IntroductionEragrostis curvula (weeping lovegrass) is a perennial forage grass in which diploid genotypes reproduce sexually whereas polyploids display pseudogamous diplosporous apomixis. Understanding the molecular basis of this reproductive system is important for the potential use of apomixis in crop improvement. Here, we applied a comparative gene expression analysis to identify and functionally assess candidate genes associated with apomictic reproduction in E. curvula.MethodsThis analysis was performed using a custom 1×1M Agilent microarray designed from floral transcriptomes of sexual and apomictic genotypes. PCR screening across 14 genotypes and a segregating mapping population was conducted to evaluate candidate gene presence. Functional assessment was carried out through heterologous expression of the candidate genes in Arabidopsis thaliana using GoldenBraid 2.0 expression cassettes.ResultsOf the 131 differentially hybridized probes identified, 130 were upregulated in apomictic plants. BLAST analyses revealed four main candidate genes, including a hypothetical protein (EcAPO1), a pre-mRNA splicing factor CWC22-like homolog (EcAPO2), a Cyclin-2A-1 (EcAPO3), and an F-box protein (EcAPO4). PCR screening showed that EcAPO1 and EcAPO2 were exclusively present in apomictic individuals, supporting their close association with the trait. Transgenic lines displayed abnormal floral and reproductive phenotypes, including homeotic transformations, supernumerary nuclei, embryo sac arrest, and altered endosperm development.DiscussionThese results indicate that E. curvula candidate genes can disrupt conserved reproductive pathways and demonstrate biological activity in a heterologous system. Together, this integrative analysis combining comparative gene expression profiling and functional assays identifies novel candidate genes associated with apomixis in E. curvula. These findings provide a foundation for dissecting the genetic control of apomixis and advance efforts toward the applied manipulation of clonal seed reproduction in crops.

Flaxseed is abundant in cyclic peptides called linusorbs, which are derived from precursor proteins through post-translational modification. Previous studies have shown that four of the five precursor proteins contain repeat structures, where the variable linusorb domain is flanked by conserved signatures. The genome of flax cultivar CDC Bethune encodes 25 additional proteins which share the linusorbs-containing repeat pattern. Gene sequence analysis revealed that these repeats have arisen through three distinct modes of tandem duplication. Driven by the genetic diversity of potential linusorbs, here we continued to characterize these linusorb-related tandem repeats (LRTRs) at the amino acid level. Similar to known linusorbs, the linusorb-like domains (LLDs) are moderately hydrophobic in contrast to the hydrophilic spacers. The conserved flanking signatures of known linusorbs remain dominant among the LLDs, indicating a conserved role in the 30 proteins. Similarities of repeat pairs (RPs) across paralogous proteins are at the level between those within proteins and those across non-paralogues, suggesting the LRTRs in protein paralogues underwent adaptive evolution after ancestral gene duplication. Positive selection was identified to episodically act on certain sites and lineages of LRTRs both within and across paralogues. By combining all the evidence, we proposed a multi-dimensional model of adaptive evolution in cyclic peptide precursor proteins for the first time, which involves the concomitance of (a) protein paralogues divergence, (b) repeat divergence within protein paralogues and (c) repeat divergence across protein paralogues. Based on a single cultivated species, the proposed model serves as a prototype for broader evolutionary studies across other Linum species.

Recent advances in nucleic acid sequencing technologies have identified new classes of molecules that allow organisms to share information. One such example is obelisks, a class of circular RNA present in cells without being encoded by host DNA. A defining feature of obelisks is that they contain a homolog of the Oblin-1 gene. Recently, additional types of RNA molecules that resemble obelisks without the inclusion of an Oblin-1 gene homolog have been reported. As such, we propose that obelisks represent a larger group of molecules we define as RNA plasmids that contain an obelisk-like structure, open reading frame(s), and behaviour more akin to plasmids. However, there is the possibility that these molecules are in fact artifacts of either library preparation or computational analyses. To provide evidence that RNA plasmids are not artifacts and bona fide nucleic acid structures within sequence datasets, we have engineered a novel bioinformatics pipeline capable of identifying novel RNA plasmids in existing metatranscriptomic short-read archive datasets. Unlike existing pipelines that search for obelisks using homology to the hypothetical obelisk protein Oblin-1, our pipeline utilizes a homology-agnostic approach, allowing identification of non-obelisk RNA plasmids. We provide evidence that these elements are circular and demonstrate their novelty by performing alignment against the entire NCBI database. We then provide support that these are not technical artifacts by analyzing how environmental variables, namely exposure to different human disease states and environmental conditions, affect their abundance. We analyze the composition of select RNA plasmids to better understand their biological relevance and how they may interact with both their host and the environment. The identification of RNA plasmids in multiple environmental datasets highlights the potential importance of this novel class of biomolecules in situ and significantly enhances our understanding of non-chromosomal genetic elements. ​

Abstract This research provides original insights into the diversity of DNA extracted from samples collected in 1978 from the Turin Shroud, revealing its biological complexity through rigorous DNA and metagenomic analyses. Our findings highlight its preservation conditions and environmental interactions, offering valuable perspectives into the identified genetic variants, which originated from multiple biological sources. Several human mitochondrial DNA (mtDNA) lineages were identified, including K1a1b1a, which matches the 1978 official collector’s mitogenome, H2a2 ( i . e . the lineage of the mtDNA reference sequence rRCS), H1b, which is common in Western Eurasia, and H33, which is prevalent in the Near East and frequent among the Druze. Moreover, the reconstructed microbiome of the Shroud reveals a rich tapestry of multiple microbes commonly found on the human epidermis, as well as archaeal communities adapted to high salinity, and fungi including molds. This is indicative of the Shroud’s preservation conditions over the centuries. Additionally, the presence of abundant Mediterranean endemic red coral, various cultivated plants ( e . g . carrot, wheat, corn, bananas, and peanuts) and domesticated animals ( e . g . cattle, pigs, chickens, dogs, and cats) provide a fascinating glimpse into the diverse biological sources of the contaminants that have accumulated on the Turin Shroud over time. Finally, radiocarbon dating of two distinct threads collected from the reliquary provides evidence of their use to repair the Shroud in the years 1534 and 1694 of the Common Era (CE). Significance statement An in-depth metagenomic analysis was conducted on several linen strands collected from different areas of the body image of the Man of the Shroud during the official sampling in 1978. Our analyses revealed several human mtDNA lineages, including one common in Western Eurasia and another prevalent in the Near East. Additionally, the diversity of animal and plant species identified details the significant environmental contamination of the Shroud that likely occurred in recent centuries, particularly following the voyages of Marco Polo and Christopher Columbus. Radiocarbon dating of two distinct textile residuals from the Shroud’s reliquary indicated a time range between 1451 and 1800 CE, overlapping with the period of its repair interventions.

This article presents a novel perspective on plant embryogenesis, fundamentally differentiating it from the animal embryo model upon which plant models have long been based to discern the genetic and molecular mechanisms. We propose a plant embryonic body plan that aligns developmental and evolutionary insights across all five embryophyte groups (bryophytes, lycophytes, monilophytes, gymnosperms, and angiosperms). This conceptual model is grounded in a Reprogramming Potential (RP) involving an activation (RP1+) -suppression (RP1-) switch (RP1+/RP1-), which integrates embryonic development in a stepwise manner across diverse embryophytes. We further explore the evolutionary trajectory of this body plan, tracing the gradual assembly of the embryophyte genetic toolkit from bryophytes to angiosperms. Key developmental processes, such as the emergence of shoot and root meristems, vascular tissues, and seeds, are also examined within an evo-devo framework. Plant phenotypic plasticity, fundamental to their adaptation and survival, is manifested in two key hallmarks: (A) the iterative, modular growth of shoot and root units, and (B) their remarkable regenerative potential. While traditionally viewed as separate phenomena, we propose a novel, integrative model that connects these hallmarks within the context of plant embryogenesis. Our “proposed universal plant embryonic body plan” reconciles the genetic and molecular mechanisms of Arabidopsis thaliana embryogenesis with the contrasting developmental patterns observed in monocots. This unified model also integrates the concept of root founder cells and collet (shoot-root junction) into an embryonic framework facilitating the study of gene regulatory networks that underpin root evolution and its architecture.

Linusorbs are cyclic peptides biosynthesized through post-translational modification of precursor proteins in flaxseed. Their precursor peptide domains are embedded in five proteins, four of which contain tandem repeats (TRs). Using the sequence patterns of the linusorb-embedded TRs, we previously mined the flax reference genome assembly and identified >280 TRs containing linusorb-like domains distributed in 25 proteins, revealing the potential diversity of linusorbs. In this work, we studied the evolution of TRs in the 30 linusorb-related genes by first verifying the gene sequences using Sanger method. Comparison of the Sanger contigs with the reference genome assembly showed widespread discrepancies in the repeat regions. We annotated the Sanger contigs and identified eight groups of paralogous genes. Pairwise comparisons were conducted among repeats within a region, across regions within a gene and across paralogous genes. Similarity matrices revealed three distinct modes of tandem duplication that differ in the number of repeats as a duplication unit. Most of the across-paralogue repeat pairs (RPs) share similarity lower than 50 %. The numbers of repeat regions and repeats also differ among most of the paralogues, suggesting that repeats diverged independently in gene paralogues. Two modes of divergence were inferred from the similarity distributions of RPs under different categories. The flanking non-repetitive regions among paralogous genes exhibited local conservation, as well as variations indicative of functional diversification. This work highlights the importance of verifying repetitive sequences in the genome assembly. Our findings about repeat duplication and divergence constitute a multi-dimensional model of repeat evolution in linusorb-related genes.

IntroductionAmong candidate genes underlying the control components of apomixis, APOLLO is known for its strong linkage to apomeiosis in the genus Boechera. The gene has “apo alleles,” which are characterized by a set of linked apomixis-specific polymorphisms, and “sex alleles.” All apomictic Boechera genotypes are heterozygous for the apo/sex alleles, whereas all sexual genotypes are homozygous for sex alleles.MethodsIn this study, native and synthetic APOLLO promoters were characterized by detecting the expression level of the β-glucuronidase (GUS) gene in Arabidopsis.ResultsComparing various flower developmental stages in transgenic lines containing different constructs with 2-kb native transgenic lines revealed that changes to the APOLLO promoter causes shifts in tissue and developmental stage specificity of GUS expression. Importantly, several apomixis-specific polymorphisms in the 5′UTR change the timing and location of GUS activity from somatic to reproductive tissues.DiscussionThese synthetic data simulate a plausible evolutionary process, whereby apomixis-specific gene activity can be achieved.

AbstractAmong candidate genes underlying the control components of apomixis, APOLLO is known for its strong linkage to apomeiosis in the genusBoechera. The gene has “apo-alleles”, which are characterized by a set of linked apomixis-specific polymorphisms, and “sex-alleles”. All apomictic□Boecheragenotypes are heterozygous for the apo/sex-alleles, while all sexual genotypes are homozygous for sex-alleles. In this study, native and synthetic APOLLO promoters were characterized by detecting the expression level of the β-glucuronidase (GUS) gene inArabidopsis. Comparing various flower developmental stages in transgenic lines containing different constructs with 2 kb native transgenic lines revealed that changes to the APOLLO promoter causes shifts in tissue- and developmental-stage specificity of GUS expression. Importantly, several apomixis-specific polymorphisms in the 5’UTR change the timing and location of GUS activity from somatic to reproductive tissues. These synthetic data simulate a plausible evolutionary process whereby apomixis-specific gene activity can be achieved.

Asexual reproduction results in offspring that are genetically identical to the mother. Among apomictic plants (reproducing asexually through seeds) many require paternal genetic contribution for proper endosperm development (pseudogamous endosperm). We examined phenotypic diversity in seed traits using a diverse panel of sexual and apomictic accessions from the genus Boechera. While genetic uniformity resulting from asexual reproduction is expected to reduce phenotypic diversity in seeds produced by apomictic individuals, pseudogamous endosperm, variable endosperm ploidy, and the deviations from 2:1 maternal:paternal genome ratio in endosperm can all contribute to increased phenotypic diversity among apomictic offspring. We characterized seed size variation in 64 diploid sexual and apomictic (diploid and triploid) Boechera lineages. In order to find out whether individual seed size was related to endosperm ploidy we performed individual seed measurements (projected area and mass) using the phenoSeeder robot system and flow cytometric seed screen. In order to test whether individual seed size had an effect on resulting fitness we performed a controlled growth experiment and recorded seedling life history traits (germination success, germination timing, and root growth rate). Seeds with triploid embryos were 33% larger than those with diploid embryos, but no average size difference was found between sexual and apomictic groups. We identified a maternal effect whereby chloroplast lineage 2 had 30% larger seeds than lineage 3, despite having broad and mostly overlapping geographic ranges. Apomictic seeds were not more uniform in size than sexual seeds, despite genetic uniformity of the maternal gametophyte in the former. Among specific embryo/endosperm ploidy combinations, seeds with tetraploid (automomous) endosperm were on average smaller, and the proportion of such seeds was highest in apomicts. Larger seeds germinated more quickly than small seeds, and lead to higher rates of root growth in young seedlings. Seed mass is under balancing selection in Boechera, and it is an important predictor of several traits, including germination probability and timing, root growth rates, and developmental abnormalities in apomictic accessions.

Background Ribosomally-synthesized cyclic peptides are widely found in plants and exhibit useful bioactivities for humans. The identification of cyclic peptide sequences and their precursor proteins is facilitated by the growing number of sequenced genomes. While previous research largely focused on the chemical diversity of these peptides across various species, there is little attention to a broader range of potential peptides that are not chemically identified. Results A pioneering study was initiated to explore the genetic diversity of linusorbs, a group of cyclic peptides uniquely occurring in cultivated flax (Linum usitatissimum). Phylogenetic analysis clustered the 5 known linusorb precursor proteins into two clades and one singleton. Preliminary tBLASTn search of the published flax genome using the whole protein sequence as query could only retrieve its homologues within the same clade. This limitation was overcome using a profile-based mining strategy. After genome reannotation, a hidden Markov Model (HMM)-based approach identified 58 repeats homologous to the linusorb-embedded repeats in 8 novel proteins, implying that they share common ancestry with the linusorb-embedded repeats. Subsequently, we developed a customized profile composed of a random linusorb-like domain (LLD) flanked by 5 conserved sites and used it for string search of the proteome, which extracted 281 LLD-containing repeats (LLDRs) in 25 proteins. Comparative analysis of different repeat categories suggested that the 5 conserved flanking sites among the non-homologous repeats have undergone convergent evolution driven by functional selection. Conclusions The profile-based mining approach is suitable for analyzing repetitive sequences. The 25 LLDR proteins identified herein represent the potential diversity of cyclic peptides within the flax genome and lay a foundation for further studies on the functions and evolution of these protein tandem repeats.

Asexual reproduction through seeds in plants (i.e., apomixis) is a heritable trait, and apomixis- linked loci have been identified in multiple species. However, direct identification of genomic elements is typically hindered as apomixis-linked loci and are commonly found in recombination-suppressed and repetitive regions. Heterochromatinized elements, such as B chromosomes and other supernumerary chromosomal DNA fragments have long been known to be associated with asexuality in both plants and animals and are prime candidate regions for the evolution of multiple apomixis factors controlling the individual elements of apomixis. Here, we examined molecular evolution, gene regulation, and chromosomal location of a male apomeiosis factor (UPG2), a long noncoding RNA gene, in sexual and apomictic Boechera with and without male apomeiosis (i.e., balanced and unbalanced apomicts). We revealed the origin of the gene in the apomixis genome on an apomixis-specific, supernumerary heterochromatic Boechera chromosome (Boe1). The UPG2 is active in the tapetum at male meiosis. We found allele classes specific to apomictic and sexual Boechera accessions and a third class that shares the features of both and points to a convergent transition state. Sex alleles are found only in some of the sexual accessions and have higher nucleotide divergence and lower transcriptional activity compared to apo alleles. These data demonstrate selective pressure to maintain the function of UPG2 for unreduced pollen formation in apomicts as the occasional transmission of the allele from unbalanced apomicts into sexual organisms that lead to pseudogenization and functional decay of copies in sexual organisms.

The mechanisms of initiation and transmission of apomixis (asexual reproduction through seeds) in natural plant populations are important for understanding the evolution of reproductive variation. Here, we used the phylogenetic diversity of the genus Boechera (Brassicaceae), together with natural diversity in pollen types produced by apomictic lines, to test whether hybridization triggers the transition to asexuality, and whether a 'triploid bridge' is required for the formation of polyploid apomicts. We performed crosses between diploid sexual recipient and diploid apomictic donor lines and tested whether the mating system (interspecific hybridization vs intraspecific outcrossing) or pollen type (haploid vs diploid) influenced the transmission of apomixis from diploid apomictic donors into sexual recipients. We used genetic markers and flow cytometric analyses of embryo and endosperm in seeds to infer the reproductive mode. Within a single generation, initiation of both diploid and polyploid apomixis in sexual Boechera can occur. Diploid apomixis is transmitted through haploid pollen (infectious asexuality) and polyploids can form through multiple pathways. The three functional elements of apomixis occasionally segregate. Variation in pollen ploidy and the segregation of apomixis elements drive reproductive diversity of hybrids and outcrosses and can be utilized for apomixis initiation in crop breeding programs.

Summary Our objective was to verify the potential of identifying different starches in Brazilian landraces of cassava using near and medium infrared analyses. The survey was conducted on 132 landraces collected throughout the Cerrado, Pantanal and Amazon biomes, in the Brazilian state of Mato Grosso, including 10 commercial starches. The results are reflective of selection for different genetic variants in small isolated producers. Infrared analysis enables rapid identification of this material and is the first step in identifying starches with different properties. The results demonstrate the potential of MIR analyses for in situ evaluation, an additional option for identifying important varieties and value the development of efforts made by small producers, whose landraces have special and economically important properties.

Cannabis sativa L. is an important yet controversial plant with a long history of recreational, medicinal, industrial, and agricultural use, and together with its sister genus Humulus, it represents a group of plants with a myriad of academic, agricultural, pharmaceutical, industrial, and social interests. We have performed a meta-analysis of pooled published genomics data, andwe present a comprehensive literature review on the evolutionary history of Cannabis and Humulus, including medicinal and industrial applications. We demonstrate that current Cannabis genome assemblies are incomplete, with ∼10% missing, 10–25% unmapped, and 45S and 5S ribosomal DNA clusters as well as centromeres/satellite sequences not represented. These assemblies are also ordered at a low resolution, and their consensus quality clouds the accurate annotation of complete, partial, and pseudogenized gene copies. Considering the importance of genomics in the development of any crop, this analysis underlines the need for a coordinated effort to quantify the genetic and biochemical diversity of this species.

SummaryHypericin is a molecule of high pharmaceutical importance that is synthesized and stored in dark glands (DGs) of St. John's Wort (Hypericum perforatum). Understanding which genes are involved in dark gland development and hypericin biosynthesis is important for the development of new Hypericum extracts that are highly demanded for medical applications. We identified two transcription factors whose expression is strictly synchronized with the differentiation of DGs. We correlated the content of hypericin, pseudohypericin, endocrocin, skyrin glycosides and several flavonoids with gene expression and DG development to obtain a revised model for hypericin biosynthesis. Here, we report for the first time genotypes which are polymorphic for the presence/total absence (G+/G−) of DGs in their placental tissues (PTs). DG development was characterized in PTs using several microscopy techniques. Fourier transform infrared microscopy was established as a novel method to precisely locate polyaromatic compounds, such as hypericin, in plant tissues. In addition, we obtained transcriptome and metabolome profiles of unprecedented resolution in Hypericum. This study addresses for the first time the development of dark glands and identifies genes that constitute strong building blocks for the further elucidation of hypericin synthesis, its manipulation in plants, its engineering in microbial systems and its applications in medical research.

Key messageReproduction in triploid plants is important for understanding polyploid population dynamics. We show that genetically identical reciprocal F1 hybrid triploids can display transgenerational epigenetic effects on viable F2 seed development.AbstractThe success or failure of reproductive outcomes from intra-species crosses between plants of different ploidy levels is an important factor in flowering plant evolution and crop breeding. However, the effects of inter-ploidy cross directions on F1 hybrid offspring fitness are poorly understood. In Arabidopsis thaliana, hybridization between diploid and tetraploid plants can produce viable F1 triploid plants. When selfed, such F1 triploid plants act as aneuploid gamete production machines where the vast majority of gametes generated are aneuploid which, following sexual reproduction, can generate aneuploid swarms of F2 progeny (Henry et al. 2009). There is potential for some aneuploids to cause gametophyte abortion and/or F2 seed abortion (Henry et al. 2009). In this study, we analyse the reproductive success of 178 self-fertilized inter-accession F1 hybrid triploids and demonstrate that the proportions of aborted or normally developed F2 seeds from the selfed F1 triploids depend upon a combination of natural variation and cross direction, with strong interaction between these factors. Single-seed ploidy analysis indicates that the embryonic DNA content of phenotypically normal F2 seeds is highly variable and that these DNA content distributions are also affected by genotype and cross direction. Notably, genetically identical reciprocal F1 hybrid triploids display grandparent-of-origin effects on F2 seed set, and hence on the ability to tolerate aneuploidy in F2 seed. There are differences between reciprocal F1 hybrid triploids regarding the proportions of normal and aborted F2 seeds generated, and also for the DNA content averages and distributions of the F2 seeds. To identify genetic variation for tolerance of aneuploidy in F2 seeds, we carried out a GWAS which identified two SNPs, termed MOT and POT, which represent candidate loci for genetic control of the proportion of normal F2 seeds obtained from selfed F1 triploids. Parental and grandparental effects on F2 seeds obtained from selfed F1 triploids can have transgenerational consequences for asymmetric gene flow, emergence of novel genotypes in polyploid populations, and for control of F2 seed set in triploid crops.

Parasitism evokes adaptive physiological changes in the host, many of which take place through gene expression changes. This response can be more or less local, depending on the organ or tissue affected by the parasite, or else systemic when the parasite affects the entire host body. The most extreme of the latter cases is intragenomic parasitism, where the parasite is present in all host nuclei as any other genomic element. Here, we show the molecular crosstalk between a parasitic chromosome (also named B chromosome) and the host genome, manifested through gene expression changes. The transcriptome analysis of 0B and 1B females of the grasshopper Eyprepocnemis plorans, validated by a microarray experiment performed on four B-lacking and five B-carrying females, revealed changes in gene expression for 188 unigenes being consistent in both experiments. Once discarded B-derived transcripts, there were 46 differentially expressed genes (30 up- and 16 downregulated) related with the adaptation of the host genome to the presence of the parasitic chromosome. Interestingly, the functions of these genes could explain some of the most important effects of B chromosomes, such as nucleotypic effects derived from the additional DNA they represent, chemical defense and detoxification, protein modification and response to stress, ovary function, and regulation of gene expression. Collectively, these changes uncover an intimate host-parasite interaction between A and B chromosomes during crucial steps of gene expression and protein function.