Genome-wide identification and analysis of the B3 gene family in Ziziphus jujuba

【Objective】Ziziphus jujuba Mill., commonly known as Chinese jujube, is an emerging superfruit with high application and market value worldwide. As a representative economic tree species in the Rhamnaceae family, it is well-known for its unique flavor and abundant nutrients, including rich vitamin C, cyclic adenosine monophosphate (cAMP), and soluble sugars. Additionally, jujube has strong tolerance to a variety of abiotic stresses, and boasts outstanding economic and ecological benefits. Originating from the middle and lower reaches of the Yellow River basin in China, jujube is a unique native germplasm resource of the country. After over 7000 years of domestication and cultivation, this species has been widely introduced and planted in nearly 50 countries across the temperate and tropical regions of five continents, forming a globally distributed cultivation pattern. To reveal the composition, structural features, and evolutionary characteristics of the B3 gene family, this study conducted a bioinformatics analysis of the B3 gene family in jujube (Z. jujuba), and analyzed its expression patterns across different tissues using transcriptome data.【Methods】Using the jujube reference genome dataset (PRJNA1014986) downloaded from the NCBI database, we adopted a stringent genome-wide bioinformatics screening strategy to identify members of the B3 gene family in jujube. We combined the hidden Markov model profile of the B3 domain (PF02362) obtained from the Pfam database with BLASTp homolo-gy search against Arabidopsis thaliana B3 protein sequences from the TAIR database (E-value ≤ 1e-10) to screen candidate ZjB3 genes. After removing redundant sequences, we used InterProScan and CDSearch tools to verify the presence of the typical B3 domain, and retained the qualified sequences as the final members of the gene family. We employed a set of professional bioinformatics tools, including DeepLoc, TBtools, MEME, PlantCARE, KaKs_Calculator, and iTOL, to perform comprehensive multidimensional analyses of the ZjB3 gene family. These systematic analyses covered chromosomal distribution visualization, identification of intragenomic collinearity and evolutionary expansion analysis, construction of a maximum-likelihood phylogenetic tree with 2000 bootstrap replicates, subfamily clustering within the gene family, prediction of conserved motif composition, characterization of exon-intron gene structure and functional domains, genome-wide identification and functional classification of promoter cis-acting elements, and selection pressure assessment of homologous gene pairs through Ka/Ks ratio calculation. Additionally, publicly available high-throughput RNA-Seq data from two jujube cultivars (S21 and Z95) covering eight different tissues (bearing shoot, leaf, stem, phloem, flower, young fruit, enlarging fruit, and mature fruit), as well as data from two other cultivars (Jinsi Xiaozao and Jinkuiwang) covering nine consecutive fruit developmental stages (S1-S9), were used to investigate the spatiotemporal expression patterns of ZjB3 genes. All raw sequencing reads were subjected to strict quality control using Fastp software to remove adapters and low-quality sequences. After that, genome alignment was performed with Hisat2, and gene expression quantification was completed using featureCounts. Finally, Log2TPM normalization was used to standardize the expression data for subsequent expression pattern analysis.【Results】A total of 64 ZjB3 genes were identified, and they are unevenly distributed across 12 chromosomes. The encoded proteins ranged from 133 to 1144 amino acids in length, with molecular weights between 15.38 and 128.26 kDa and isoelectric points between 4.05 and 9.97. Subcellular localization prediction showed that all 64 ZjB3 proteins are localized in the nucleus. Phylogenetic analysis classified these genes into five subfamilies: (ABI3, ARF, HSI, RAV, and REMConserved motif analysis revealed significant differences in motif composition among different subfamilies. Functional domain analysis showed that all B3 family members contain a typical B3 domain, and some subfamily members also have additional functional domains. Gene structure analysis revealed clear subfamily-specific characteristics. Promoter cis-regulatory element analysis indicated that this gene family is enriched in elements associated with hormone and light responses. Selection pressure analysis of homologous gene pairs within this gene family showed that all Ka/Ks values were less than 1, suggesting that the entire family is under strong purifying selection, with highly conserved sequences and functions and no distinct positive selection signals. Based on transcriptome data from public databases, we analyzed the expression patterns of B3 gene family members in different jujube tissues. The results showed that members of the jujube B3 gene family exhibit distinct expression patterns across different tissues, indicating that these genes are involved in regulating different growth and developmental stages of jujube. Further clustering analysis revealed that genes within the same subfamily have similar expression patterns, indicating potential synergistic effects among these genes.【Conclusion】Our genome- wide characterization identified 64 ZjB3 genes distributed across 12 jujube chromosomes. These genes can be divided into five distinct subfamilies, all of which have the conserved characteristic of localizing to the nucleus. Gene structure, conserved motifs, and domain compositions showed obvious divergence specific to different subfamilies. Moreover, the entire ZjB3 gene family experienced strong purifying selection during its evolutionary process, which indicates a high level of functional conservation. The abundant hormone- and light-responsive cis-elements in the promoters suggested that ZjB3 genes may participate in multiple regulatory pathways controlling jujube growth and development. Furthermore, the tissue-specific and stage-dependent expression patterns of ZjB3 genes indicate that these genes play crucial and diverse roles in regulating jujube tissue growth and fruit development. These findings would provide a fundamental basis for further functional verification and exploration of the evolutionary mechanism of the B3 gene family in jujube.