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Abstract Eucalyptus grandis is a hardwood tree used worldwide as pure species or hybrid partner to breed fast-growing plantation forestry crops that serve as feedstocks of timber and lignocellulosic biomass for pulp, paper, biomaterials, and biorefinery products. The current v2.0 genome reference for the species served as the first reference for the genus and has helped drive the development of molecular breeding tools for eucalypts. Using PacBio HiFi long reads and Omni-C proximity ligation sequencing, we produced an improved, haplotype-phased assembly (v4.0) for TAG0014, an early-generation selection of E. grandis. The 2 haplotypes are 571 Mbp (HAP1) and 552 Mbp (HAP2) in size and consist of 37 and 46 contigs scaffolded onto 11 chromosomes (contig N50 of 28.9 and 16.7 Mbp), respectively. These haplotype assemblies are 70–90 Mbp smaller than the diploid v2.0 assembly but capture all except one of the 22 telomeres, suggesting that substantial redundant sequence was included in the previous assembly. A total of 35,929 (HAP1) and 35,583 (HAP2) gene models were annotated, of which 438 and 472 contain long introns (>10 kbp) in gene models previously (v2.0) identified as multiple smaller genes. These and other improvements have increased gene annotation completeness levels from 93.8 to 99.4% in the v4.0 assembly. We found that 6,493 and 6,346 genes are within tandem duplicate arrays (HAP1 and HAP2, respectively, 18.4 and 17.8% of the total) and >43.8% of the haplotype assemblies consists of repeat elements. Analysis of synteny between the haplotypes and the E. grandis v2.0 reference genome revealed extensive regions of collinearity, but also some major rearrangements, and provided a preview of population and pangenome variation in the species. 

Genomic characterization of Cannabis sativa has accelerated rapidly in the last decade as sequencing costs have decreased and public and private interest in the species has increased. Here, we present seven new chromosome-level haplotype-phased genomes of C. sativa. All of these genotypes were alive at the time of publication, and several have numerous years of associated phenotype data. We performed a k-mer-based pangenome analysis to contextualize these assemblies within over 200 existing assemblies. This allowed us to identify unique haplotypes and genomic diversity among Cannabis sativa genotypes. We leveraged linkage maps constructed from F2 progeny of two of the assembled genotypes to characterize the recombination rate across the genome showing strong periphery-biased recombination. Lastly, we re-aligned a bulk segregant analysis dataset for the major-effect flowering locus Early1 to several of the new assemblies to evaluate the impact of reference bias on the mapping results and narrow the locus to a smaller region of the chromosome. These new assemblies, combined with the continued propagation of the genotypes, will contribute to the growing body of genomic resources for C. sativa to accelerate future research efforts. 

Abstract Ancient whole-genome duplications (WGDs) are believed to facilitate novelty and adaptation by providing the raw fuel for new genes. However, it is unclear how recent WGDs may contribute to evolvability within recent polyploids. Hybridization accompanying some WGDs may combine divergent gene content among diploid species. Some theory and evidence suggest that polyploids have a greater accumulation and tolerance of gene presence-absence and genomic structural variation, but it is unclear to what extent either is true. To test how recent polyploidy may influence pangenomic variation, we sequenced, assembled, and annotated twelve complete, chromosome-scale genomes of Camelina sativa, an allohexaploid biofuel crop with three distinct subgenomes. Using pangenomic comparative analyses, we characterized gene presence-absence and genomic structural variation both within and between the subgenomes. We found over 75% of ortholog gene clusters are core in Camelina sativa and <10% of sequence space was affected by genomic structural rearrangements. In contrast, 19% of gene clusters were unique to one subgenome, and the majority of these were Camelina-specific (no ortholog in Arabidopsis). We identified an inversion that may contribute to vernalization requirements in winter-type Camelina, and an enrichment of Camelina-specific genes with enzymatic processes related to seed oil quality and Camelina’s unique glucosinolate profile. Genes related to these traits exhibited little presence-absence variation. Our results reveal minimal pangenomic variation in this species, and instead show how hybridization accompanied by WGD may benefit polyploids by merging diverged gene content of different species. 

Abstract Cotton (Gossypium hirsutumL.) is the key renewable fibre crop worldwide, yet its yield and fibre quality show high variability due to genotype-specific traits and complex interactions among cultivars, management practices and environmental factors. Modern breeding practices may limit future yield gains due to a narrow founding gene pool. Precision breeding and biotechnological approaches offer potential solutions, contingent on accurate cultivar-specific data. Here we address this need by generating high-quality reference genomes for three modern cotton cultivars (‘UGA230’, ‘UA48’ and ‘CSX8308’) and updating the ‘TM-1’ cotton genetic standard reference. Despite hypothesized genetic uniformity, considerable sequence and structural variation was observed among the four genomes, which overlap with ancient and ongoing genomic introgressions from ‘Pima’ cotton, gene regulatory mechanisms and phenotypic trait divergence. Differentially expressed genes across fibre development correlate with fibre production, potentially contributing to the distinctive fibre quality traits observed in modern cotton cultivars. These genomes and comparative analyses provide a valuable foundation for future genetic endeavours to enhance global cotton yield and sustainability. 

Abstract Cultivated pear consists of several Pyrus species with Pyrus communis (European pear) representing a large fraction of worldwide production. As a relatively recently domesticated crop and perennial tree, pear can benefit from genome-assisted breeding. Additionally, comparative genomics within Rosaceae promises greater understanding of evolution within this economically important family. Here, we generate a fully phased chromosome-scale genome assembly of P. communis ‘d’Anjou.’ Using PacBio HiFi and Dovetail Omni-C reads, the genome is resolved into the expected 17 chromosomes, with each haplotype totaling nearly 540 Megabases and a contig N50 of nearly 14 Mb. Both haplotypes are highly syntenic to each other and to the Malus domestica ‘Honeycrisp’ apple genome. Nearly 45,000 genes were annotated in each haplotype, over 90% of which have direct RNA-seq expression evidence. We detect signatures of the known whole-genome duplication shared between apple and pear, and we estimate 57% of d’Anjou genes are retained in duplicate derived from this event. This genome highlights the value of generating phased diploid assemblies for recovering the full allelic complement in highly heterozygous crop species. 

Abstract Background Cotton fibers provide a powerful model for studying cell differentiation and elongation. Each cotton fiber is a singular and elongated cell derived from epidermal-layer cells of a cotton seed. Efforts to understand this dramatic developmental shift have been impeded by the difficulty of separation between fiber and epidermal cells. Results Here we employed laser-capture microdissection (LCM) to separate these cell types. RNA-seq analysis revealed transitional differences between fiber and epidermal-layer cells at 0 or 2 days post anthesis. Specifically, down-regulation of putative cell cycle genes was coupled with upregulation of ribosome biosynthesis and translation-related genes, which may suggest their respective roles in fiber cell initiation. Indeed, the amount of fibers in cultured ovules was increased by cell cycle progression inhibitor, Roscovitine, and decreased by ribosome biosynthesis inhibitor, Rbin-1. Moreover, subfunctionalization of homoeologs was pervasive in fiber and epidermal cells, with expression bias towards 10% more D than A homoeologs of cell cycle related genes and 40–50% more D than A homoeologs of ribosomal protein subunit genes. Key cell cycle regulators were predicted to be epialleles in allotetraploid cotton. MYB-transcription factor genes displayed expression divergence between fibers and ovules. Notably, many phytohormone-related genes were upregulated in ovules and down-regulated in fibers, suggesting spatial-temporal effects on fiber cell development. Conclusions Fiber cell initiation is accompanied by cell cycle arrest coupled with active ribosome biosynthesis, spatial-temporal regulation of phytohormones and MYB transcription factors, and homoeolog expression bias of cell cycle and ribosome biosynthesis genes. These valuable genomic resources and molecular insights will help develop breeding and biotechnological tools to improve cotton fiber production. 

ABSTRACT Yellow monkeyflowers (Mimulus guttatuscomplex, Phrymaceae) are a powerful system for studying ecological adaptation, reproductive variation, and genome evolution. To initiate pan‐genomics in this group, we present four chromosome‐scale assemblies and annotations of accessions spanning a broad evolutionary spectrum: two from a singleM. guttatuspopulation, one from the closely related selfing speciesM. nasutus, and one from a more divergent speciesM. tilingii. All assemblies are highly complete and resolve centromeric and repetitive regions. Comparative analyses reveal such extensive structural variation in repeat‐rich, gene‐poor regions that large portions of the genome are unalignable across accessions. As a result, thisMimuluspan‐genome is primarily informative in genic regions, underscoring limitations of resequencing approaches in such polymorphic taxa. We document gene presence–absence, investigate the recombination landscape using high‐resolution linkage data, and quantify nucleotide diversity. Surprisingly, pairwise differences at fourfold synonymous sites are exceptionally high—even in regions of very low recombination—reaching ~3.2% within a singleM. guttatuspopulation, ~7% within the interfertileM. guttatusspecies complex (approximately equal to SNP divergence between great apes and Old World monkeys), and ~7.4% between that complex and the reproductively isolatedM. tilingii. Genome‐wide patterns of nucleotide variation show little evidence of linked selection, and instead suggest that the concentration of genes (and likely selected sites) in high‐recombination regions may buffer diversity loss. These assemblies, annotations, and comparative analyses provide a robust genomic foundation forMimulusresearch and offer new insights into the interplay of recombination, structural variation, and molecular evolution in highly diverse plant genomes.