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Plant genome-scale resources are being generated at an increasing rate as sequencing technologies continue to improve and raw data costs continue to fall; however, the cost of downstream analyses remains large. This has resulted in a considerable range of genome assembly and annotation qualities across plant genomes due to their varying sizes, complexity, and the technology used for the assembly and annotation. To effectively work across genomes, researchers increasingly rely on comparative genomic approaches that integrate across plant community resources and data types. Such efforts have aided the genome annotation process and yielded novel insights into the evolutionary history of genomes and gene families, including complex non-model organisms. The essential tools to achieve these insights rely on gene family analysis at a genome-scale, but they are not well integrated for rapid analysis of new data, and the learning curve can be steep. Here we present PlantTribes2, a scalable, easily accessible, highly customizable, and broadly applicable gene family analysis framework with multiple entry points including user provided data. It uses objective classifications of annotated protein sequences from existing, high-quality plant genomes for comparative and evolutionary studies. PlantTribes2 can improve transcript models and then sort them, either genome-scale annotations or individual gene coding sequences, into pre-computed orthologous gene family clusters with rich functional annotation information. Then, for gene families of interest, PlantTribes2 performs downstream analyses and customizable visualizations including, (1) multiple sequence alignment, (2) gene family phylogeny, (3) estimation of synonymous and non-synonymous substitution rates among homologous sequences, and (4) inference of large-scale duplication events. We give examples of PlantTribes2 applications in functional genomic studies of economically important plant families, namely transcriptomics in the weedy Orobanchaceae and a core orthogroup analysis (CROG) in Rosaceae. PlantTribes2 is freely available for use within the main public Galaxy instance and can be downloaded from GitHub or Bioconda. Importantly, PlantTribes2 can be readily adapted for use with genomic and transcriptomic data from any kind of organism.

The rapid development of sequencing technologies has led to a deeper understanding of plant genomes. However, direct experimental evidence connecting genes to important agronomic traits is still lacking in most non-model plants. For instance, the genetic mechanisms underlying plant architecture are poorly understood in pome fruit trees, creating a major hurdle in developing new cultivars with desirable architecture, such as dwarfing rootstocks in European pear (Pyrus communis). An efficient way to identify genetic factors for important traits in non-model organisms can be to transfer knowledge across genomes. However, major obstacles exist, including complex evolutionary histories and variable quality and content of publicly available plant genomes. As researchers aim to link genes to traits of interest, these challenges can impede the transfer of experimental evidence across plant species, namely in the curation of high-quality, high-confidence gene models in an evolutionary context. Here we present a workflow using a collection of bioinformatic tools for the curation of deeply conserved gene families of interest across plant genomes. To study gene families involved in tree architecture in European pear and other rosaceous species, we used our workflow, plus a draft genome assembly and high-quality annotation of a second P. communis cultivar, ‘d’Anjou.’ Our comparative gene family approach revealed significant issues with the most recent ‘Bartlett’ genome - primarily thousands of missing genes due to methodological bias. After correcting assembly errors on a global scale in the ‘Bartlett’ genome, we used our workflow for targeted improvement of our genes of interest in both P. communis genomes, thus laying the groundwork for future functional studies in pear tree architecture. Further, our global gene family classification of 15 genomes across 6 genera provides a valuable and previously unavailable resource for the Rosaceae research community. With it, orthologs and other gene family members can be easily identified across any of the classified genomes. Importantly, our workflow can be easily adopted for any other plant genomes and gene families of interest.

We assess relationships among 192 species in all 12 monocot orders and 72 of 77 families, using 602 conserved single-copy (CSC) genes and 1375 benchmarking single-copy ortholog (BUSCO) genes extracted from genomic and transcriptomic datasets. Phylogenomic inferences based on these data, using both coalescent-based and supermatrix analyses, are largely congruent with the most comprehensive plastome-based analysis, and nuclear-gene phylogenomic analyses with less comprehensive taxon sampling. The strongest discordance between the plastome and nuclear gene analyses is the monophyly of a clade comprising Asparagales and Liliales in our nuclear gene analyses, versus the placement of Asparagales and Liliales as successive sister clades to the commelinids in the plastome tree. Within orders, around six of 72 families shifted positions relative to the recent plastome analysis, but four of these involve poorly supported inferred relationships in the plastome-based tree. In Poales, the nuclear data place a clade comprising Ecdeiocoleaceae+Joinvilleaceae as sister to the grasses (Poaceae); Typhaceae, (rather than Bromeliaceae) are resolved as sister to all other Poales. In Commelinales, nuclear data place Philydraceae sister to all other families rather than to a clade comprising Haemodoraceae+Pontederiaceae as seen in the plastome tree. In Liliales, nuclear data place Liliaceae sister to Smilacaceae, and Melanthiaceae are placed sister to all other Liliales except Campynemataceae. Finally, in Alismatales, nuclear data strongly place Tofieldiaceae, rather than Araceae, as sister to all the other families, providing an alternative resolution of what has been the most problematic node to resolve using plastid data, outside of those involving achlorophyllous mycoheterotrophs. As seen in numerous prior studies, the placement of orders Acorales and Alismatales as successive sister lineages to all other extant monocots. Only 21.2% of BUSCO genes were demonstrably single-copy, yet phylogenomic inferences based on BUSCO and CSC genes did not differ, and overall functional annotations of the two sets were very similar. Our analyses also reveal significant gene tree-species tree discordance despite high support values, as expected given incomplete lineage sorting (ILS) related to rapid diversification. Our study advances understanding of monocot relationships and the robustness of phylogenetic inferences based on large numbers of nuclear single-copy genes that can be obtained from transcriptomes and genomes.

Disentangling the phylogenetic relationships of taxonomically complex plant groups is often mired by challenges associated with recent speciation, hybridization, complex mating systems, and polyploidy. Here, we perform a phylogenomic analysis of eyebrights (Euphrasia), a group renowned for taxonomic complexity, with the aim of documenting the extent of phylogenetic discordance at both deep and at shallow phylogenetic scales. We generate whole-genome sequencing data and integrate this with prior genomic data to perform a comprehensive analysis of nuclear genomic, nuclear ribosomal (nrDNA), and complete plastid genomes from 57 individuals representing 36 Euphrasia species. The species tree analysis of 3,454 conserved nuclear scaffolds (46 Mb) reveals that at shallow phylogenetic scales postglacial colonization of North Western Europe occurred in multiple waves from discrete source populations, with most species not being monophyletic, and instead combining genomic variants from across clades. At a deeper phylogenetic scale, the Euphrasia phylogeny is structured by geography and ploidy, and partially by taxonomy. Comparative analyses show Southern Hemisphere tetraploids include a distinct subgenome indicative of independent polyploidy events from Northern Hemisphere taxa. In contrast to the nuclear genome analyses, the plastid genome phylogeny reveals limited geographic structure, while the nrDNA phylogeny is informative of some geographic and taxonomic affinities but more thorough phylogenetic inference is impeded by the retention of ancestral polymorphisms in the polyploids. Overall our results reveal extensive phylogenetic discordance at both deeper and shallower nodes, with broad-scale geographic structure of genomic variation but a lack of definitive taxonomic signal. This suggests that Euphrasia species either have polytopic origins or are maintained by narrow genomic regions in the face of extensive homogenizing gene flow. Moreover, these results suggest genome skimming will not be an effective extended barcode to identify species in groups such as Euphrasia, or many other postglacial species groups.

Estimating maturity in pome fruits is a critical task that directs virtually all postharvest supply chain decisions. This is especially important for European pear (Pyrus communis) cultivars because losses due to spoilage and senescence must be minimized while ensuring proper ripening capacity is achieved (in part by satisfying a fruit chilling requirement). Reliable methods are lacking for accurate estimation of pear fruit maturity, and because ripening is maturity dependent it makes predicting ripening capacity a challenge. In this study of the European pear cultivar ‘d’Anjou’, we sorted fruit at harvest based upon on-tree fruit position to build contrasts of maturity. Our sorting scheme showed clear contrasts of maturity between canopy positions, yet there was substantial overlap in the distribution of values for the index of absorbance difference (IAD), a non-destructive spectroscopic measurement that has been used as a proxy for pome fruit maturity. This presented an opportunity to explore a contrast of maturity that was more subtle than IAD could differentiate, and thus guided our subsequent transcriptome analysis of tissue samples taken at harvest and during storage. Using a novel approach that tests for condition-specific differences of co-expressed genes, we discovered genes with a phased character that mirrored our sorting scheme. The expression patterns of these genes are associated with fruit quality and ripening differences across the experiment. Functional profiles of these co-expressed genes are concordant with previous findings, and also offer new clues, and thus hypotheses, about genes involved in pear fruit quality, maturity, and ripening. This work may lead to new tools for enhanced postharvest management based on activity of gene co-expression modules, rather than individual genes. Further, our results indicate that modules may have utility within specific windows of time during postharvest management of ‘d’Anjou’ pear.

Phtheirospermum (Orobanchaceae), a hemiparasitic genus of Eastern Asia, is characterized by having long and viscous glandular hairs on stems and leaves. Despite this unifying character, previous phylogenetic analyses indicate that Phtheirospermum is polyphyletic, with Phtheirospermum japonicum allied with tribe Pedicularideae and members of the Ph. tenuisectum complex allied with members of tribe Rhinantheae. However, no analyses to date have included broad phylogenetic sampling necessary to test the monophyly of Phtheirospermum species, and to place these species into the existing subfamiliar taxonomic organization of Orobanchaceae. Two other genera of uncertain phylogenetic placement are Brandisia and Pterygiella, also both of Eastern Asia. In this study, broadly sampled phylogenetic analyses of nrITS and plastid DNA revealed hard incongruence between these datasets in the placement of Brandisia. However, both nrITS and the plastid datasets supported the placement of Ph. japonicum within tribe Pedicularideae, and a separate clade consisting of the Ph. tenuisectum complex and a monophyletic Pterygiella. Analyses were largely in agreement that Pterygiella, the Ptheirospermum complex, and Xizangia form a clade not nested within any of the monophyletic tribes of Orobanchaceae recognized to date. Ph. japonicum, a model species for parasitic plant research, is widely distributed in Eastern Asia. Despite this broad distribution, both nrITS and plastid DNA regions from a wide sampling of this species showed high genetic identity, suggesting that the wide species range is likely due to a recent population expansion. The Ph. tenuisectum complex is mainly distributed in the Hengduan Mountains region. Two cryptic species were identified by both phylogenetic analyses and morphological characters. Relationships among species of the Ph. tenuisectum complex and Pterygiella remain uncertain. Estimated divergence ages of the Ph. tenuisectum complex corresponding to the last two uplifts of the Qinghai–Tibet Plateau at around 8.0–7.0 Mya and 3.6–1.5 Mya indicated that the development of a hot-dry valley climate during these uplifts may have driven species diversification in the Ph. tenuisectum complex.