions of more than 600 kb were identified. By comparing the EBW genome with those of J. regia and J. microcarpa, we detected InDel sizes of 34.9 Mb in J. regia and 18.3 Mb in J. microcarpa, respectively. Transcriptomic analysis of differentially expressed genes identified five presumed NBS-LRR (NUCLEOTIDE BINDING SITE-LEUCINE-RICH REPEAT) genes were upregulated during the development of walnut husks and shells compared to developing embryos. We also identified candidate genes with essential roles in seed oil synthesis, including FAD (FATTY ACID DESATURASE) and OLE (OLEOSIN). Our work advances the understanding of fatty acid bioaccumulation and disease resistance in nut crops, and also provides an essential resource for conducting genomics-enabled breeding in walnut.

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.

Machine learning (ML) reproducibility needs to be informed with reliable evaluation measures. However, routine image classification is evaluated using metrics that are highly sensitive to class prevalence. Consequently, the reproducibility of ML models remains unclear due to class imbalance-induced noise. We suggest regularly using class imbalance-resistant evaluation metrics, including balanced accuracy, area under precision-recall curve, and image classification efficacy, for the evaluation of the reproducibility of ML models. Each of these evaluation metrics is conceptually consistent with and logically complements the others, and their joint use can help explain different aspects of classification performance at the whole-class level and individual class level. These metrics can be used for the validation, testing, and/or transfer of ML classifiers. Comprehensive analysis using these metrics as a routine approach strengthens the reproducibility of ML models.

Walnut (Juglans) species are economically important hardwood trees cultivated worldwide for both edible nuts and high-quality wood. Broad-scale assessments of species diversity, evolutionary history, and domestication are needed to improve walnut breeding. In this study, we sequenced 309 walnut accessions from around the world, including 55 Juglans relatives, 98 wild Persian walnuts (J. regia), 70 J. regia landraces, and 86 J. regia cultivars. The phylogenetic tree indicated that J. regia samples (section Dioscaryon) were monophyletic within Juglans. The core areas of genetic diversity of J. regia germplasm were southwestern China and southern Asia near the Qinghai-Tibet Plateau and the Himalayas, and the uplift of the Himalayas was speculated to be the main factor leading to the current population dynamics of Persian walnut. The pattern of genomic variation in terms of nucleotide diversity, linkage disequilibrium, single nucleotide polymorphisms, and insertions/deletions revealed the domestication and selection footprints in Persian walnut. Selective sweep analysis, GWAS, and expression analysis further identified two transcription factors, JrbHLH and JrMYB6, that influence the thickness of the nut diaphragm as loci under selection during domestication. Our results elucidate the domestication and selection footprints in Persian walnuts and provide a valuable resource for the genomics-assisted breeding of this important crop.

Frost damage is among the major limitations to reforestation and forest restoration projects worldwide. Investigations of environmental and genetic effects on frost resistance have focused on boreal and temperate tree species rather than tropical trees. Koa (Acacia koa A. Gray) is a valuable tropical hardwood tree species endemic to the Hawaiian Islands, USA. Koa occurs across a wide elevational gradient, and newly planted trees are subject to winter frost at high elevations. We sought to determine whether different koa populations show variation in freeze hardiness as a cold-tolerance mechanism, and whether exposure to hardening conditions prior to frost exposure can modify koa cold-tolerance adaptation. Seeds from 13 populations of koa (Acacia koa A. Gray) were collected across an elevational range (603–2050 m) on the Island of Hawai’i. Four-month-old seedlings grown from the 13 population seed sources were divided into control (non-acclimated) and cold-acclimated treatments, maintained at 26 °C/22 °C (day/night) or exposed to gradually decreasing temperatures to 8 °C/4 °C (day/night), respectively. After six weeks, control and cold-acclimated seedlings from each population were tested for freeze tolerance by electrolyte leakage at five test temperatures ranging from 5 °C (control) to −20 °C. Treatment effects were mainly observed at the lowest test temperatures (−15 and −20 °C). A higher index of cold damage occurred in the non-acclimated seedlings for most of the populations. Several of our higher elevation populations showed greater cold tolerance than populations from lower elevations, particularly when cold-acclimated. Our results suggest that cold acclimation may increase frost hardiness in a tropical forest tree species, and that there is likely some adaptive variation in frost tolerance among populations from different elevations. Cold acclimation could be a useful tool to prepare koa seedlings to be planted in high-elevation sites prone to freezing winter temperatures.

Chloroplast (cp) DNA genomes are traditional workhorses for studying the evolution of species and reconstructing phylogenetic relationships in plants. Species of the genus Castanea (chestnuts and chinquapins) are valued as a source of nuts and timber wherever they grow, and chestnut species hybrids are common. We compared the cp genomes of C. mollissima, C. seguinii, C. henryi, and C. pumila. These cp genomes ranged from 160,805 bp to 161,010 bp in length, comprising a pair of inverted repeat (IR) regions (25,685 to 25,701 bp) separated by a large single-copy (LSC) region (90,440 to 90,560 bp) and a small single-copy (SSC) region (18,970 to 19,049 bp). Each cp genome encoded the same 113 genes; 82–83 protein-coding genes, 30 transfer RNA genes, and four ribosomal RNA genes. There were 18 duplicated genes in the IRs. Comparative analysis of cp genomes revealed that rpl22 was absent in all analyzed species, and the gene ycf1 has been pseudo-genized in all Chinese chestnuts except C. pumlia. We analyzed the repeats and nucleotide substitutions in these plastomes and detected several highly variable regions. The phylogenetic analyses based on plastomes confirmed the monophyly of Castanea species.

Quercus rubra half sib progenies (N = 93) expressed high levels of variance for both growth and stem form traits in three locations in Indiana, USA at age 11 or 12. Height, diameter, and volume were measured and sweep, branch angle, forking, and branch retention were rated using a (+ / −) system. Families selected for volume showed no unfavorable increases in sweep or branch angle and only a slight increase in branch retention. Northern red oak (Quercus rubra L.) is the most planted hardwood in the central USA. Red oak seeds from sources with improved growth and form are unavailable in the Central Hardwood Region, in part because the absence of agreed methods for stem form evaluation diminishes the effectiveness of selection. To identify red oak families improved for growth and to determine if a simple + / − rating system could identify red oak families with improved form. We evaluated 93 open-pollinated families of 11 or 12-year-old red oak growing in three sites in central Indiana (USA) for height, diameter, volume, and four traits rated as + / − : sweep, branch angle, forking, and branch retention. Family × location effects were significant for all quantitative traits but not for any binary traits; differences among families were significant for all traits at all sites. Heritabilities for most traits were high. Selection of the top 20 families for volume at each site resulted in no change in population means for sweep or branch angle and only a small increase in the retention of large limbs. A + / − rating system to evaluate hardwood form can help breeders deliver improved red oak to landowners when more complex systems are impractical.

The utility of seventeen Microsatellite (SSR) markers and fifteen inter simple sequence repeats (ISSR) markers for the identification of twenty eight ramets of 11 varieties of walnut ( Juglans regia ) was explored. Thirty nine individual genomes were screened using 61 and 38 scorable fragments from SSR and ISSR markers, respectively. The least polymorphic SSR locus was WGA004 (two alleles) and the most polymorphic (5 alleles) was WGA276. Polymorphism information content values ranged from 0.08 (WGA004) to 0.43 (WGA032) in SSR markers and from 0.11 (AGA (AC)7) to 0.49 (CAC(TGT)5) in ISSR markers, with an average of 0.29 and 0.19, respectively. In most cases, grafted varieties with identical names also had the same microsatellites profile. The principal coordinate analysis and clustering (UPGMA) based on the combined marker set emphasized two failures in grafting or off-types, ramets identified as Serr 4 (S4) and Vina 1 (V1). The presence of two off-type ramets in the walnut research orchard emphasizes the importance of using molecular certification for proving true-to-type of walnut orchards. Using 13 polymorphic SSRs, we tabulated a DNA fingerprint chart of 11 walnut varieties. Except for ‘Chandler’, each cultivar could be distinguished using a combination of only two SSR loci. The 13 SSRs markers evaluated in this study could be used in future to identify clones produced from the varieties.

By comparing the phylogeography of Juglans regia and J. mandshurica, we found that two walnut species, even when sympatric, rarely introgress, suggesting that strong hybridization barriers exist between these species. The biogeographic investigation of temperate walnut (Juglans) trees is of great interest because of their ecological and economical importance. Our goal was to perform an in-depth investigation of the genetic and phylogeographic history of J. regia and J. mandshurica, two walnut species that are sympatric in parts of their ranges, including tests for gene flow and incomplete lineage sorting. We employed a 234 bp locus of mitochondrial DNA, a 1.8 Kbp locus of chloroplast DNA, 3 nuclear loci totaling 1740 bp in length, and 17 EST-SSRs. We sampled 559 individuals, 332 of J. regia and 227 of J. mandshurica, from 69 locations. We found that J. regia and J. mandshurica, even when sympatric, rarely introgress, suggesting that strong barriers to hybridization exist between these species. Niche overlap analyses for the two species found that they occupy distinct ecological niches and that the sympatric populations may be the result of recent postglacial population expansion.

The advantages of clonal forestry have been well described, but little progress has been made in the identification of phenotypes best suited to this method in high-value hardwood species. The genetic variation within, clonal repeatability (broad-sense heritability) of, and Pearson's correlations among phenological, morphological, physiological, and growth traits (N = 22) of black walnut (Juglans nigra L.) were investigated using 25 grafted clonal genotypes. The trees were grown at wide spacing (4.6 m x 6.1 m) in an intensively managed plantation in north-central Indiana, USA. Clonal effect was significant (p < 0.05) for most traits except gas exchange variables. Many phenological traits showed high clonal repeatability (Rc2 > 0.70), including foliation dates, first female bloom, first pollen shed, and crown retention rate (autumnal defoliation). Some traits showed moderate repeatability (0.35 < R-c(2) < 0.7), such as branch angle, branch frequency, tree diameter, height, anthracnose severity, and foliar carbon and nitrogen concentration. Net CO2 assimilation rate and stomatal conductance had low repeatability (R-c(2) < 0.35). We observed a previously unreported association between sexual morph and tree stem growth, i.e., protandrous trees generally grew faster than protogynous trees. This association between sexual morph and stem growth may be an evolutionary consequence of differences in reproductive costs in protandrous versus protogynous trees, and needs to be tested in other monoecious woody species. Overall, our analyses indicated that the following traits are associated with rapid stem growth: 1) early foliation; 2) early male flowering or late female flowering, i.e., more likely a protandrous variety; 3) large average branch diameter; 4) high branch frequency; 5) small (more vertical) average branch angle; 6) early leaf fall; 7) low laterseason foliar carbon concentration; and 8) large number of small fruits and seeds. These traits may be used, with further testing, to define a biomass ideotype for J. nigra.

Manchurian walnut ( Juglans mandshurica Maxim.) is a synonym of J . cathayensis , a diploid, vulnerable, temperate deciduous tree valued for its wood and nut. It is also valued as a rootstock for Juglans regia because of its reported tolerance of lesion nematode. Reference genomes are available for several Juglans species, our goal was to produce a de novo, chromosome‐level assembly of the J . mandshurica genome. Here, we reported an improved assembly of J . mandshurica with a contig N50 size of 6.49 Mb and a scaffold N50 size of 36.1 Mb. The total genome size was 548 Mb encoding 29,032 protein coding genes which were annotated. The collinearity analysis showed that J . mandshurica and J. regia originated from a common ancestor, with both species undergoing two WGD events. A genomic comparison showed that J . mandshurica was missing 1657 genes found in J. regia , and J . mandshurica includes 2827 genes not found in of the J. regia genome. The J . mandshurica contained 1440 unique paralogues that were highly enriched for flavonoid biosynthesis, phenylpropanoid biosynthesis, and plant‐pathogen interaction. Four gene families related to disease resistance notable contraction (rapidly evolving; LEA , WAK , PPR , and PR ) in J . mandshurica compared to eight species. JmaPR10 and JmaPR8 contained three orthologous gene pairs with J. regia that were highly expressed in root bark. JmaPR10 is a strong candidate gene for lesion nematodes resistance in J . mandshurica . The J . mandshurica genome should be a useful resource for study of the evolution, breeding, and genetic variation in walnuts ( Juglans ).

American elm, Ulmus americana L., was widely cultivated in the USA and Canada as a landscape tree. Despite its importance in landscaping and horticulture, its genome is poorly characterized. We assembled the chloroplast genomes of two American elm genotypes (RV16 and Am. 57845); to our knowledge, this is the first description of sequencing and assembly of this species. The complete chloroplast genome of U. americana ranged from 158,935 to 158,993 bp and it contains 127 genes, namely 85 protein-coding genes, 34 tRNA genes, and 8 rRNA genes. Between the two American elm chloroplasts we sequenced, we identified 240 high-quality sequence variants (SNPs and indels). To evaluate the phylogeny of American elm, we compared the chloroplast genomes of the two American elms with seven Asian elm species and twelve other chloroplast genomes available through the NCBI database. As expected, Ulmus was closely related to Morus and Cannabis, as all three genera are assigned to the Urticales. We clarified the timing of the divergence of American elm from the available Asian elms, the divergence within these Asian elms, and all the species' relative ages. Comparison of the chloroplasts of American elm with the available Asian elms revealed that trnH was absent from American elm but not most Asian elms; conversely, petB, petD, psbL, trnK, and rps16 are present in the American elm but absent from all Asian elms analyzed. ycf15 was present in both American and Asian elms but absent from members of closely related genera. The complete chloroplast genome of U. americana will provide useful genetic resources for characterizing the genetic diversity of U. americana and potentially help to conserve natural populations of American elm.