Ji Qi

In flowering plants, anther morphogenesis and microspore mother cell (MMC) specification of the male reproductive organ are critical for fertility and seed-setting or yield, but their underlying mechanisms in soybean (Glycine max) remain largely uncharacterized. Here, we cytologically established a staged developmental framework of early soybean anthers that were collected to generate a high-resolution single-nucleus RNA-seq atlas. We further characterized 11 major cell types annotated by cluster-specific marker genes and validated by RNA in situ hybridization. Pseudotime trajectory analysis reconstructed the differentiation process of microsporocytes, tapetum, and middle layer from archesporial cells, and established a regulatory network governing germline development. Parallel comparison of mitotic dividing cells (DCs) and meiotic MMCs revealed divergent transcriptional and cell-cycle programs. DCs follow a linear mitotic cascade with smooth, continuous transcriptional regulation, whereas MMCs undergo lineage-specific transcriptional reprogramming with an oscillatory activation of cell-cycle regulators to orchestrate meiotic transitions and progression. By comparing the transcriptional regulatory network of DCs and MMCs, we identified a pair of C2H2 zinc finger factors GmKNUa/b, the homologs of Arabidopsis KNUCKLES, as a key switch for MMC differentiation in soybean. The CRISPR-Cas9-edited Gmknua/b double mutants lack meiocytes, and GmKNUa/b might trigger the mitosis-to-meiosis transition by repressing the persistent mitotic cell cycle. Taken together, these results reveal the cellular signatures and molecular basis of early soybean anther morphogenesis and germline specialization, and provide candidate targets for manipulating germline fate and male fertility in soybean.

Meiotic recombination in reproductive cells is initiated by DNA double-strand breaks (DSBs) and requires the expression of meiosis-specific genes, yet how the transcription of those genes is regulated in either meiotic or somatic cells remains unclear. Here, using a representative meiosis-specific DMC1 gene promoter as a bait, we identified two DREAM factors TCX5 and TCX6 required for direct repression of DMC1 in somatic cells. Transcriptomic analyses show broad ectopic up-regulation of meiotic genes in tcx5/6 leaves . Consistently, tcx5/6 somatic cells form DSBs and exhibit hypersensitivity to DNA damage compared to wild type. Further genetic analyses reveal that SPO11-1 is dispensable for ectopic meiotic gene activation but essential for the DNA damage response in tcx5/6 somatic cells. Last, we demonstrated that TCX5/6 repress meiotic gene expression through H3K4me3 modification. This finding uncovers an unexpected mechanism for suppressing meiotic recombination in somatic cells, thus preventing genetic diversity and genome instability in organisms.

Ovule, as the developmental precursor to seed, represents a key evolutionary innovation in seed plants. However, its origin and evolutionary trajectory have been debated for over a century, largely due to the disconnects between morphological information gained from fossils and the regulatory mechanisms of ovule development inferred from extant angiosperms. The core regulatory networks shared by gymnosperms and angiosperms may serve as a bridge in terms of the ovule developmental process. We employed spatial transcriptome sequencing to profile dynamic gene expression during ovule development in four representative gymnosperm species ( Ginkgo biloba , Gnetum montanum , Pinus tabuliformis, and Cycas panzhihuaensis ). Our analyses reveal distinct trajectories of cell-type differentiation during formation of tissues, uncovering critical genes and pathways involved in ovule primordium initiation, chalaza formation, and subsequently development of integument and nucellus. The spatiotemporal expression patterns of key regulatory genes, particularly those associated with proximal–distal (PD) polarity establishment, strongly support the hypothesis that early formed chalaza regions function as a meristematic zone, giving rise to the formation of integument and nucellus in a manner reminiscent of the shoot apical meristem activity. Moreover, gene expression patterns in the developing integument reveal well-defined PD and adaxial–abaxial polarity highly conserved across gymnosperm lineages. These findings suggest that integument development originated through the recruitment of preexisting regulatory networks into a core developmental module, providing clues for understanding the molecular mechanisms of integument formation underlying the origin of seed plant ovules.

Summary: Bamboo with its remarkable growth rate and economic significance, offers an ideal system to investigate the molecular basis of organogenesis in rapidly growing plants, particular in monocots, where gene regulatory networks governing the maintenance and differentiation of shoot apical and intercalary meristems remain a subject of controversy. We employed both spatial and single‐nucleus transcriptome sequencing on 10× platform to precisely dissect the gene functions in various tissues and early developmental stages of bamboo shoots. Our comprehensive analysis reveals distinct cell trajectories during shoot development, uncovering critical genes and pathways involved in procambium differentiation, intercalary meristem formation, and vascular tissue development. Spatial and temporal expression patterns of key regulatory genes, particularly those related to hormone signaling and lipid metabolism, strongly support the hypothesis that intercalary meristem origin from surrounded parenchyma cells. Specific gene expressions in intercalary meristem exhibit regular and dispersed distribution pattern, offering clues for understanding the intricate molecular mechanisms that drive the rapid growth of bamboo shoots. The single‐nucleus and spatial transcriptome analysis reveal a comprehensive landscape of gene activity, enhancing the understanding of the molecular architecture of organogenesis and providing valuable resources for future genomic and genetic studies relying on identities of specific cell types.

Poaceae members shared a whole-genome duplication called rho. However, little is known about the evolutionary pattern of the rho-derived duplicates among Poaceae lineages and implications in adaptive evolution. Here we present phylogenomic/phylotranscriptomic analyses of 363 grasses covering all 12 subfamilies and report nine previously unknown whole-genome duplications. Furthermore, duplications from a single whole-genome duplication were mapped to multiple nodes on the species phylogeny; a whole-genome duplication was likely shared by woody bamboos with possible gene flow from herbaceous bamboos; and recent paralogues of a tetraploid Oryza are implicated in tolerance of seawater submergence. Moreover, rho duplicates showing differential retention among subfamilies include those with functions in environmental adaptations or morphogenesis, including ACOT for aquatic environments (Oryzoideae), CK2β for cold responses (Pooideae), SPIRAL1 for rapid cell elongation (Bambusoideae), and PAI1 for drought/cold responses (Panicoideae). This study presents a Poaceae whole-genome duplication profile with evidence for multiple evolutionary mechanisms that contribute to gene retention and losses.

Transcriptome sequencing technologies have revolutionized the field of phylogenomics by facilitating the identification of homologous genes for species without whole genome sequences. To infer complex evolution relationship among eukaryotes, it is essential to obtain complete sequences of protein‐coding genes to provide informative mutations. However, transcriptomes in eukaryotes often consist of a large number of duplicated genes and alternative isoforms, bringing great challenges for developing effective tools to obtain complete coding gene sequences. Here, we present a net‐flow based assembler TransMCL, which aims to assemble fragmented transcripts into complete sequences while eliminating redundant isoforms during homologue clustering. By employing Markov clustering strategies and homologous gene guidance, TransMCL can accurately assemble genes for multiple organisms in an affordable time frame, making it well‐suited for phylogenomic studies based on transcriptomic data. Our results demonstrate that TransMCL can assemble 89.95%–92.95% of the total expressed genes into near‐complete transcripts on benchmark plant/animal datasets. Furthermore, applying TransMCL to multiple transcriptomes in a single run enhances the completeness of genes, even in the absence of guidance homologues from closely related species. These findings highlight the potential of TransMCL in phylogenomic studies, enabling the comprehensive characterization of gene families at the whole genome scale. By overcoming the challenges of complex gene contents in eukaryotes, TransMCL can significantly enhance our understanding of evolution of gene families across species.

Advances in spatial transcriptomics enlarge the use of single cell technologies to unveil the expression landscape of the tissues with valuable spatial context. Here, we propose an unsupervised and manifold learning-based algorithm, Spatial Transcriptome based cEll typE cLustering (STEEL), which identifies domains from spatial transcriptome by clustering beads exhibiting both highly similar gene expression profiles and close spatial distance in the manner of graphs. Comprehensive evaluation of STEEL on spatial transcriptomic datasets from 10X Visium platform demonstrates that it not only achieves a high resolution to characterize fine structures of mouse brain but also enables the integration of multiple tissue slides individually analyzed into a larger one. STEEL outperforms previous methods to effectively distinguish different cell types/domains of various tissues on Slide-seq datasets, featuring in higher bead density but lower transcript detection efficiency. Application of STEEL on spatial transcriptomes of early-stage mouse embryos (E9.5–E12.5) successfully delineates a progressive development landscape of tissues from ectoderm, mesoderm and endoderm layers, and further profiles dynamic changes on cell differentiation in heart and other organs. With the advancement of spatial transcriptome technologies, our method will have great applicability on domain identification and gene expression atlas reconstruction.

Whole-genome duplication events have long been discovered throughout the evolution of eukaryotes, contributing to genome complexity and biodiversity and leaving traces in the descending organisms. Therefore, an accurate and rapid phylogenomic method is needed to identify the retained duplicated genes on various lineages across the target taxonomy. Here, we present Tree2GD, an integrated method to identify large-scale gene duplication events by automatically perform multiple procedures, including sequence alignment, recognition of homolog, gene tree/species tree reconciliation, Ks distribution of gene duplicates and synteny analyses. Application of Tree2GD on 2 datasets, 12 metazoan genomes and 68 angiosperms, successfully identifies all reported whole-genome duplication events exhibited by these species, showing effectiveness and efficiency of Tree2GD on phylogenomic analyses of large-scale gene duplications. Tree2GD is written in Python and C++ and is available at https://github.com/Dee-chen/Tree2gd Supplementary data are available at Bioinformatics online.