PanKmer: k-mer-based and reference-free pangenome analysis.

SUMMARY: Pangenomes are replacing single reference genomes as the definitive representation of DNA sequence within a species or clade. Pangenome analysis predominantly leverages graph-based methods that require computationally intensive multiple genome alignments, do not scale to highly complex eukaryotic genomes, limit their scope to identifying structural variants (SVs), or incur bias by relying on a reference genome. Here, we present PanKmer, a toolkit designed for reference-free analysis of pangenome datasets consisting of dozens to thousands of individual genomes. PanKmer decomposes a set of input genomes into a table of observed k-mers and their presence-absence values in each genome. These are stored in an efficient k-mer index data format that encodes SNPs, INDELs, and SVs. It also includes functions for downstream analysis of the k-mer index, such as calculating sequence similarity statistics between individuals at whole-genome or local scales. For example, k-mers can be "anchored" in any individual genome to quantify sequence variability or conservation at a specific locus. This facilitates workflows with various biological applications, e.g. identifying cases of hybridization between plant species. PanKmer provides researchers with a valuable and convenient means to explore the full scope of genetic variation in a population, without reference bias. AVAILABILITY AND IMPLEMENTATION: PanKmer is implemented as a Python package with components written in Rust, released under a BSD license. The source code is available from the Python Package Index (PyPI) at https://pypi.org/project/pankmer/ as well as Gitlab at https://gitlab.com/salk-tm/pankmer. Full documentation is available at https://salk-tm.gitlab.io/pankmer/.

Genomics of turions from the Greater Duckweed reveal its pathways for dormancy and re-emergence strategy.

Over 15 families of aquatic plants are known to use a strategy of developmental switching upon environmental stress to produce dormant propagules called turions. However, few molecular details for turion biology have been elucidated due to the difficulties in isolating high-quality nucleic acids from this tissue. We successfully developed a new protocol to isolate high-quality transcripts and carried out RNA-seq analysis of mature turions from the Greater Duckweed Spirodela polyrhiza. Comparison of turion transcriptomes to that of fronds, the actively growing leaf-like tissue, were carried out. Bioinformatic analysis of high confidence, differentially expressed transcripts between frond and mature turion tissues revealed major pathways related to stress tolerance, starch and lipid metabolism, and dormancy that are mobilized to reprogram frond meristems for turion differentiation. We identified the key genes that are likely to drive starch and lipid accumulation during turion formation, as well as those in pathways for starch and lipid utilization upon turion germination. Comparison of genome-wide cytosine methylation levels also revealed evidence for epigenetic changes in the formation of turion tissues. Similarities between turions and seeds provide evidence that key regulators for seed maturation and germination were retooled for their function in turion biology.

The genome and preliminary single-nuclei transcriptome of Lemna minuta reveals mechanisms of invasiveness.

The ability to trace every cell in some model organisms has led to the fundamental understanding of development and cellular function. However, in plants the complexity of cell number, organ size, and developmental time makes this a challenge even in the diminutive model plant Arabidopsis (Arabidopsis thaliana). Duckweed, basal nongrass aquatic monocots, provide an opportunity to follow every cell of an entire plant due to their small size, reduced body plan, and fast clonal growth habit. Here we present a chromosome-resolved genome for the highly invasive Lesser Duckweed (Lemna minuta) and generate a preliminary cell atlas leveraging low cell coverage single-nuclei sequencing. We resolved the 360 megabase genome into 21 chromosomes, revealing a core nonredundant gene set with only the ancient tau whole-genome duplication shared with all monocots, and paralog expansion as a result of tandem duplications related to phytoremediation. Leveraging SMARTseq2 single-nuclei sequencing, which provided higher gene coverage yet lower cell count, we profiled 269 nuclei covering 36.9% (8,457) of the L. minuta transcriptome. Since molecular validation was not possible in this nonmodel plant, we leveraged gene orthology with model organism single-cell expression datasets, gene ontology, and cell trajectory analysis to define putative cell types. We found that the tissue that we computationally defined as mesophyll expressed high levels of elemental transport genes consistent with this tissue playing a role in L. minuta wastewater detoxification. The L. minuta genome and preliminary cell map provide a paradigm to decipher developmental genes and pathways for an entire plant.

Changes in ambient temperature are the prevailing cue in determining Brachypodium distachyon diurnal gene regulation.

Plants are continuously exposed to diurnal fluctuations in light and temperature, and spontaneous changes in their physical or biotic environment. The circadian clock coordinates regulation of gene expression with a 24 h period, enabling the anticipation of these events. We used RNA sequencing to characterize the Brachypodium distachyon transcriptome under light and temperature cycles, as well as under constant conditions. Approximately 3% of the transcriptome was regulated by the circadian clock, a smaller proportion than reported in most other species. For most transcripts that were rhythmic under all conditions, including many known clock genes, the period of gene expression lengthened from 24 to 27 h in the absence of external cues. To functionally characterize the cyclic transcriptome in B. distachyon, we used Gene Ontology enrichment analysis, and found several terms significantly associated with peak expression at particular times of the day. Furthermore, we identified sequence motifs enriched in the promoters of similarly phased genes, some potentially associated with transcription factors. When considering the overlap in rhythmic gene expression and specific pathway behavior, thermocycles was the prevailing cue that controlled diurnal gene regulation. Taken together, our characterization of the rhythmic B. distachyon transcriptome represents a foundational resource with implications in other grass species.

Contrasting a reference cranberry genome to a crop wild relative provides insights into adaptation, domestication, and breeding.

Cranberry (Vaccinium macrocarpon) is a member of the Heath family (Ericaceae) and is a temperate low-growing woody perennial native to North America that is both economically important and has significant health benefits. While some native varieties are still grown today, breeding programs over the past 50 years have made significant contributions to improving disease resistance, fruit quality and yield. An initial genome sequence of an inbred line of the wild selection 'Ben Lear,' which is parent to multiple breeding programs, provided insight into the gene repertoire as well as a platform for molecular breeding. Recent breeding efforts have focused on leveraging the circumboreal V. oxycoccos, which forms interspecific hybrids with V. macrocarpon, offering to bring in novel fruit chemistry and other desirable traits. Here we present an updated, chromosome-resolved V. macrocarpon reference genome, and compare it to a high-quality draft genome of V. oxycoccos. Leveraging the chromosome resolved cranberry reference genome, we confirmed that the Ericaceae has undergone two whole genome duplications that are shared with blueberry and rhododendron. Leveraging resequencing data for 'Ben Lear' inbred lines, as well as several wild and elite selections, we identified common regions that are targets of improvement. These same syntenic regions in V. oxycoccos, were identified and represent environmental response and plant architecture genes. These data provide insight into early genomic selection in the domestication of a native North American berry crop.

Underwater CAM photosynthesis elucidated by Isoetes genome.

To conserve water in arid environments, numerous plant lineages have independently evolved Crassulacean Acid Metabolism (CAM). Interestingly, Isoetes, an aquatic lycophyte, can also perform CAM as an adaptation to low CO2 availability underwater. However, little is known about the evolution of CAM in aquatic plants and the lack of genomic data has hindered comparison between aquatic and terrestrial CAM. Here, we investigate underwater CAM in Isoetes taiwanensis by generating a high-quality genome assembly and RNA-seq time course. Despite broad similarities between CAM in Isoetes and terrestrial angiosperms, we identify several key differences. Notably, Isoetes may have recruited the lesser-known 'bacterial-type' PEPC, along with the 'plant-type' exclusively used in other CAM and C4 plants for carboxylation of PEP. Furthermore, we find that circadian control of key CAM pathway genes has diverged considerably in Isoetes relative to flowering plants. This suggests the existence of more evolutionary paths to CAM than previously recognized.