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  1. Abstract

    Whitebark pine (WBP, Pinus albicaulis) is a white pine of subalpine regions in the Western contiguous United States and Canada. WBP has become critically threatened throughout a significant part of its natural range due to mortality from the introduced fungal pathogen white pine blister rust (WPBR, Cronartium ribicola) and additional threats from mountain pine beetle (Dendroctonus ponderosae), wildfire, and maladaptation due to changing climate. Vast acreages of WBP have suffered nearly complete mortality. Genomic technologies can contribute to a faster, more cost-effective approach to the traditional practices of identifying disease-resistant, climate-adapted seed sources for restoration. With deep-coverage Illumina short reads of haploid megagametophyte tissue and Oxford Nanopore long reads of diploid needle tissue, followed by a hybrid, multistep assembly approach, we produced a final assembly containing 27.6 Gb of sequence in 92,740 contigs (N50 537,007 bp) and 34,716 scaffolds (N50 2.0 Gb). Approximately 87.2% (24.0 Gb) of total sequence was placed on the 12 WBP chromosomes. Annotation yielded 25,362 protein-coding genes, and over 77% of the genome was characterized as repeats. WBP has demonstrated the greatest variation in resistance to WPBR among the North American white pines. Candidate genes for quantitative resistance include disease resistance genes known as nucleotide-binding leucine-rich repeat receptors (NLRs). A combination of protein domain alignments and direct genome scanning was employed to fully describe the 3 subclasses of NLRs. Our high-quality reference sequence and annotation provide a marked improvement in NLR identification compared to previous assessments that leveraged de novo-assembled transcriptomes.

     
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  2. 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. 
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  3. Understanding the genomic and environmental basis of cold adaptation is key to understand how plants survive and adapt to different environmental conditions across their natural range. Univariate and multivariate genome-wide association (GWAS) and genotype-environment association (GEA) analyses were used to test associations among genome-wide SNPs obtained from whole-genome resequencing, measures of growth, phenology, emergence, cold hardiness, and range-wide environmental variation in coastal Douglas-fir (Pseudotsuga menziesii). Results suggest a complex genomic architecture of cold adaptation, in which traits are either highly polygenic or controlled by both large and small effect genes. Newly discovered associations for cold adaptation in Douglas-fir included 130 genes involved in many important biological functions such as primary and secondary metabolism, growth and reproductive development, transcription regulation, stress and signaling, and DNA processes. These genes were related to growth, phenology and cold hardiness and strongly depend on variation in environmental variables such degree days below 0c, precipitation, elevation and distance from the coast. This study is a step forward in our understanding of the complex interconnection between environment and genomics and their role in cold-associated trait variation in boreal tree species, providing a baseline for the species’ predictions under climate change. 
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  4. SUMMARY

    Drought is a major limitation for survival and growth in plants. With more frequent and severe drought episodes occurring due to climate change, it is imperative to understand the genomic and physiological basis of drought tolerance to be able to predict how species will respond in the future. In this study, univariate and multitrait multivariate genome‐wide association study methods were used to identify candidate genes in two iconic and ecosystem‐dominating species of the western USA, coast redwood and giant sequoia, using 10 drought‐related physiological and anatomical traits and genome‐wide sequence‐capture single nucleotide polymorphisms. Population‐level phenotypic variation was found in carbon isotope discrimination, osmotic pressure at full turgor, xylem hydraulic diameter, and total area of transporting fibers in both species. Our study identified new 78 new marker × trait associations in coast redwood and six in giant sequoia, with genes involved in a range of metabolic, stress, and signaling pathways, among other functions. This study contributes to a better understanding of the genomic basis of drought tolerance in long‐generation conifers and helps guide current and future conservation efforts in the species.

     
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  5. Abstract The giant sequoia (Sequoiadendron giganteum) of California are massive, long-lived trees that grow along the U.S. Sierra Nevada mountains. Genomic data are limited in giant sequoia and producing a reference genome sequence has been an important goal to allow marker development for restoration and management. Using deep-coverage Illumina and Oxford Nanopore sequencing, combined with Dovetail chromosome conformation capture libraries, the genome was assembled into eleven chromosome-scale scaffolds containing 8.125 Gbp of sequence. Iso-Seq transcripts, assembled from three distinct tissues, was used as evidence to annotate a total of 41,632 protein-coding genes. The genome was found to contain, distributed unevenly across all 11 chromosomes and in 63 orthogroups, over 900 complete or partial predicted NLR genes, of which 375 are supported by annotation derived from protein evidence and gene modeling. This giant sequoia reference genome sequence represents the first genome sequenced in the Cupressaceae family, and lays a foundation for using genomic tools to aid in giant sequoia conservation and management. 
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  6. Abstract

    Sequencing, assembly, and annotation of the 26.5 Gbp hexaploid genome of coast redwood (Sequoia sempervirens) was completed leading toward discovery of genes related to climate adaptation and investigation of the origin of the hexaploid genome. Deep-coverage short-read Illumina sequencing data from haploid tissue from a single seed were combined with long-read Oxford Nanopore Technologies sequencing data from diploid needle tissue to create an initial assembly, which was then scaffolded using proximity ligation data to produce a highly contiguous final assembly, SESE 2.1, with a scaffold N50 size of 44.9 Mbp. The assembly included several scaffolds that span entire chromosome arms, confirmed by the presence of telomere and centromere sequences on the ends of the scaffolds. The structural annotation produced 118,906 genes with 113 containing introns that exceed 500 Kbp in length and one reaching 2 Mb. Nearly 19 Gbp of the genome represented repetitive content with the vast majority characterized as long terminal repeats, with a 2.9:1 ratio of Copia to Gypsy elements that may aid in gene expression control. Comparison of coast redwood to other conifers revealed species-specific expansions for a plethora of abiotic and biotic stress response genes, including those involved in fungal disease resistance, detoxification, and physical injury/structural remodeling and others supporting flavonoid biosynthesis. Analysis of multiple genes that exist in triplicate in coast redwood but only once in its diploid relative, giant sequoia, supports a previous hypothesis that the hexaploidy is the result of autopolyploidy rather than any hybridizations with separate but closely related conifer species.

     
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  7. Abstract 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. 
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