The introduction of the chestnut blight fungus (Cryphonectria parasitica) resulted in the American chestnut’s (Castanea dentata) functional extinction from forests of the eastern USA. This prompted several efforts to create blight tolerant American chestnuts, including hybridization with related chestnut species, backcross breeding, irradiation of chestnut seeds, and transgenesis. Characterizing establishment and growth among these chestnut varieties is necessary to plan effective breeding and reintroduction programs. We assessed incremental height and diameter growth rates and survival for five chestnut varieties (non-transgenic American, Asian hybrid, backcross, transgenic Darling 54 American, and irradiated American) and red oak from 2019 to 2023 in open field and shelterwood silvicultural treatment plots at three sites (Virginia, Pennsylvania, and New York). Blight performance was not evaluated. All varieties exhibited greater growth and survival in the open field than shelterwood (Cohen’s d = 0.55 growth, 0.22 survival). Backcross seedlings exhibited similar growth and survival compared to non-transgenic American chestnut. Transgenic American chestnuts in most open field plots had lower height growth and survival, but similar diameter growth compared to non-transgenic American chestnut, while transgenic chestnuts in shelterwood plots had equivalent performance to non-transgenic varieties. Thus, both varieties in active development for enhancing American chestnut blight tolerance (D54 transgenic and backcross) exhibited responses dependent on location and initial size. Notable differences existed among state sites, particularly for growth in NY, and survival in PA and VA. These results suggest that selection of optimal American chestnut varieties for restoration may be context dependent.

More than a century after two introduced pathogens killed billions of American chestnut trees, introgression of resistance alleles from Chinese chestnuts has contributed to the recovery of self-sustaining populations. However, progress has been slow because of the complex genetic architecture of resistance. To better understand blight resistance, we compared reference genomes, gene expression responses, and stem metabolite profiles of the resistant Chinese and susceptible American chestnut species. To accelerate resistance breeding, we conducted large-scale phenotyping and genotyping in hybrids of these species. Simulation and inoculation experiments suggest that significant resistance gains are possible through selectively breeding trees with an average of 70 to 85% American chestnut ancestry. The resources developed in this work are foundational for breeding to create diverse restoration populations with sufficient disease resistance and competitive growth.

Co-adaptation of cytoplasmic and nuclear genomes is critical to physiological function for many species. Despite this understanding, hybridization can disrupt co-adaptation, leading to a mismatch between maternally inherited cytoplasmic genomes and biparentally inherited nuclear genomes. Few studies have examined the consequences of cytonuclear interactions to physiological function across environments. Here, we quantify the degree of co-introgression between chloroplast and nuclear-chloroplast (N-cp) genes across repeated hybrid zones and its consequences to physiological function across environments. We use whole-genome resequencing and common garden experiments with clonally replicated genotypes sampled across the natural hybrid zone between Populus trichocarpa and P. balsamifera. Geographic clines were used to test for co-introgression of the chloroplast genome with N-cp and non-interacting nuclear genes. Co-introgression of the chloroplast alongside N-cp genes was limited, although contact-zone specific patterns point to the importance of regional differences. Combining ancestry estimates with phenotypic data across common gardens revealed that mismatches between chloroplast and nuclear ancestry can influence physiological performance, but the strength and direction of these effects vary depending on the environment. Overall, this study highlights the importance of cytonuclear interactions to adaptation, and the context-dependent role the environment may play influencing that interaction.

Over a century after two introduced pathogens decimated American chestnut populations, breeding programs continue to incorporate resistance from Chinese chestnut to recover self-sustaining populations. Due to complex genetics of chestnut blight resistance, it is challenging to obtain trees with sufficient resistance and competitive growth. We developed high quality reference genomes for Chinese and American chestnut and leveraged large disease phenotype and genotype datasets to develop accurate genomic selection. Inoculation and simulation results indicate that resistance may be substantially increased in trees that inherited 70% to 100% of their genome from American chestnut. To facilitate gene editing, we integrated multiple lines of evidence to discover candidate alleles for blight resistance and susceptibility. These genomic resources provide a strong foundation to accelerate restoration of this iconic tree. ### Competing Interest Statement The authors have declared no competing interest.

The use of genetic data for timber species and population assignment is a powerful tool for combating the illegal timber trade, but the challenges of extracting DNA from timber have prevented the routine use of genetics as a supply chain management tool. To overcome these challenges, we explored the feasibility of focused ultrasound extraction (FUSE) for rapid DNA release from timber. Using high-pressure ultrasound pulses, FUSE generates a cavitation bubble cloud that disintegrates samples into acellular debris, resulting in the mechanical release of DNA. In this work, FUSE was applied to white oak (Quercus alba) timber shavings to test the feasibility of using FUSE for timber DNA extraction for the first time. Results showed that FUSE processing disintegrated the tissue samples and released significant quantities of DNA. After five minutes of tissue processing DNA quantities of 0.21 ± 0.02ng/mg, 0.99 ± 0.32ng/mg, and 0.14 ± 0.01ng/mg, were released from medium, coarse, and combination shaving groups, respectively. Amplification and sequencing of regions within the matK and rbcL chloroplast genes confirmed that the quality of DNA prepared with FUSE was suitable for PCR and short-read sequencing applications. Overall, these results show that FUSE can serve as a DNA sample preparation method capable of releasing high-quality DNA from timber in a fraction of the time required by conventional extraction methods. Based on the improved efficiency of DNA release with FUSE, ongoing work aims to develop this technology into portable systems that can be used to rapidly prepare timber samples for genetic species identification.

American chestnut ( Castanea dentata) is a deciduous tree species of eastern North America that was decimated by the introduction of the chestnut blight fungus ( Cryphonectria parasitica) in the early 20th century. Although millions of American chestnuts survive as root collar sprouts, these trees rarely reproduce. Thus, the species is considered functionally extinct. American chestnuts with improved blight resistance have been developed through interspecific hybridization followed by conspecific back- crossing, and by genetic engineering. Incorporating adaptive genomic diversity into these backcross families and transgenic lines is important for restoring the species across broad climatic gradients. To develop sampling recommendations for ex situ conservation of wild adaptive genetic variation, we coupled whole- genome resequencing of 384 stump sprouts with genotype-environment association analyses and found that the species range can be subdivided into three seed zones characterized by relatively homogeneous adaptive allele frequencies. We estimated that 21 to 29 trees per seed zone will need to be conserved to capture most extant adaptive diversity. We also resequenced the genomes of 269 backcross trees to understand the extent to which the breeding program has already captured wild adaptive diversity, and to estimate optimal reintroduction sites for specific families on the basis of their adaptive portfolio and future climate projections. Taken together, these results inform the development of an ex situ germplasm conservation and breeding plan to target blight- resistant breeding populations to specific environments and provides a blueprint for developing restoration plans for other imperiled tree species.

Closely related species often use the same genes to adapt to similar environments. However, we know little about why such genes possess increased adaptive potential and whether this is conserved across deeper evolutionary lineages. Adaptation to climate presents a natural laboratory to test these ideas, as even distantly related species must contend with similar stresses. Here, we re-analyse genomic data from thousands of individuals from 25 plant species as diverged as lodgepole pine and Arabidopsis (similar to 300 Myr). We test for genetic repeatability based on within-species associations between allele frequencies in genes and variation in 21 climate variables. Our results demonstrate significant statistical evidence for genetic repeatability across deep time that is not expected under randomness, identifying a suite of 108 gene families (orthogroups) and gene functions that repeatedly drive local adaptation to climate. This set includes many orthogroups with well-known functions in abiotic stress response. Using gene co-expression networks to quantify pleiotropy, we find that orthogroups with stronger evidence for repeatability exhibit greater network centrality and broader expression across tissues (higher pleiotropy), contrary to the 'cost of complexity' theory. These gene families may be important in helping wild and crop species cope with future climate change, representing important candidates for future study.

Population demographic changes, alongside landscape, geographic and climate heterogeneity, can influence the timing, stability and extent of introgression where species hybridise. Thus, quantifying interactions across diverged lineages, and the relative contributions of interspecific genetic exchange and selection to divergence at the genome-wide level is needed to better understand the drivers of hybrid zone formation and maintenance. We used seven latitudinally arrayed transects to quantify the contributions of climate, geography and landscape features to broad patterns of genetic structure across the hybrid zone of Populus trichocarpa and P. balsamifera and evaluated the demographic context of hybridisation over time. We found genetic structure differed among the seven transects. While ancestry was structured by climate, landscape features influenced gene flow dynamics. Demographic models indicated a secondary contact event may have influenced contemporary hybrid zone formation with the origin of a putative hybrid lineage that inhabits regions with higher aridity than either of the ancestral groups. Phylogenetic relationships based on chloroplast genomes support the origin of this hybrid lineage inferred from demographic models based on the nuclear data. Our results point towards the importance of climate and landscape patterns in structuring the contact zones between P. trichocarpa and P. balsamifera and emphasise the value whole genome sequencing can have to advancing our understanding of how neutral processes influence divergence across space and time.

Rapid advances in DNA sequencing technologies made it cost-effective to get high-throughput genomic sequence data. Applications in whole genome resequencing presented new approaches to investigate natural variation and adaptation of species. The genus Populus in the Salicaceae family includes economically and ecologically critical species that are dioecious, wind-pollinated, and predominantly distributed in river ecosystems. Poplar serves as a model for the research on the evolutionary genomics of forest tree species. In this study, we aimed to present an example of a comprehensive SNP calling pipeline, demonstrating its application in elucidating genetic diversity and population structure from whole genome resequencing data. The results discovered genetic variants from whole genome resequencing data of poplar species. The pipeline involved sequence preprocessing, alignment to the reference genome, and variant calling.

The American chestnut ( Castanea dentata ) is a functionally extinct tree species that was decimated by an invasive fungal pathogen in the early 20 th century. Disease resistant chestnuts have been developed through hybridization and genetic modification, but these populations may lack the adaptive genomic diversity necessary to restore the species across its climatically diverse historical range. An understanding of the genomic architecture of local adaptation in wild American chestnut and identification of seed zones for germplasm conservation are necessary in order to deploy locally adapted, disease-resistant American chestnut populations. Here, we characterize the genomic basis of climate adaptation in remnant wild American chestnut, define seed zones based on climate envelopes and adaptive diversity, and make sampling recommendations for germplasm conservation. Whole genome re-sequencing of 384 trees coupled with genotype-environment association methods suggest the species range can be most parsimoniously subdivided into three seed zones characterized by relatively homogeneous allele frequencies relative to rangewide adaptive diversity. Using these data, we developed a method to estimate the number of samples required from each seed zone to recapitulate standing adaptive diversity in each seed zone, and found that on average, 21-29 trees will need to be conserved to capture 95% of the wild adaptive diversity. Taken together, these results will inform the development of an ex situ germplasm conservation and breeding plan to develop locally adapted blight-resistant American chestnut populations, and provide a blueprint for developing restoration plans for other imperiled tree species.

Summary Quantifying the mechanisms underlying hybrid zone formation requires an evaluation of both neutral and nonneutral evolutionary processes. Population demographic changes, alongside landscape heterogeneity and climate adaptation, can influence the timing, stability, and extent of introgression where species hybridize. Thus, quantifying interactions across diverged lineages, and the relative contributions of interspecific genetic exchange and selection to divergence at the genome-wide level is needed. We quantified the contributions of climate and geography to the genetic structure across replicate contact zones between Populus trichocarpa and P. balsamifera, evaluating the demographic context of hybridization and the role of genetic connectivity over time. We found genomic structure differed among the seven latitudinally arrayed transects. While ancestry was strongly structured by climate, geography appears to have restricted gene flow in certain regions of the hybrid zone. The best fitting demographic model indicated high rates of gene flow alongside selection have likely influenced the establishment and maintenance of the hybrid zone. Our results point towards the importance of climate in structuring the contact zones between P. trichocarpa and P. balsamifera and emphasize the value of sampling replicated zones of contact to understand how neutral and non-neutral processes influence hybrid zone formation across space and time.

Abstract Premise Sample preparation in genomics is a critical step that is often overlooked in molecular workflows and impacts the success of downstream genetic applications. This study explores the use of a recently developed focused ultrasound extraction (FUSE) technique to enable the rapid release of DNA from plant tissues for genetic analysis. Methods FUSE generates a dense acoustic cavitation bubble cloud that pulverizes targeted tissue into acellular debris. This technique was applied to leaf samples of American chestnut (Castanea dentata), tulip poplar (Liriodendron tulipifera), red maple (Acer rubrum), and chestnut oak (Quercus montana). Results We observed that FUSE can extract high quantities of DNA in 9–15 min, compared to the 30 min required for control DNA extraction methods. FUSE extracted DNA quantities of 24.33 ± 6.51 ng/mg and 35.32 ± 9.21 ng/mg from American chestnut and red maple, respectively, while control methods yielded 6.22 ± 0.87 ng/mg and 11.51 ± 1.95 ng/mg, respectively. The quality of the DNA released by FUSE allowed for successful amplification and next‐generation sequencing. Discussion These results indicate that FUSE can improve DNA extraction efficiency for leaf tissues. Continued development of this technology aims to adapt to field‐deployable systems to increase the cataloging of genetic biodiversity, particularly in low‐resource biodiversity hotspots.

Closely-related species often use the same genes to adapt to similar environments 1,2 . However, we know little about why such genes possess increased adaptive potential, and whether this is conserved across deeper evolutionary time. Classic theory suggests a “cost of complexity”: adaptation should occur via genes affecting fewer traits to reduce deleterious side-effects (i.e. lower pleiotropy) 3 . Adaptation to climate presents a natural laboratory to test this theory, as even distantly-related species must contend with similar stresses 4 . Here, we re-analyse genomic data from thousands of individuals from 25 plant species to identify a suite of 108 genes enriched for signatures of repeated local adaptation to climate. This set includes many genes with well-known functions in abiotic stress response, identifying key genes that repeatedly drive adaptation in species as distantly-related as lodgepole pine and Arabidopsis (~ 300 My). Using gene co-expression networks to quantify each gene’s pleiotropy, we find enrichment for greater network centrality/interaction strength and broader expression across tissues (i.e. higher pleiotropy), contrary to the ”cost of complexity” theory. These genes may be particularly important in helping both wild and crop species cope with future climate change, representing a set of important candidates for future study.

Changes in telomere length are increasingly used to indicate species' response to environmental stress across diverse taxa. Despite this broad use, few studies have explored telomere length in plants. Thus, evaluation of new approaches for measuring telomeres in plants is needed. Rapid advances in sequencing approaches and bioinformatic tools now allow estimation of telomere content from whole-genome sequencing (WGS) data, a proxy for telomere length. While telomere content has been quantified extensively using quantitative polymerase chain reaction (qPCR) and WGS in humans, no study to date has compared the effectiveness of WGS in estimating telomere length in plants relative to qPCR approaches. In this study, we use 100 Populus clones re-sequenced using short-read Illumina sequencing to quantify telomere length comparing three different bioinformatic approaches (Computel, K-seek and TRIP) in addition to qPCR. Overall, telomere length estimates varied across different bioinformatic approaches, but were highly correlated across methods for individual genotypes. A positive correlation was observed between WGS estimates and qPCR, however, Computel estimates exhibited the greatest correlation. Computel incorporates genome coverage into telomere length calculations, suggesting that genome coverage is likely important to telomere length quantification when using WGS data. Overall, telomere estimates from WGS provided greater precision and accuracy of telomere length estimates relative to qPCR. The findings suggest WGS is a promising approach for assessing telomere length and, as the field of telomere ecology evolves, may provide added value to assaying response to biotic and abiotic environments for plants needed to accelerate plant breeding and conservation management.

We evaluated an alternative small stem assay (AltSSA) for blight resistance in backcross hybrid chestnut trees (Castanea dentata/mollissima). Whereas standard small stem assays (SSAs) are done by inoculating small incisions in stems, in our AltSSA, 4- to 5-mm stems are cut off, and the exposed (living) stem tips are inoculated with discs of Cryphonectria parasitica inoculum and temporarily covered with plastic sleeves. Intended primarily for forward selection, this method was designed to be easy to implement, to consistently induce cankering, and to better enable seedling recovery via the development of lateral shoots from the lower stem. After 90+ days, cankers are evaluated and removed, and seedlings are prepared for out-planting. Previous results showed that AltSSAs performed at least as well as a common SSA method in distinguishing resistant and susceptible types. In this follow-up analysis of 35 lines of backcross seedlings studied in 2020 and 2021, we showed that mean orange zone canker length (OZCL) and a multifactor principal components analysis-based blight resistance index gave results consistent with predictions derived from two methods of blight resistance phenotyping and percentage of American chestnut ancestry of the parents of each line. As expected, based upon the apparent polygenic inheritance of blight resistance in backcross chestnut trees, mean OZCL of backcross families ranged from intermediate (F-1 hybrid-level) to low (wild-type American chestnut-level). Consistent with prior results, canker production was near 100%, survivorship after out-planting was very high, and postinoculation stem dieback was not apparently related to the stem tip inoculations. Altogether, these results suggest that the AltSSA is a viable method for early detection of relative blight resistance in seedlings and may enable a reduction in the numbers of trees out-planted and placed under care for long-term evaluation and breeding. Thus, the AltSSA can prevent time, resources, and orchard space from being used on susceptible trees.

Black cottonwood (Populus trichocarpa) is a species of economic interest and an outstanding study model. The aspen borer (Saperda calcarata) causes irreversible damage to poplars and other riparian species in North America. The insect can produce multiple effects ranging from the presence of some galleries in the stem to tree death. Despite the ecological and commercial importance of this tree–insect interaction, the genetic mechanisms underlying the response of P. trichocarpa to S. calcarata are scarcely understood. In this study, a common garden trial of P. trichocarpa provenances, established in Davis, California, was assessed at the second year of growth, regarding the infestation of S. calcarata from a natural outbreak. A genome-wide association study (GWAS) was conducted using 629k of exonic SNPs to assess the relationship between genomic variation and insect attack. Tree architecture, in terms of stem number per plant, and the wood metabolome were also included. Insect attack was independent of the number of stems per tree. The performed GWAS identified three significantly associated SNP markers (q-value < 0.2) belonging to the same number of gene models, encoding proteins involved in signal transduction mechanisms and secondary metabolite production, including that of R-mandelonitrile lyase, Chromodomain-helicase-DNA-binding family protein, and Leucine-rich repeat protein. These results are aligned with the current knowledge of defensive pathways in plants and trees, helping to expand the understanding of the defensive response mechanisms of black cottonwood against wood borer insects.

American chestnut (Castanea dentata) was once the most economically and ecologically important hardwood species in the eastern United States. In the first half of the 20th century, an exotic fungal pathogen-Cryphonectria parasitica-decimated the species, killing billions of chestnut trees. Two approaches to developing blight-resistant American chestnut populations show promise, but both will require introduction of adaptive genomic diversity from wild germplasm to produce diverse, locally adapted restoration populations. Here we characterize population structure, demographic history, and genomic diversity in a range-wide sample of 384 wild American chestnuts to inform conservation and breeding with blight-resistant varieties. Population structure analyses suggest that the chestnut range can be roughly divided into northeast, central, and southwest populations. Within-population genomic diversity estimates revealed a clinal pattern with the highest diversity in the southwest, which likely reflects bottleneck events associated with Quaternary glaciation. Finally, we identified genomic regions under positive selection within each population, which suggests that defence against fungal pathogens is a common target of selection across all populations. Taken together, these results show that American chestnut underwent a postglacial expansion from the southern portion of its range leading to three extant genetic populations. These populations will serve as management units for breeding adaptive genetic variation into the blight-resistant tree populations for targeted reintroduction efforts.

American chestnut ( Castanea dentata ) was once the most economically and ecologically important hardwood species in the United States. In the first half of the 20th century, an exotic fungal pathogen – Cryphonectria parasitica – decimated the species, killing approximately four billion trees. Two approaches to developing blight resistant American chestnut populations show promise, but both will require introduction of adaptive genomic diversity from wild germplasm to produce diverse, locally adapted reforestation populations. Here we characterize population structure, demographic history, and genomic diversity in a range-wide sample of 384 wild American chestnuts to inform conservation and breeding with blight resistant varieties. Population structure analyses with DAPC and ADMIXTURE suggest that the chestnut range can be roughly divided into northeast, central, and southwest populations. Within-population genomic diversity estimates revealed a clinal pattern with the highest diversity in the southwest, which likely reflects bottleneck events associated with Quaternary glaciation. Finally, we identified genomic regions under positive selection within each population, which suggests that defense against fungal pathogens is a common target of selection across all populations. Taken together, these results show that American chestnut underwent a postglacial expansion from the southern portion of its range leading to three extant populations. These populations will serve as management units for breeding adaptive genetic variation into the blight-resistant tree populations for targeted reintroduction efforts.

Premise An informatics approach was used for the construction of an Axiom genotyping array from heterogeneous, high‐throughput sequence data to assess the complex genome of loblolly pine ( Pinus taeda ). Methods High‐throughput sequence data, sourced from exome capture and whole genome reduced‐representation approaches from 2698 trees across five sequence populations, were analyzed with the improved genome assembly and annotation for the loblolly pine. A variant detection, filtering, and probe design pipeline was developed to detect true variants across and within populations. From 8.27 million variants, a total of 642,275 were evaluated and 423,695 of those were screened across a range‐wide population. Results The final informatics and screening approach delivered an Axiom array representing 46,439 high‐confidence variants to the forest tree breeding and genetics community. Based on the annotated reference genome, 34% were located in or directly upstream or downstream of genic regions. Discussion The Pita50K array represents a genome‐wide resource developed from sequence data for an economically important conifer, loblolly pine. It uniquely integrates independent projects that assessed trees sampled across the native range. The challenges associated with the large and repetitive genome are addressed in the development of this resource.