README FILE – Supplementary Data for Evolutionary Dynamics of the Proanthocyanidin Biosynthesis Gene LAR This file was generated on August 21st, 2025 by Maria F. Marin-Recinos and Boas Pucker. A. GENERAL INFORMATION Title of the dataset: Supplementary data for Evolutionary Dynamics of the Proanthocyanidin Biosynthesis Gene LAR Brief description of the research project and its aims: This dataset contains supplementary files associated with the manuscript "Evolutionary Dynamics of the Proanthocyanidin Biosynthesis Gene LAR", which investigates the evolutionary divergence, sequence variation, promoter motif composition, and coexpression patterns of LAR1 and LAR2 genes across multiple plant lineages. Supplementary materials include statistical analysis outputs, gene expression data, polypeptide sequences, sequence conservation values, cis-regulatory element data, BAM files with aligned RNA-seq reads around the LAR1 and LAR2 loci, and synteny results. Author Information A. Investigator Contact Information Name: Maria F. Marin-Recinos Institution: Plant Biotechnology and Bioinformatics, Institute for Cellular and Molecular Botany - IZMB, University of Bonn. Address: Kirschalle 1, 53115 Bonn, Germany. Email: marmarin@uni-bonn.de B. Project Supervisor (Principal Investigator) Name: Prof. Dr. Boas Pucker Institution: Plant Biotechnology and Bioinformatics, Institute for Cellular and Molecular Botany - IZMB, University of Bonn. Address: Kirschalle 1, 53115 Bonn, Germany. Email: pucker@uni-bonn.de C. In case of questions related to this dataset, please contact: Name: Prof. Dr. Boas Pucker Institution: Plant Biotechnology and Bioinformatics, Institute for Cellular and Molecular Botany - IZMB, University of Bonn. Address:Kirschalle 1, 53115 Bonn, Germany. Email: pucker@uni-bonn.de Date of data collection: 2023-06-21 – 2025-08-05 Funding sources: This work was supported by the BMBF-funded de.NBI Cloud within the German Network for Bioinformatics Infrastructure (de.NBI) (031A532B, 031A533A, 031A533B, 031A534A, 031A535A, 031A537A, 031A537B, 031A537C, 031A537D, 031A538A). Language of the dataset: English B. DATA & FILE OVERVIEW The supplementary materials are provided as six Additional file groups. Files are named according to the format: Additional_file_X (single file in group X) Additional_file_XA, XB, XC… (multiple files in group X) Additional files: - Additional_file_1 Type: Multiple files (A and B): PDF and CSV tables Description: LMM/EMM results for LAR paralog expression and the corresponding underlying expression matrix. Additional_file_1A-statistic_analysis — PDF document with the complete results of the Linear Mixed Models (LMM) and Estimated Marginal Means (EMM) statistical analyses. Includes violin plots summarizing variation in gene expression of LAR paralogs across tissues for the following species: Taxus chinensis, Ginkgo biloba, Pinus pinaster, Picea sitchensis, Thuja plicata, Vitis vinifera, Theobroma cacao, Moringa oleifera, Gossypium hirsutum, Actinidia chinensis, Eucalyptus grandis, Populus trichocarpa, Carya illinoinensis, and Morella rubra. Additional_file_1B-GE__LAR — CSV tables containing transcript-level expression data in Transcripts per Million (TPM) for LAR1, LAR2 in dicots species, and LARI and LARII in gymnosperm species across different plant tissues. Contains 14 tables (one per species) corresponding to the species listed in Additional_file_1A-statistic_analysis. Each csv table includes: Per-sample TPM values across tissues. Gene identifiers (NCBI/Phytozome IDs). Short description of the dataset at the top of the file. Additional_file_1C-phylogenetic_tree - PDF document with the complete phylogenetic tree that includes non-collapsed clades and full labels. Additional_file_1D-gene-positions - CSV table listing the genomic positions of LAR1 and LAR2 genes. - Additional_file_2 Type: Multiple files (A to F) related to polypeptide sequence differences and logo plot analysis. Additional_file_2A – CSV files with containing the output tables from sequence comparison using the tool: Divergent Amino Acids (DivAAs) (https://github.com/k-georgi/DivAAs): Additional_file_2A_Gymnosperm_LAR_scores: Positional scores, most common residues or codon per group, and conservation rates for gymnosperm LARI and LARII sequences. Additional_file_2A_Dicots_LAR_scores: Positional scores, most common residues or codon per group, and conservation rates for dicot LAR1 and LAR2 sequences. Note: In DivAAs output tables, 0 values indicate no conservation score or absence of a residue at that position, while NaN values indicate positions that could not be mapped to a reference sequence (e.g., alignment gaps or missing data). Additional_file_2B_conservation_barplot – PDF document containing bar plots results, obtained from DivAAS, showing the amino acid conservation values within specific LAR sequence groups: Gymnosperms and Dicots. Additional_file_2C_AA_gymnosperm_LARI – FASTA file containing polypeptide sequences for LARI in gymnosperms. Additional_file_2D_AA_gymnosperm_LARII – FASTA file containing polypeptide sequences for LARII in gymnosperms. Additional_file_2E_AA_dicot_LAR1 – FASTA file containing polypeptide sequences for LAR1 in dicots. Additional_file_2F_AA_dicot_LAR2 – FASTA file containing polypeptide sequences for LAR2 in dicots. - Additional_file_3 Type: CSV Description: Six CSV files containing results of coexpression analysis for LAR1 and LAR2 in Vitis vinifera, Theobroma cacao, and Moringa oleifera. Each table shows the Spearman correlation coefficient, adjusted p-value, gene expression level (GE), and functional annotation (including Medicago and Arabidopsis homologs and associated database links). Filenames follow the convention: Additional_file_3-coexp_GE__LAR<1,2>.csv - Additional_file_4 Type: Multiple files (A to E) related to promoter analysis and cis-regulatory element identification. Additional_file_4A – Six CSV files containing the output from PLACE database searches for predicted cis-regulatory elements (CREs) in the promoter regions of LAR1 and LAR2 genes. Additionally, three CSV files include frequency tables summarizing the number of occurrences of each cis-element in LAR gene promoters across Vitis vinifera, Theobroma cacao, and Moringa oleifera. Filenames follow the convention: Additional_file_4A-CRE__LAR<1,2>.csv or Additional_file_4A-frequency_CRE_.csv Additional_file_4B_SRA_accessions – CSV file listing the SRA accessions (SRR IDs) for RNA-seq datasets used in promoter validation and co-expression analysis. Additional_file_4C_promoters_UTR – FASTA file containing promoter and 5’ untranslated region (UTR) sequences for LAR1 and LAR2 genes from all species analyzed. Additional_file_4D_LAR_reads_mapping_Tcacao – BAM file containing the RNA-seq read mapping to Theobroma cacao LAR1 and LAR2 loci for promoter analysis. Additional_file_4E_LAR_reads_mapping_Moleifera – BAM file containing the RNA-seq read mapping to Moringa oleifera LAR1 and LAR2 loci for promoter analysis. - Additional_file_5 Type: XLSX Description: CSV files summarizing synteny connections for LAR1 and LAR2 genes across multiple plant species. Additional_file_5_sinteny_LAR1.csv - Synteny connections for LAR1 (indicated with *) across five plant species (Vitis vinifera, Theobroma cacao, Moringa oleifera, Carica papaya, and Bretschneidera sinensis). A dot (.) indicates absence of a corresponding syntenic gene in that species. Additional_file_5_sinteny_LAR1_brassicales.csv - Synteny connections for LAR1 in Brassicales (indicated with *), listing syntenic genes only among the Brassicales species: Moringa oleifera, Carica papaya, Bretschneidera sinensis, Capsella rubella, Boechera stricta, Arabidopsis thaliana, Arabidopsis halleri, Eutrema salsugineum, and Brassica rapa. A dot (.) indicates absence of a corresponding syntenic gene in that species. Additional_file_5_sinteny_LAR2.csv - Synteny connections for LAR2 (indicated with *) across the same five species as in Additional_file_5_sinteny_LAR1.csv. A dot (.) indicates absence of a corresponding syntenic gene in that species. - Additional_file_6 Type: Multiple FASTA files Description: Polypeptide and coding sequence (CDS) sequences for Leucoanthocyanidin reductase (LAR), Anthocyanindin reductase (ANR), and Dihydroflavonol 4-reductase (DFR) in multiple species used in phylogenetic and sequence conservation analyses. Additional_file_6A_AA_LAR_ANR_DFR: FASTA file containing polypeptide sequences of LAR, ANR, and DFR. Additional_file_6B_CDS_LAR_ANR_DFR: FASTA file containing coding sequences (CDS) of LAR, ANR, and DFR. C. SHARING/ACCESS INFORMATION Data derived from another source: No Licenses: CC BY 4.0 Links to related publications: https://doi.org/10.1101/2025.08.06.668978 Other publicly accessible locations: n/a D. METHODOLOGICAL INFORMATION Data collection, structure, and processing: RNA-seq datasets (public SRAs) were retrieved from NCBI and mapped to reference genomes using HISAT2 (https://github.com/DaehwanKimLab/hisat2). Gene expression was quantified in Transcripts per-Million (TPM) using kallisto (https://github.com/pachterlab/kallisto). Coexpression analysis were generated using CoExp (https://github.com/bpucker/CoExp). Sequence conservation was calculated with the tool: Divergent Amino Acids (DivAAs)(https://github.com/k-georgi/DivAAs), generating positional scores and conservation values. Promoter cis-elements were identified using the PLACE database (https://www.dna.affrc.go.jp/PLACE/). Synteny was inferred using JCVI (https://github.com/tanghaibao/jcvi). Note: Details about the methods used for the collection and analysis of the data can be found in the Methods section of: https://doi.org/10.1101/2025.08.06.668978 Abbreviations defined: LAR - Leucoanthocyanidin Reductase TPM – Transcripts Per Million LMM - Linear Mixed Models EMM - Estimated Marginal Means DivAAs – Divergent Amino Acids ANR – Anthocyanidin Reductase DFR – Dihydroflavonol 4-Reductase CRE – Cis-Regulatory Element TSS – Transcription Start Site CDS - Coding Sequence GE - Gene Expression People involved in data collection, processing, analysis and/or submission: all authors File formats: PDF, XLSX, TXT, FASTA