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Paired Single-Cell Transcriptome and DNA Barcode Detection in Zebrafish Using ScarTrace.

ScarTrace is a CRISPR/Cas9-based genetic lineage tracing method that allows for uniquely barcoding the DNA of single cells at a target GFP sequence during developing zebrafish embryos. Single cells from barcoded adult zebrafish can be isolated from various tissues (e.g., marrow, brain, eyes, fins), and their transcriptome and barcode sequences are captured by single-cell cDNA amplification and genomic DNA nested PCR, respectively. Computationally, cell type and barcode identification permit clone tracing and lineage tree reconstruction of tissues to unravel fate decisions during embryogenesis.

Animals↗

Uncovering hidden complexity in the Apis mellifera mitotranscriptome: a polyadenylation-centered perspective.

Mitochondrial transcription is gaining increasing attention as researchers seek to better understand the full coding potential of mitochondrial DNA (mtDNA). Emerging evidence suggests that mtDNA may encode additional elements beyond classical oxidative phosphorylation genes, pointing to a more complex transcriptional architecture than previously recognized. In this study, we explored the mitochondrial transcriptome of Apis mellifera (Insecta: Hymenoptera), with a particular focus on polyadenylation-associated features. Our analysis revealed that both sense and antisense transcripts undergo polyadenylation, although transcript abundance and poly(A) tail lengths varied markedly across mitochondrial genes. Several transcripts exhibited alternative isoforms, either extended or truncated, frequently including intergenic regions. These regions may represent functional non-coding elements or structural variants rather than conventional untranslated regions (UTRs). Interestingly, some transcripts also contained non-templated nucleotide additions particularly cytosine residues immediately upstream of the poly(A) tails. Monocistronic units that included portions of downstream intergenic regions were among the most abundantly represented, suggesting a possible regulatory role for these sequences. To experimentally validate our in silico findings, we performed RT-qPCR to assess relative gene expression and applied 3' RACE-PCR to define transcript boundaries. These approaches confirmed the presence of multiple transcript isoforms and supported the involvement of polyadenylation in shaping mitochondrial RNA diversity. Together, our findings reveal a previously underappreciated level of complexity in the A. mellifera mitochondrial transcriptome and highlight the potential regulatory significance of polyadenylation dynamics and intergenic region transcription.

Animals↗

Transcriptome analysis of the pectoral fin degeneration in half-smooth tongue sole (Cynoglossus semilaevis).

Appendage degeneration is a notable morphological feature of some teleosts with specialized benthic lifestyles. The half-smooth tongue sole (Cynoglossus semilaevis) undergoes severe pectoral fin regression during metamorphosis. However, the molecular basis underlying rapid pectoral fin degeneration remains unclear. Here, we performed time-series transcriptome sequencing on pectoral fins at pre-metamorphosis, metamorphosis peak and post-metamorphosis to characterize the molecular changes associated with pectoral fin degeneration. Transcriptional dynamics and functional enrichment showed that no significant enrichment of classical apoptosis-related transcriptional pathways was detected during pectoral fin degeneration. Instead, sustained downregulation of twist1b, identified as a transcriptomic candidate, together with significant upregulation of ssh1, coupled with enrichment of lysosome and ubiquitin-proteasome system (UPS) pathways, suggested enhanced tissue remodeling during pectoral fin degeneration. Temporal expression clustering revealed heterochronic misalignment in the developmental gene expression: upstream initiator tbx5 was upregulated at early metamorphosis, while downstream maintenance signal fgf10 decreased synchronously. Distal patterning gene hoxd12a exhibited premature expression and rapid decay, losing sustained late-phase expression. Moreover, transient elevation of gli3 during metamorphosis may contribute to restricted distal fin growth. We conclude that pectoral fin degeneration in C. semilaevis is associated with heterochronic disruption of developmental signaling and extensive tissue remodeling. This study provides transcriptomic insights into pectoral fin degeneration in tongue soles and establishes a basis for future functional studies of appendage reduction in teleosts.

Animals↗

Integrative Multiomics and Drug Sensitivity Profiling Reveal Potential Biomarkers and Therapeutic Strategies in Pediatric Solid Tumors.

UNLABELLED: Cure rates for childhood malignancies using established therapy protocols have increased to an average of 80% but have reached a plateau. Moreover, survival rates are particularly low for some pediatric tumors-such as high-risk group 3 medulloblastomas, osteosarcomas, Ewing sarcomas, high-risk neuroblastomas, and high-grade gliomas-and dismal for patients with relapsed malignancies. A functional drug response profiling platform for pediatric solid and brain tumors has been established within the INFORM program to identify patient-specific vulnerabilities and biomarkers and to unravel molecular mechanisms associated with drug response profiles for clinical translation. In this study, we performed a multiomics analysis using drug sensitivity profiles, as well as genomic and transcriptomic data, of 81 pediatric solid tumor samples. The integrative analysis suggested two multiomics signatures associated with drug sensitivity. One signature distinguished neuroblastoma samples with sensitivity to navitoclax, a BCL2 family inhibitor. A second signature was specific to a subset of Wilms tumors harboring the SIX1 (Q177R) hotspot mutation that displayed high expression of MGAM, PTPN14, STAT4, and KDM2B and high sensitivity to MEK inhibitors. A patient-specific causal interaction network analysis suggested possible molecular interactions between MEK inhibitors and the SIX1 mutation in Wilms tumor samples. In conclusion, the integration of drug sensitivity profiling and multiomics data revealed potential biomarkers that may be associated with drug sensitivity in pediatric solid tumors. Patient-specific causal interaction network analysis further elucidated the interaction between inhibitors and signature biomarkers, providing insights that may inform clinical translation. SIGNIFICANCE: The combination of multiomics analysis and drug sensitivity profiling identified two signatures related to drug sensitivity in pediatric solid tumors, contributing to the advancement of functional precision medicine and personalized treatment strategies. This article is part of a special series: Driving Cancer Discoveries with Computational Research, Data Science, and Machine Learning/AI .

Humans↗

Generating correlated data for omics simulation.

Simulation of realistic omics data is a key input for benchmarking studies that help users obtain optimal computational pipelines. Omics data involves large numbers of measured features on each sample and these measures are generally correlated with each other. However, simulation too often ignores these correlations, perhaps due to computational and statistical hurdles of doing so. To alleviate this, we describe three approaches for generating omics-scale data with correlated measures which mimic real datasets. These approaches are all based on a Gaussian copula approach with a covariance matrix that decomposes into a diagonal part and a low-rank part. This decomposition allows for extremely efficient simulation, overcoming a hurdle for adoption of past methods. We use these approaches to demonstrate the importance of including correlation in two benchmarking applications. First, we show that variance of results from the popular DESeq2 method increases when dependence is included. Second, we demonstrate that CYCLOPS, a method for inferring circadian time of collection from transcriptomics, improves in performance when given gene-gene dependencies in some circumstances. We provide an R package, dependentsimr, that has efficient implementations of these methods and can generate dependent data with arbitrary marginal distributions, including discrete (binary, ordered categorical, Poisson, negative binomial), continuous (normal), or with an empirical distribution.

Computer Simulation↗

SpatioMark: quantifying the impact of spatial proximity on cell phenotype.

MOTIVATION: As research advances in spatially resolving the biological archetype of various diseases, technologies that capture the spatial relationships between cells are demonstrating increasing value. Whilst there are an increasing number of analytical methods being developed to identify the complex web of interactions between cells, the downstream impacts of these cell-cell relationships are under explored. RESULTS: We present SpatioMark, a statistical framework that simplifies the assessment of gene or protein expression changes within a cell type that are associated with the spatial proximity to other cell types. We demonstrate its performance across spatial proteomics and transcriptomics datasets. We link identified relationships with differences in patient survival. We highlight key challenges in identifying changes in molecular markers associated with the localization of cells. We propose correction strategies that reduce artefact-induced relationships. AVAILABILITY AND IMPLEMENTATION: SpatioMark is implemented in the Statial R package on Bioconductor: https://bioconductor.org/packages/release/bioc/html/Statial.html.

Humans↗

Gene expression profiling using a novel method: amplified differential gene expression (ADGE).

Amplified differential gene expression (ADGE) is a novel technique, designed to profile gene expression of the whole transcriptome or to compare expression of a set of genes between two samples. ADGE employs hybridization to quadratically amplify the ratio of an expressed gene between control and tester samples before displaying. The subtle structures of adapters and primers are designed for displaying the amplified ratio of an expressed gene between two samples. Four selective nucleotides at the 3' end of primers are used to increase PCR efficiency for targeted molecules and to improve detection of PCR products. Double PCR with the same pair of primers expands the detection range, especially for genes of low abundance. Integration of these steps makes ADGE sensitive and accurate. Application to drug resistant human tumor cell lines showed that ADGE accurately profiled expression levels for induced, repressed or unchanged genes. The qualitative expression patterns for ADGE were verified with RT-PCR.

DNA Primers↗

Proteomic hub proteins CDKN2B, TRAPPC2L, WFS1, and ARPP19 drive biochemical recurrence and metastatic progression in prostate cancer: Protein macromolecule action.

The biological characteristics and metastasis mechanism of prostate cancer are complex, involving the important role of many proteins in cell transcriptional regulation. This study focused on the role of the proteomic hub proteins CDKN2B, TRAPPC2L, WFS1 and ARPP19 in the biochemical recurrence and metastasis progression of prostate cancer. Cross-platform transcriptome integration and differential expression analysis were used to evaluate transcriptome characteristics in a prostate cancer cohort. Functional enrichment analysis was performed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation, and weighted gene co-expression network analysis (WGCNA) was used to investigate cancer progression subtypes. It was found that prostate cancer progression showed significant transcriptome heterogeneity, and low-expression genes dominated. We reveal the important role of epithelial-immune interactions and inflammatory signaling in transcriptional remodeling in prostate cancer. The co-expression network topology analysis showed that the immune-metabolic center module plays a central role in cancer progression. CDKN2B was identified as a key transcriptional determinant in prostate cancer typing, while TRAPPC2L and WFS1 acted as core transcriptional regulators, driving metastatic heterogeneity. ARPP19 and LOC650152 also show important transcriptional driving effects in advanced prostate cancer.

Humans↗

Transcriptome-Proteome Analysis of Human Naive and Memory B Cell Subsets Reveals Isotype and Subclass-Specific Phenotypes.

Antibodies produced by B cells aid in the recognition and clearance of pathogens and are the cornerstone of vaccination strategies. Humans produce nine different antibody isotypes, and their effector functions differ according to the type of antigen and route of exposure. Phenotypic variation between isotype-switched B cell subsets is expected but not studied in detail. To obtain a molecular definition of isotype-defined cell identity, we performed proteomics and transcriptomics on isotype-defined populations of human naive and memory B cells (MBCs): CD27-IgM+IgD+, CD27+CD38lo/-IgM+IgD+, CD27+CD38lo/-IgM+IgD-, and IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4 MBCs (CD27+CD38lo/-Ig+). Combined proteome and transcriptome analysis revealed that mRNA and protein expression profiles separate isotype-defined B cell subsets according to their differentiation status. mRNA and protein expression levels correlated reasonably well for many genes. IgG4-switched B cells were most distinct from naive B cells in terms of mRNA as well as protein expression profiles. Besides a distinct expression profile of cytokine and Fc receptors, we identified a high expression of IgE-coding mRNA in IgG4-switched B cells. SDR16C5 was identified as uniquely upregulated in IgG4-switched B cells. Taken together, this study highlights the distinct phenotypic profile of IgG4-switched B cells.

Humans↗

Comprehensive transcriptomics and proteomics analysis of neointima formation in human saphenous vein: implications for bypass graft disease.

Human saphenous veins (SVs) are widely used as grafts in coronary artery bypass (CABG) surgery but often fail due to neointima formation. Little is known, however, regarding the cellular, transcriptomic, and proteomic dynamics of neointima formation in human veins. Here, we performed transcriptomics and proteomics analysis in an ex vivo tissue culture model of neointima formation in human SVs procured for CABG surgery. Histological examination demonstrated significant elastin degradation and neointima formation (indicated by increased neointima area and neointima-to-media ratio) in SVs subjected to tissue culture. Analysis of data from 72 patients suggests that the progression of SV remodeling and neointima formation differs according to sex and body mass index, which is negatively associated with neointima formation in males only. RNA sequencing demonstrated upregulation of proinflammatory and proliferation-related genes during neointima formation and identified novel processes, including increased cellular stress and DNA damage responses, reflecting tissue trauma associated with vein harvesting. Proteomic analysis identified upregulated extracellular matrix-related and coagulation/thrombosis proteins and downregulated metabolic proteins. Spatial transcriptomics, used to infer regionally enriched gene expression, suggested dynamic alterations in fibroblast and vascular smooth muscle cell (VSMC) states during neointima formation. Specifically, we identified the emergence of HES1+ and matrix metalloproteinase 2- and 14-positive (MMP2+/MMP14+) expression in VSMCs and fibroblasts, respectively, during neointima formation. Furthermore, our data suggest that MIR647, identified through screening, maintains VSMC contractile gene expression. Our findings suggest dynamic transcriptomic and proteomic changes during neointima formation in human veins and provide useful mechanistic information for the pathogenesis of SV graft disease.NEW & NOTEWORTHY Using multiomics and spatial transcriptomics, we uncover dynamic molecular and cellular changes driving neointima proliferation in human saphenous veins, the most common conduit for bypass surgery. Our study highlights sex- and body mass index-associated differences, novel fibroblast and smooth muscle cell states, and a role for microRNA-647 in preserving vascular contractile phenotype. These findings provide new insight into the mechanisms of vein graft failure and may guide future strategies to improve coronary bypass outcomes.

Humans↗

Transcriptomic insights into the molecular mechanism of antifouling agent-induced settlement inhibition in the Pacific oyster Crassostrea gigas.

Marine biofouling remains a persistent challenge to maritime industries and marine ecosystems worldwide. In this study, we systematically evaluated the acute toxicity, settlement inhibitory efficacy, and underlying molecular mechanisms of an N-oleyl-1,3-propanediamine-based antifouling agent using pediveliger larvae of the Pacific oyster Crassostrea gigas. The 96 h-LC50 of the agent was determined to be 0.81 mg/L, and exposure to 1.68 mg/L achieved complete larval settlement inhibition without inducing significant acute toxicity. Transcriptomic analysis identified 791 differentially expressed genes, dominated by downregulated genes associated with ribosomal function, translation, cell adhesion, and cytoskeletal organization. The agent exerts its inhibitory effect primarily through the global suppression of protein synthesis, disruption of cell-substrate adhesion and cytoskeletal integrity, and induction of proteotoxic stress responses. These findings reveal a multi-pathway molecular mechanism underlying antifouling agent-induced settlement inhibition in oyster larvae and provide key molecular biomarkers to support the development of eco-friendly antifouling technologies.

Animals↗

Effect of Tertiary Lymphoid Structures on Immune Cell Infiltration in the Tumor Microenvironment and Prognosis in Lung Adenocarcinoma.

Tertiary lymphoid structures (TLSs) modulate immune responses in various solid tumors, but their comprehensive role in lung adenocarcinoma (LUAD) remains unclear. In this study, we analyzed RNA-seq data from 539 LUAD patients in The Cancer Genome Atlas (TCGA) and microarray data from 223 samples from the Gene Expression Omnibus (GEO, GSE13213, and GSE37745). TLS signatures were evaluated via unsupervised consensus clustering based on 12 chemokine transcriptome signatures. The relationships between TLS and clinical characteristics, tumor microenvironment (TME) cell infiltration, and prognosis were assessed using ESTIMATE and CIBERSORT. A prognostic model was established using LASSO regression and validated with external datasets. Additionally, H&E and IHC analyses were performed to explore associations between intratumoral TLS density, immune-related molecular expression, and patient prognosis in LUAD. Consensus clustering of the TCGA cohort revealed two distinct LUAD patient clusters according to TLS abundance. Cluster 1 exhibited greater immune cell infiltration, more favorable prognosis, and increased expression of immune checkpoint molecules. We developed a prognostic model comprising eight survival-associated genes that act as independent prognostic factors for patient survival. H&E/IHC analyses revealed that TLS density-regardless of pathological stage-was associated with better prognosis; higher intratumoral TLS density/proportion was also related to more favorable outcomes. IHC confirmed that survival-associated genes (CD5, HLA-DMB, and P2RY13) are independent prognostic indicators in LUAD. Our study demonstrated the close relationship between TLS signatures and an active immune microenvironment, highlighting their potential as independent prognostic indicators in LUAD.

Humans↗

Spatial biology reveals altered macrophage states in immunosuppressed non-melanoma skin cancer.

Immunosuppressed patients with non-melanoma skin cancer experience worse clinical outcomes, yet the tumor immune microenvironment associated with systemic immunosuppression remains incompletely defined. Using integrated single-cell, spatial transcriptomic, multiplex immunofluorescence, and spatial epigenomic profiling across immunocompetent and immunosuppressed tumors, we found that overall immune-cell composition was largely preserved despite differences in immune-cell distribution, spatial organization, and T cell clonality. Immunosuppressed tumors demonstrated reduced intratumoral macrophage densities, decreased T cell clonal diversity, altered antigen-presenting cell and T cell spatial interactions, and distinct fibroblast- and macrophage-associated spatial niches. Multi-cohort validation across complementary spatial and single-cell platforms identified consistent alterations in innate-adaptive immune organization in immunosuppressed tumors. Together, these findings define spatial and functional remodeling of the tumor immune microenvironment under systemic immunosuppression and provide a framework for future therapeutic investigation in high-risk patients.

Humans↗

Transcriptome analysis of brown adipose tissue in Brandt's vole treated with tannic acid under cold exposure.

BACKGROUND: Tannic acid (TA) is a hydrolysable plant secondary metabolite known to influence multiple physiological processes in animals; however, its role in regulating brown adipose tissue (BAT) thermogenesis remains poorly understood. Notably, the overwinter food caches of Brandt's voles predominantly consist of Artemisia species, which are rich in TA. This study aimed to determine whether TA contributes to cold tolerance in Brandt's voles by activating BAT thermogenesis. Adult male voles were administered TA, after which the masses of BAT and inguinal white adipose tissue (iWAT) were measured, and temperature changes in BAT, the body surface, and the rectum were recorded following exposure to - 20 °C. In addition, transcriptomic analyses of BAT were performed, and the expression and protein levels of key thermogenic markers were assessed. RESULTS: The results showed that TA reduced iWAT mass while exerting minimal effects on BAT mass. TA-treated voles exhibited significantly elevated temperatures in BAT, the body surface, and the rectum after cold exposure. Histological analyses revealed that TA treatment reduced adipocyte area in iWAT while increasing the number of nuclei in brown adipocytes in BAT. In BAT, differentially expressed genes (DEGs) in voles receiving a low TA dose were significantly enriched in pathways related to fat digestion and absorption and peroxisome proliferator-activated receptor (PPAR) signaling. In contrast, DEGs in voles administered a high TA dose were predominantly associated with brown adipocyte differentiation and the upregulation of cold-induced thermogenesis. Moreover, TA administration increased the expression of FFAR4 and UCP1, as well as the protein levels of PGC-1α, PPARγ, and UCP1 following cold exposure. CONCLUSIONS: Collectively, these findings demonstrate that TA enhances cold tolerance in Brandt's voles by promoting thermogenic gene expression and stimulating brown adipocyte differentiation in BAT, providing novel insights into the role of plant secondary metabolites in mammalian cold adaptation and herbivore-plant interactions.

Animals↗

Integration of methylome and transcriptome reveals age-associated signatures of stage-specific dynamics and regulatory remodeling in dogs.

Aging in mammals is characterized by widespread yet coordinated epigenetic alterations. However, integrative analyses of DNA methylation and gene expression in dogs remain largely unexplored, particularly within genetically homogeneous single-breed populations. To elucidate the molecular signatures of canine aging, we profiled the genome-wide methylome and transcriptome of clinically healthy beagle dogs representing three distinct age groups. Global methylation levels were highly conserved across individuals, yet both methylation and gene expression variability increased progressively with aging. This epigenetic drift was not stochastic but structured by genomic context, as reduced CpG-gene associations indicated a gradual loss of epigenetic control over transcription. We further observed stage-related methylation change patterns across the three age groups, including early-shift, late-shift, and progressive patterns. These groups showed partially overlapping but distinct hallmark associations, suggesting links to different age-related biological processes. Together, these findings indicate that canine epigenetic aging involves multifaceted molecular changes across adulthood and support dogs as a useful model for investigating conserved molecular signatures of aging.

Beagle dog↗

Systematic Dissection of Key Driver Perturbation Signatures in Single Cells via ECCITE-seq.

CRISPR screens, such as expanded CRISPR-compatible cellular indexing of transcriptomes and epitopes by sequencing (ECCITE-seq), enable the simultaneous measurement of transcriptomes, gRNA identity, and cell-surface protein expression at single-cell resolution to systematically interrogate gene function. This platform provides a powerful and scalable experimental approach for validating disease-associated regulators identified by large-scale association studies and other computational methods, including network-based analyses of multi-omics data. Here, as an example application, we describe an ECCITE-seq framework to characterize the transcriptomic consequences of perturbing multiple neuronal key driver genes associated with Alzheimer's disease (AD) in human-induced pluripotent stem cell (hiPSC)-derived neurons. More broadly, by integrating customized pooled gRNA libraries with different CRISPR effectors across multiple cell types, this approach allows for the assessment of the regulatory impact of candidate genes implicated in development and disease processes.

Humans↗

A transcriptome-wide approach for rapid pathotype discrimination of Puccinia striiformis f. sp. tritici in north-western India.

Stripe rust of wheat caused by Puccinia striiformis f. sp. tritici (Pst) remains a major constraint to wheat production in India due to the rapid evolution and frequent emergence of virulent pathotypes. Rapid and reliable discrimination of Pst pathotypes is essential for effective resistance deployment and surveillance. In the present study, transcriptome-wide simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were exploited to develop and validate molecular markers for pathotype-specific detection of Pst pathotypes prevalent in North India (110S119, 238S119, 46S119, 110S84 and 78S84). Microsatellite mining from 6103 core orthologous clusters comprising 51,127 transcripts mined 14,634 SSR loci, from which 93 primer pairs were synthesized. However, only three SSR markers exhibited polymorphism indicating limited discrimination potential of expressed sequence-derived (EST) SSRs for pathotype differentiation. In contrast, SNP discovery through stringent variant calling and filtration yielded 186 pathotype-specific homokaryotic SNPs, of which 56 high-confidence loci were selected for Kompetitive Allele-Specific PCR (KASP) assay development. A total of 48 KASP markers were synthesized and 14 demonstrated clear pathotype- or cluster-specific polymorphism representing substantially higher resolution than SSR markers. The high SNP-to-KASP conversion efficiency (~ 95%) and reproducible fluorescence-based clustering emphasize the robustness of KASP assay. Comparative evaluation revealed that SNP-based KASP markers provide superior discriminatory capacity for closely related Pst pathotypes and represent a promising complementary molecular approach for rapid identification of predominant Indian Pst pathotypes. The validated marker panel developed in this study can complement conventional virulence phenotyping and field pathogenomics approaches for surveillance of currently known pathotypes, while continued refinement may accommodate future changes in pathogen populations.

India↗

Integrated transcriptomic, transcriptional factors, and protein interaction reveal the regulatory mechanisms of flowering time in rice (Oryza sativa L.).

Appropriate flowering time is important for rice regional adaptation and optimum rice production, but little is known about the omics of heading date in rice. Here, we studied omics including transcriptome, proteome and transcriptional factors to identify regulatory genes related to flowering time. A total of 1402 differentially expressed genes (DEGs, 721 up-regulated and 681 down-regulated) were detected in wild and mutant. These transcripts are classified according to biological processes, cellular components, and molecular functions. Among these differentially expressed genes, many transcription factor genes demonstrated multiple regulatory pathways involved in flowering time. Gene expression analysis showed that Os03g0122600 (OsMADS50), Os08g0105000 (Ehd3), Os06g0275000 (Hd1) were expressed higher and Os06g0199500 (OsHAL3), Os06g0498800 (OsMFT1), Os08g0105000 (Ehd3), Os06g0157700 (Hd3a), and Os02g0731700 (Ghd2), were expressed lower in wild compared to mutant, which are the key genes that regulate the flowering in rice. In addition, Ghd7 interacted with Os10g30860 and Os12g08260 using yeast two-hybrid assay. We identified 28 potential Ghd7 transcriptional regulators using the transcription factor-centered yeast one hybrid (TF-Centered Y1H) assay. Taken together, this study developed a new set of genomic resources to identify and characterize genes, proteins, and motifs associated with flowering time.

Oryza↗