IntroductionGene expression in plants is inherently dynamic, shifting rapidly during development and in response to environmental stimuli. However, many essential cellular processes require remarkable transcriptomic stability. The identity, genomic features, and evolutionary conservation of stably expressed genes (SEGs) in plants across broad evolutionary scales remain poorly understood.ResultsWe analyzed comprehensive transcriptome atlases from five species representing major clades of angiosperms (Arabidopsis thaliana, Fagopyrum esculentum, Solanum lycopersicum, Phalaenopsis equestris, and Zea mays) to identify genes with consistently low expression variation across organs, developmental stages, and environmental conditions. We identified 672 orthogroups that are universally stable across angiosperms; these are predominantly single-copy genes enriched for fundamental cellular functions including DNA repair, mRNA splicing, and translation. Validation using independent datasets and additional species confirmed that stability is maintained across diverse genotypes, conditions, and species. SEGs tend to be longer, have more exons, and exhibit lower GC content than other expressed genes. Using multiomics information available for Arabidopsis, we also explored epigenetic and functional characteristics associated with stability. Loss-of-function mutations in SEGs result in lethality far more frequently than in ubiquitously expressed genes, and protein abundance is similarly stable. SEGs have a significantly higher level of gene body methylation and H3K4me1 marks.ConclusionsOur results reveal several genomic and epigenetic features that distinguish SEGs from ubiquitously expressed genes. They also suggest that SEG stability is not governed by specific regulatory motifs, but is rather “by default” state driven by a distinctive chromatin architecture.

Species of the Apiaceae family produce furanocoumarins—photosensitizing substances widely used in pharmacology. Among them, certain species of the genus Heracleum, commonly known as “giant hogweeds”, are particularly renowned for their photosensitizing properties. While the metabolic composition of these plants has been studied extensively, their genomics remains largely unexplored, as does the biosynthesis of furanocoumarins within the genus. The reasons behind the increased photosensitivity of giant hogweeds compared to other Heracleum species remain unclear. A comparison of the genomic features and furanocoumarin content of the aboveground organs in two Heracleum species was carried out: H. sosnowskyi, a giant hogweed with strong photosensitizing activity, and H. sibiricum, a non-invasive and “harmless” species. Both species produce all known angular and linear compounds of the furanocoumarin synthesis pathway (from umbelliferone to isopimpinellin). The genomes of both species possess homologues of genes involved in the biosynthesis of both angular and linear furanocoumarins. However, the two species differ significantly in the quantity of those compounds. H. sibiricum synthesizes angular furanocoumarins in a much lower level, probably due to lack of the gene for umbelliferon-8-dimetylallyl transferase enzyme. H. sosnowskyi accumulates significantly higher level of xanthotoxin. Interpopulation differences were observed in both the compound content and the expression level of biosynthetic genes. This study represents the first comparative analysis of genomic and metabolic data for closely related Heracleum species in relation to furanocoumarin biosynthesis. • H. sosnowskyi contains all compounds of linear/angular furocoumarin synthesis pathway • H. sibiricum does not synthesize angular furanocoumarins in above-ground organs • H. sosnowskyi contains more highly expressed homologues of 5- and 8-MOP synthesis • Genomes of both species contain all homologues for furanocoumarin synthesis pathway • Furanocoumarin synthesis is activated in different populations and climate conditions

The genus Capsella serves as a model for understanding speciation, hybridization, and genome evolution in plants. Here, we present a chromosome-scale genome assembly of Capsella orientalis, the maternal progenitor of a cosmopolitan allotetraploid C. bursa-pastoris. Using nanopore sequencing and data on chromatin contacts (Hi-C), we assembled the genome into eight pseudo-chromosomes with high contiguity, evidenced by a benchmarking universal single-copy orthologs (BUSCO) completeness score of 99.3%. Comparative analysis with C. rubella and C. bursa-pastoris revealed overall synteny, except for 2 Mb inversion on chromosome 4 of C. rubella. Comparative genome analysis highlighted the conservation of gene content and structural integrity in the C. orientalis-derived subgenome of C. bursa-pastoris, with the exception of a 1.8 Mb region absent in O subgenome but present in C. orientalis. The genome annotation includes 27,675 protein-coding genes, with most exhibiting one-to-one orthology with Arabidopsis thaliana. Notably, 2,155 genes showed no similarity to A. thaliana ones. These results establish a robust genomic resource for C. orientalis, facilitating future studies on polyploid evolution, gene regulation, and species divergence within Capsella.

Seed shattering is a crucial adaptive trait in wild plant species but is undesirable in domesticated crops. In this study, we investigated the anatomical and molecular mechanisms underlying seed shattering in common buckwheat (Fagopyrum esculentum) and explored its genetic control. Previous research has shown that seed shattering in F. esculentum follows a monogenic inheritance pattern. To pinpoint the genomic region associated with this trait, we analyzed an F₂ progeny derived from a cross between wild ancestor of common buckwheat - Fagopyrum esculentum ssp. ancestrale and the cultivated variety - Fagopyrum esculentum cv. Dasha. Anatomical analysis revealed that both shattering and non-shattering phenotypes possess an abscission zone (AZ); however, in non-shattering plants, the AZ remains underdeveloped and inactive. Histological observations indicated that AZ formation begins during early bud development. Transcriptomic analysis across four developmental stages (early bud, late bud, flower, and fruit) identified abscisic acid regulation, ethylene signaling, and cell wall modification as key factors in AZ differentiation. Unexpectedly, many genes typically associated with abscission layer activation showed increased expression at the flower stage rather than the fruit stage, suggesting that AZ activation occurs earlier than anticipated. We also provided preliminary data on the localization of the gene responsible for shattering in wild buckwheat and reviewed candidate genes. Our results provide new insights into the structure of abscission zone in buckwheat and into molecular mechanisms underlying seed shattering in buckwheat. We identified key pathways involved in abscission zone differentiation and performed the initial localization of the causative gene.

BACKGROUND:As genomes of many eukaryotic species, especially plants, are large and complex, their de novo sequencing and assembly is still a difficult task despite progress in sequencing technologies. An alternative to genome assembly is the assembly of transcriptome, the set of RNA products of the expressed genes. While a bunch of de novo transcriptome assemblers exists, the challenges of transcriptomes (the existence of isoforms, the uneven expression levels across genes) complicates the generation of high-quality assemblies suitable for downstream analyses. RESULTS:We developed Trans2express - a web-based tool and a pipeline of de novo hybrid transcriptome assembly and postprocessing based on rnaSPAdes with a set of subsequent filtrations. The pipeline was tested on Arabidopsis thaliana cDNA sequencing data obtained using Illumina and Oxford Nanopore Technologies platforms and three non-model plant species. The comparison of structural characteristics of the transcriptome assembly with reference Arabidopsis genome revealed the high quality of assembled transcriptome with 86.1% of Arabidopsis expressed genes assembled as a single contig. We tested the applicability of the transcriptome assembly for gene expression analysis. For both Arabidopsis and non-model species the results showed high congruence of gene expression levels and sets of differentially expressed genes between analyses based on genome and based on the transcriptome assembly. CONCLUSIONS:We present Trans2express - a protocol for de novo hybrid transcriptome assembly aimed at recovering of a single transcript per gene. We expect this protocol to promote the characterization of transcriptomes and gene expression analysis in non-model plants and web-based tool to be of use to a wide range of plant biologists.

Background Capsella bursa-pastoris , a cosmopolitan weed of hybrid origin, is an emerging model object for the study of early consequences of polyploidy, being a fast growing annual and a close relative of Arabidopsis thaliana . The development of this model is hampered by the absence of a reference genome sequence. Results We present here a subgenome-resolved chromosome-scale assembly and a genetic map of the genome of Capsella bursa-pastoris . It shows that the subgenomes are mostly colinear, with no massive deletions, insertions, or rearrangements in any of them. A subgenome-aware annotation reveals the lack of genome dominance—both subgenomes carry similar number of genes. While most chromosomes can be unambiguously recognized as derived from either paternal or maternal parent, we also found homeologous exchange between two chromosomes. It led to an emergence of two hybrid chromosomes; this event is shared between distant populations of C. bursa-pastoris . The whole-genome analysis of 119 samples belonging to C. bursa-pastoris and its parental species C. grandiflora/rubella and C. orientalis reveals introgression from C. orientalis but not from C. grandiflora/rubella . Conclusions C. bursa-pastoris does not show genome dominance. In the earliest stages of evolution of this species, a homeologous exchange occurred; its presence in all present-day populations of C. bursa-pastoris indicates on a single origin of this species. The evidence coming from whole-genome analysis challenges the current view that C. grandiflora/rubella was a direct progenitor of C. bursa-pastoris ; we hypothesize that it was an extinct (or undiscovered) species sister to C. grandiflora/rubella .

The sleeping chironomid Polypedilum vanderplanki is capable of anhydrobiosis, a striking example of adaptation to extreme desiccation. Tolerance to complete desiccation in this species is associated with emergence of multiple paralogs of protective genes. One of the gene families highly expressed under anhydrobiosis and involved in this process is protein-L-isoaspartate (D-aspartate) O-methyltransferases (PIMTs). Recently, another closely related midge was discovered, Polypedilum pembai, which is able not only to tolerate desiccation but also to survive multiple desiccation-rehydration cycles. To investigate the evolution of anhydrobiosis in these species, we sequenced and assembled the genome of P. pembai and compared it with P. vanderplanki and also performed a population genomics analysis of several populations of P. vanderplanki and one population of P. pembai. We observe positive selection and radical changes in the genetic architecture of the PIMT locus between the two species, including its amplification in the P. pembai lineage. In particular, PIMT-4, the most highly expressed of these PIMTs, is present in six copies in the P. pembai; these copies differ in expression profiles, suggesting possible sub- or neofunctionalization. The nucleotide diversity of the genomic region carrying these new genes is decreased in P. pembai, but not in the orthologous region carrying the ancestral gene in P. vanderplanki, providing evidence for a selective sweep associated with postduplication adaptation in the former. Overall, our results suggest an extensive relatively recent and likely ongoing adaptation of the mechanisms of anhydrobiosis.

Interspecific gene comparisons are the keystones for many areas of biological research and are especially important for the translation of knowledge from model organisms to economically important species. Currently they are hampered by the low resolution of methods based on sequence analysis and by the complex evolutionary history of eukaryotic genes. This is especially critical for plants, whose genomes are shaped by multiple whole genome duplications and subsequent gene loss. This requires the development of new methods for comparing the functions of genes in different species. Here, we report ISEEML (Interspecific Similarity of Expression Evaluated using Machine Learning)–a novel machine learning-based algorithm for interspecific gene classification. In contrast to previous studies focused on sequence similarity, our algorithm focuses on functional similarity inferred from the comparison of gene expression profiles. We propose novel metrics for expression pattern similarity–expression score (ES)–that is suitable for species with differing morphologies. As a proof of concept, we compare detailed transcriptome maps of Arabidopsis thaliana, the model species, Zea mays (maize) and Fagopyrum esculentum (common buckwheat), which are species that represent distant clades within flowering plants. The classifier resulted in an AUC of 0.91; under the ES threshold of 0.5, the specificity was 94%, and sensitivity was 72%.

Pusa sibirica, the Baikal seal, is the only extant, exclusively freshwater, pinniped species. The pending issue is, how and when they reached their current habitat—the rift lake Baikal, more than three thousand kilometers away from the Arctic Ocean. To explore the demographic history and genetic diversity of this species, we generated a de novo chromosome-length assembly, and compared it with three closely related marine pinniped species. Multiple whole genome alignment of the four species compared with their karyotypes showed high conservation of chromosomal features, except for three large inversions on chromosome VI. We found the mean heterozygosity of the studied Baikal seal individuals was relatively low (0.61 SNPs/kbp), but comparable to other analyzed pinniped samples. Demographic reconstruction of seals revealed differing trajectories, yet remarkable variations in Ne occurred during approximately the same time periods. The Baikal seal showed a significantly more severe decline relative to other species. This could be due to the difference in environmental conditions encountered by the earlier populations of Baikal seals, as ice sheets changed during glacial–interglacial cycles. We connect this period to the time of migration to Lake Baikal, which occurred ~3–0.3 Mya, after which the population stabilized, indicating balanced habitat conditions.

AbstractBackgroundCapsella bursa-pastoris, a cosmopolitan weed of hybrid origin, is an emerging model object for the study of early consequences of polyploidy, being a fast growing annual and a close relative ofArabidopsis thaliana. The development of this model is hampered by the absence of a reference genome sequence.Resultswe present here a subgenome-resolved chromosome-scale assembly and a genetic map of the genome ofCapsella bursa-pastoris. It shows that the subgenomes are mostly colinear, with no massive deletions, insertions or rearrangements in any of them. A subgenome-aware annotation reveals the lack of genome dominance – both subgenomes carry similar number of genes. While most chromosomes can be unambiguously recognized as derived from either paternal or maternal parent, we also found homeologous exchange between two chromosomes. It led to an emergence of two hybrid chromosomes; this event is shared between distant populations ofC. bursa-pastoris. The whole-genome analysis of 119 samples belonging toC. bursa-pastorisand its parental speciesC. grandiflora/rubellaandC. orientalisreveals introgression fromC. orientalisbut not fromC. grandiflora/rubella.ConclusionsC. bursa-pastorisdo not show genome dominance. In the earliest stages of evolution of this species a homeologous exchange occurred; its presence in all present-day populations ofC. bursa-pastorisindicates on a single origin of this species. The evidence coming from whole-genome analysis challenges the current view thatC. grandiflora/rubellawas a direct progenitor ofC. bursa-pastoris; we hypothesize that it was an extinct (or undiscovered) species sister toC. grandiflora/rubella.

Heracleum sosnowskyi, belonging to a group of giant hogweeds, is a plant with large effects on ecosystems and human health. It is an invasive species that contributes to the deterioration of grassland ecosystems. The ability of H. sosnowskyi to produce linear furanocoumarins (FCs), photosensitizing compounds, makes it very dangerous. At the same time, linear FCs are compounds with high pharmaceutical value used in skin disease therapies. Despite this high importance, it has not been the focus of genetic and genomic studies. Here, we report a chromosome-scale assembly of Sosnowsky's hogweed genome. Genomic analysis revealed an unusually high number of genes (55106) in the hogweed genome, in contrast to the 25-35 thousand found in most plants. However, we did not find any traces of recent whole-genome duplications not shared with its confamiliar, Daucus carota (carrot), which has approximately thirty thousand genes. The analysis of the genomic proximity of duplicated genes indicates on tandem duplications as a main reason for this increase. We performed a genome-wide search of the genes of the FC biosynthesis pathway and surveyed their expression in aboveground plant parts. Using a combination of expression data and phylogenetic analysis, we found candidate genes for psoralen synthase and experimentally showed the activity of one of them using a heterologous yeast expression system. These findings expand our knowledge on the evolution of gene space in plants and lay a foundation for further analysis of hogweed as an invasive plant and as a source of FCs.

An increased frequency of B-cell lymphomas is observed in human immunodeficiency virus-1 (HIV-1)-infected patients, although HIV-1 does not infect B cells. Development of B-cell lymphomas may be potentially due to the action of the HIV-1 Tat protein, which is actively released from HIV-1-infected cells, on uninfected B cells. The exact mechanism of Tat-induced B-cell lymphomagenesis has not yet been precisely identified. Here, we ectopically expressed either Tat or its TatC22G mutant devoid of transactivation activity in the RPMI 8866 lymphoblastoid B cell line and performed a genome-wide analysis of host gene expression. Stable expression of both Tat and TatC22G led to substantial modifications of the host transcriptome, including pronounced changes in antiviral response and cell cycle pathways. We did not find any strong action of Tat on cell proliferation, but during prolonged culturing, Tat-expressing cells were displaced by non-expressing cells, indicating that Tat expression slightly inhibited cell growth. We also found an increased frequency of chromosome aberrations in cells expressing Tat. Thus, Tat can modify gene expression in cultured B cells, leading to subtle modifications in cellular growth and chromosome instability, which could promote lymphomagenesis over time.

MOTIVATION:Advances in high-throughput sequencing have illuminated the complexity of transcriptome landscape in eukaryotes. An inherent part of this complexity is the presence of multiple isoforms generated by the alternative splicing and the use of alternative transcription start and polyadenylation sites. However, currently available tools have limited capacity to infer full-length isoforms. RESULTS:We developed a new pipeline, FLIC (full-length isoform constructor). FLIC is based on the long-read transcriptome data and integrates several key features: (1) utilizing biological replicate concordance to filter out noise and artifacts; (2) employing peak calling to precisely identify transcription start and polyadenylation sites; (3) enabling robust isoform reconstruction with minimal reliance on existing annotations. We evaluated FLIC using a dedicated set of real and simulated data of Arabidopsis thaliana cDNA sequencing. Results demonstrate that FLIC accurately reconstructs known and novel isoforms, outperforming existing tools, especially in the absence of reference annotations. A direct comparison with CAGE, currently regarded as the gold standard for transcription start site identification, shows that FLIC is equally accurate, while being much less time-consuming. Thus, FLIC provides a valuable tool for comprehensive transcript characterization, particularly for non-model organisms or when dealing with incomplete or inaccurate annotations. AVAILABILITY AND IMPLEMENTATION:FLIC is available at https://github.com/albidgy/FLIC.

Over the past decade, molecular phylogenetics has reshaped our understanding of the fungal tree of life by unraveling a hitherto elusive diversity of the protistan relatives of Fungi. Aphelida constitutes one of these novel deep branches that precede the emergence of osmotrophic fungal lifestyle and hold particular significance as the pathogens of algae. Here, we obtain and analyze the genomes of aphelid species Amoeboaphelidium protococcarum and Amoeboaphelidium occidentale. Genomic data unmask the vast divergence between these species, hidden behind their morphological similarity, and reveal hybrid genomes with a complex evolutionary history in two strains of A. protococcarum. We confirm the proposed sister relationship between Aphelida and Fungi using phylogenomic analysis and chart the reduction of characteristic proteins involved in phagocytic activity in the evolution of Holomycota. Annotation of aphelid genomes demonstrates the retention of actin nucleation-promoting complexes associated with phagocytosis and amoeboid motility and also reveals a conspicuous expansion of receptor-like protein kinases, uncharacteristic of fungal lineages. We find that aphelids possess multiple carbohydrate-processing enzymes that are involved in fungal cell wall synthesis but do not display rich complements of algal cell-wall-processing enzymes, suggesting an independent origin of fungal plant-degrading capabilities. Aphelid genomes show that the emergence of Fungi from phagotrophic ancestors relied on a common cell wall synthetic machinery but required a different set of proteins for digestion and interaction with the environment.

Naturally occurring mutants whose phenotype recapitulates the changes that distinguish closely related species are of special interest from the evolutionary point of view. They can give a key about the genetic control of the changes that led to speciation. In this study, we described lepidium-like (lel), a naturally occurring variety of an allotetraploid species Capsella bursa-pastoris that is characterized by the typical loss of all four petals. In some cases, one or two basal flowers in the raceme had one or two small petals. The number and structure of other floral organs are not affected. Our study of flower development in the mutant showed that once initiated, petals either cease further development and cannot be traced in anthetic flowers or sometimes develop to various degrees. lel plants showed an earlier beginning of floral organ initiation and delayed petal initiation compared to the wild-type plants. lel phenotype has a wide geographical distribution, being found at the northern extremity of the species range as well as in the central part. The genetic analysis of inheritance demonstrated that lel phenotype is controlled by two independent loci. While the flower in the family Cruciferae generally has a very stable structure (i.e., four sepals, four petals, six stamens, and two carpels), several deviations from this ground plan are known, in particular in the genus Lepidium, C. bursa-pastoris is an emerging model for the study of polyploidy (which is also very widespread in Cruciferae); the identification and characterization of the apetalous mutant lays a foundation for further research of morphological evolution in polyploids.

Common buckwheat (Fagopyrum esculentum) is an important non-cereal grain crop and a prospective component of functional food. Despite this, the genomic resources for this species and for the whole family Polygonaceae, to which it belongs, are scarce. Here, we report the assembly of the buckwheat genome using long-read technology and a high-resolution expression atlas including 46 organs and developmental stages. We found that the buckwheat genome has an extremely high content of transposable elements, including several classes of recently (0.5–1 Mya) multiplied TEs (“transposon burst”) and gradually accumulated TEs. The difference in TE content is a major factor contributing to the three-fold increase in the genome size of F. esculentum compared with its sister species F. tataricum. Moreover, we detected the differences in TE content between the wild ancestral subspecies F. esculentum ssp. ancestrale and buckwheat cultivars, suggesting that TE activity accompanied buckwheat domestication. Expression profiling allowed us to test a hypothesis about the genetic control of petaloidy of tepals in buckwheat. We showed that it is not mediated by B-class gene activity, in contrast to the prediction from the ABC model. Based on a survey of expression profiles and phylogenetic analysis, we identified the MYB family transcription factor gene tr_18111 as a potential candidate for the determination of conical cells in buckwheat petaloid tepals. The information on expression patterns has been integrated into the publicly available database TraVA: http://travadb.org/browse/Species=Fesc/. The improved genome assembly and transcriptomic resources will enable research on buckwheat, including practical applications.

The vast diversity of Orchidaceae together with sophisticated adaptations to pollinators and other unique features make this family an attractive model for evolutionary and functional studies. The sequenced genome of Phalaenopsis equestris facilitates Orchidaceae research. Here, we present an RNA-seq-based transcriptome map of P. equestris that covers 19 organs of the plant, including leaves, roots, floral organs and the shoot apical meristem. We demonstrated the high quality of the data and showed the similarity of the P. equestris transcriptome map with the gene expression atlases of other plants. The transcriptome map can be easily accessed through our database Transcriptome Variation Analysis (TraVA) for visualizing gene expression profiles. As an example of the application, we analyzed the expression of Phalaenopsis "orphan"genes-those that do not have recognizable similarity with the genes of other plants. We found that approximately half of these genes were not expressed; the ones that were expressed were predominantly expressed in reproductive structures.

Fagopyrum esculentum(common buckwheat) is an important agricultural non-cereal grain plant. Despite extensive genetic studies, the information on its mitochondrial genome is still lacking. Using long reads generated by single-molecule real-time technology coupled with circular consensus sequencing (CCS) protocol, we assembled the buckwheat mitochondrial genome and detected that its prevalent form consists of 10 circular chromosomes with a total length of 404 Kb. In order to confirm the presence of a multipartite structure, we developed a new targeted assembly tool capable of processing long reads. The mitogenome contains all genes typical for plant mitochondrial genomes and long inserts of plastid origin (similar to 6.4% of the total mitogenome length). Using this new information, we characterized the genetic diversity of mitochondrial and plastid genomes in 11 buckwheat cultivars compared with the ancestral subspecies,F. esculentumssp.ancestrale. We found it to be surprisingly low within cultivars: Only three to six variations in the mitogenome and one to two in the plastid genome. In contrast, the divergence withF. esculentumssp.ancestraleis much higher: 220 positions differ in the mitochondrial genome and 159 in the plastid genome. The SNPs in the plastid genome are enriched in non-synonymous substitutions, in particular in the genes involved in photosynthesis:psbA,psbC, andpsbH. This presumably reflects the selection for the increased photosynthesis efficiency as a part of the buckwheat breeding program.

Shepherd’s purse (Capsella bursa-pastoris) is a cosmopolitan annual weed and a promising model plant for studying allopolyploidization in the evolution of angiosperms. Though plant mitochondrial genomes are a valuable source of genetic information, they are hard to assemble. At present, only the complete mitogenome of C. rubella is available out of all species of the genus Capsella. In this work, we have assembled the complete mitogenome of C. bursa-pastoris using high-precision PacBio SMRT third-generation sequencing technology. It is 287,799 bp long and contains 32 protein-coding genes, 3 rRNAs, 25 tRNAs corresponding to 15 amino acids, and 8 open reading frames (ORFs) supported by RNAseq data. Though many repeat regions have been found, none of them is longer than 1 kbp, and the most frequent structural variant originated from these repeats is present in only 4% of the mitogenome copies. The mitochondrial DNA sequence of C. bursa-pastoris differs from C. rubella, but not from C. orientalis, by two long inversions, suggesting that C. orientalis could be its maternal progenitor species. In total, 377 C to U RNA editing sites have been detected. All genes except cox1 and atp8 contain RNA editing sites, and most of them lead to non-synonymous changes of amino acids. Most of the identified RNA editing sites are identical to corresponding RNA editing sites in A. thaliana.