The melon (Cucumis melo) DHL92 v4.0 genome sequence, protein sequences, and annotation file were downloaded from the CuGenDB database (http://cucurbitgenomics.org/, accessed on 8 July 2025). The amino acid sequences of Arabidopsis DA1 and DA1-related (DAR) proteins were obtained from TAIR (https://www.arabidopsis.org/, accessed on 8 July 2025). We used two methods to identify CmDA1 proteins. First, a Hidden Markov Model (HMM) search was performed using the DA1 domain profile (PF12315) from Pfam (https://www.ebi.ac.uk/interpro/entry/pfam/, accessed on 8 July 2025) (Potter et al., 2018). The melon proteome was searched with HMMER, retaining sequences with E-values ≤ 1e-5 (Ye et al., 2006). Second, a BLASTP search was conducted using the Arabidopsis DA1 and DAR sequences as queries against the melon proteome, with an E-value cutoff of 1e-5. Candidate sequences from both methods were combined. We removed redundant sequences and alternative transcripts, keeping only the longest transcript for each gene. The resulting sequences were verified for the presence of UIM and LIM domains using SMART (http://smart.embl-heidelberg.de/, accessed on 8 July 2025) and NCBI CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 8 July 2025). Sequences lacking these domains were discarded, yielding the final CmDA1 gene family members. Physicochemical properties of CmDA1 proteins, including amino acid number, molecular weight, and theoretical pI, were predicted using the ProtParam tool on ExPASy (https://web.expasy.org/protparam/, accessed on 8 July 2025).

Protein sequences of DA1 family members were retrieved from Arabidopsis thaliana, Cucumis melo, Oryza sativa (Rice Genome Annotation Project, http://rice.uga.edu), Cucumis sativus (v3.0, http://cucurbitgenomics.org), and Citrullus lanatus (Watermelon Genome Database, http://www.watermelondb.cn). Multiple sequence alignment was performed using ClustalW within MEGA software (v11.0.13). A phylogenetic tree was constructed using the Neighbor-Joining (NJ) method with the following parameters: 1000 bootstrap replicates, amino acid p-distance model, and partial deletion for gap treatment (site coverage cutoff of 50%) (Kumar et al., 2016). The resulting tree was visualized and annotated using the iTOL online tool (https://itol.embl.de) (Letunic and Bork, 2021). The identified CmDA1 genes were systematically named based on their phylogenetic clustering patterns and established nomenclature from related species.

The chromosomal positions of the identified CmDA1 genes were extracted from the melon genome annotation file (GFF3) and visualized using the ‘Gene Location Visualize from GTF/GFF’ function in TBtools (v2.310). Conserved motifs in the CmDA1 protein sequences were identified using the Multiple Expectation Maximization for Motif Elicitation (MEME) online tool (http://meme-suite.org/tools/meme, accessed on 10 July 2025) with the following parameters: maximum number of motifs set to 10, and motif width restricted to 6–100 amino acids (Bailey et al., 2009). Protein domains were predicted using the NCBI Conserved Domain Database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 10 July 2025) (Marchler-Bauer et al., 2015). Gene structure features (exon-intron organization) were derived from the comparison of the genomic DNA and coding sequences of the CmDA1 genes. Finally, the phylogenetic tree, conserved motifs, protein domains, and gene structures were integrated and visualized using the “Gene Structure View (Advanced)” module in TBtools (V2.310).

Intraspecific synteny and gene duplication events (including segmental and tandem duplications) among the CmDA1 genes were analyzed using the One-Step MCScanX tool in TBtools with default parameters (Wang et al., 2012). The results, including chromosomal distributions, gene density, and syntenic relationships, were integrated and visualized in a Circos plot using the Advanced Circos module within TBtools (v2.310) (Krzywinski et al., 2009).

For interspecific synteny analysis, whole-genome protein sequences, and annotation files of Cucumis melo, Arabidopsis, and Oryza sativa were compared using the One-Step MCScanX module in TBtools. The analysis was performed with an E-value threshold of 1e-3 and the number of BLAST hits set to 10. Syntenic relationships were visualized using the Multiple Synteny Plot module, with CmDA1 gene family members highlighted to identify orthologous gene pairs among the three species.

The 2,000 bp promoter sequences upstream of the transcription start sites of CmDA1 genes were extracted using TBtools (v2.310). Cis-regulatory elements in these promoter regions were identified using the PlantCARE online database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 12 July 2025). The cis-regulatory elements identified by PlantCARE were visualized using the “Simple BioSequence Viewer” module in TBtools (v2.310).

To investigate the expression patterns of CmDA1 genes, we utilized publicly available RNA-seq data from BioProject PRJDB6414 (Yano et al., 2018). Our analysis included two main aspects: tissue-specific expression across eight distinct tissues (roots, stems, upper stems, young leaves, mature leaves, flowers, fruits, and tendrils) and developmental expression during fruit maturation at nine time points from 2 to 50 days after pollination. The raw sequencing data underwent comprehensive processing through an established bioinformatics pipeline. Initially, sequence data were converted to FASTQ format using fastq-dump (v3.0.10), followed by quality assessment and filtering with fastp (v0.24.1). Cleaned reads were then aligned to the melon reference genome using HISAT2 (v2.1.0) with appropriate strand-specific parameters. The resulting alignment files were processed with SAMtools (v1.21) for format conversion, and gene expression levels were calculated as FPKM values using custom R scripts. We constructed separate heatmaps with TBtools (v2.310) to display the expression patterns of CmDA1 genes.

We used the melon inbred line HN15, which was developed by our research group. The mature fruits of the ‘HN15’ inbred line typically reach a single fruit weight of 1000–1500 g. Plants were cultivated under controlled greenhouse conditions with a 16/8−h light/dark cycle and temperatures maintained at 28°C during the day and 18°C at night. To examine the expression of the four CmDA1 genes, fruit flesh samples were harvested at four developmental stages: 7, 15, 22, and 30 days after pollination (DAP). Roots, stems, and leaves were sampled at the flowering stage. For each tissue type and fruit stage, three biological replicates were taken. All collected samples were immediately frozen in liquid nitrogen and stored at −80°C until RNA extraction.

For RNA extraction, we employed the Spectrum™ Plant Total RNA Kit (Merck KGaA) following the manufacturer’s protocol. RNA quality and concentration were verified using a NanoDrop 2000 spectrophotometer. We then synthesized first-strand cDNA from 1 µg of total RNA using the SweScript All-in-One First-Strand cDNA Synthesis Kit (TRANS, G3337). Specific primers for CmDA1 were designed with Primer Premier 5 software to generate 150–300 bp amplicons.

The qRT–PCR reactions were conducted in 20 µL volumes containing 10 µL of 2× SYBR Green qPCR Master Mix (no ROX) (TRANS, G3320), 1 µL of cDNA template, and 2 µL each of forward and reverse primers. Amplification was performed on a CFX96 Real-Time PCR Detection System (Bio-Rad, USA) using the following program: 95°C for 30 s, then 40 cycles of 95°C for 15 s and 60°C for 30 s. We used the melon EF1α gene as an internal control and calculated relative expression levels according to the 2^-ΔΔCT method (Zhang et al., 2018). The experiment included three biological replicates, each with three technical replicates. Statistical analysis was performed using one-way ANOVA in SPSS (v26.0), and graphical representations were prepared with GraphPad Prism 8. All primer sequences are provided in Supplementary Table S1.

We identified four DA1 genes in the melon genome through a genome-wide analysis. Based on phylogenetic relationships with Arabidopsis, these genes were named CmDA1, CmDA1-1, CmDA1-2, and CmDA1-3. Analysis of the physicochemical properties revealed variations among the four CmDA1 proteins (Table 1). The coding sequence lengths ranged from 1,281 bp to 1,443 bp, encoding proteins of 426 to 480 amino acids. Molecular weights varied from 47.84 kDa to 55.41 kDa, while theoretical isoelectric points spanned from 4.53 to 6.84, indicating differential charge characteristics. Chromosomal mapping showed that the four CmDA1 genes were distributed across chromosomes 3, 8, 10, and 11, with each gene located on a separate chromosome (Figure 1).

To elucidate the phylogenetic relationships within the DA1 family, a multiple sequence alignment was performed using ClustalW with protein sequences from Cucumis melo (melon), Cucumis sativus (cucumber), Citrullus lanatus (watermelon), Arabidopsis thaliana, and Oryza sativa (rice). A subsequent phylogenetic tree was constructed using the Neighbor-Joining method in MEGA 11 with 1,000 bootstrap replicates. Genome-wide identification revealed four DA1 genes in melon, five in cucumber, and five in watermelon.

The resulting phylogenetic tree classified all DA1 proteins into three distinct subfamilies (I–III) (Figure 2). Subfamily I comprised eleven members, including two from melon (CmDA1 and CmDA1-1), two from cucumber (CsaV3_2G028370 and CsaV3_2G016950), two from watermelon (ClG42_02g0132100 and ClG42_08g0170100.1), two from Arabidopsis (AtDA1 and AtDAR1), and three from rice (OsDA1, Os12g0596800, and Os03g0626600). Notably, CmDA1 clustered most closely with the cucumber gene CsaV3_2G028370, while CmDA1–1 formed a closest clade with CsaV3_2G016950. Subfamily II contained five members, featuring CmDA1–2 from melon, which showed close relationships with CsaV3_5G008440 from cucumber and ClG42_07g0035000 from watermelon. Subfamily III included eight members, consisting of five from Arabidopsis and one each from melon (CmDA1-3), cucumber (CsaV3_6G042590), and watermelon. Within this subfamily, CmDA1–3 shared the closest phylogenetic relationship with the cucumber ortholog CsaV3_6G042590.

To investigate the evolutionary relationships of DA1 genes across species, we performed collinearity analyses among Arabidopsis, Cucumis melo, and Oryza sativa (Figure 3). Interspecies analysis revealed five syntenic gene pairs between melon and Arabidopsis and two syntenic pairs between melon and O. sativa, suggesting potential functional importance of these conserved genes during melon evolution. Furthermore, intraspecies analysis within the melon genome identified only one syntenic relationship between CmDA1 and CmDA1-3 (Figure 4), indicating limited gene duplication events in the evolutionary history of the DA1 family in melon.

To elucidate the structural characteristics of the CmDA1 proteins, we analyzed their conserved motifs using the MEME suite (Figure 5A). Five motifs (1, 2, 6, 7, and 8) were consistently present across all four members, suggesting their fundamental role in DA1 protein function. Distinct distribution patterns were observed for other motifs: motifs 3 and 5 were exclusively shared by CmDA1, CmDA1-2, and CmDA1-3, while motif 4 was uniquely conserved in CmDA1, CmDA1-1, and CmDA1-2. These conserved yet differentiated motif patterns imply both functional conservation and potential specialization among CmDA1 members.

Gene structure analysis revealed substantial diversity in exon-intron organization (Figure 5B). All CmDA1 genes possess non-coding UTR regions and exhibit considerable variation in exon numbers, ranging from 8 (CmDA1-1) to 12 (CmDA1-2), with CmDA1 and CmDA1–3 containing 11 and 9 exons, respectively. Correspondingly, intron numbers varied from 7 to 11. The observed structural divergence in gene architecture suggests the possibility of functional differentiation within the CmDA1 family.

Analysis of the 2.0 kb promoter regions upstream of the CmDA1 genes identified various cis-acting regulatory elements (Figure 6). All promoters contained core promoter elements around the -30 region and multiple hormone-responsive elements, including gibberellin-, auxin-, and MeJA-responsive motifs. The presence of these elements suggests potential regulation of CmDA1 genes by phytohormones known to influence cell division and expansion. Additional motifs such as AT-rich DNA binding protein sites and endosperm expression-related elements were also detected. These findings indicate that CmDA1 genes may be regulated by a complex network involving hormonal and developmental signals.

To explore the spatial and temporal expression profiles of the CmDA1 genes, we analyzed transcriptome data from eight distinct tissues and nine stages of fruit development. The expression patterns uncovered both conserved and divergent regulatory strategies among the four CmDA1 genes (Figure 7A). In the tissue-specific analysis, each gene displayed unique expression preferences. CmDA1 was predominantly expressed in roots and flowers, implying potential functions in root development and reproductive processes. CmDA1–1 exhibited broad expression, with high transcript levels in seven of the eight tissues examined, except for upper stems. CmDA1–2 showed preferential expression in roots and stems, indicating a possible role in vegetative growth regulation. Notably, CmDA1–3 was highly expressed in six of the eight tissues, excluding young and mature leaves, suggesting it may act as a central regulator across multiple organs.

During fruit flesh development from 2 to 50 days after pollination (DAP), the four genes displayed distinct temporal expression dynamics (Figure 7B). CmDA1 transcript levels gradually declined throughout fruit development, pointing to a potential early-stage role. CmDA1–1 maintained consistently high expression from 15 to 43 DAP, with relatively lower expression at both earlier (2–8 DAP) and later (50 DAP) stages, indicating a mid-to-late phase-specific regulatory pattern. CmDA1–2 exhibited peak expression at 15 and 43 DAP, with lower expression at other time points, indicative of stage-specific regulation. In contrast, CmDA1–3 maintained consistently high expression during fruit development, with a marked upward trend from 2 to 36 DAP, underscoring its potential as a key persistent regulator of fruit maturation. Collectively, the distinct spatiotemporal expression profiles suggest functional divergence among the CmDA1 members, and the broad tissue distribution combined with sustained expression during fruit development identifies CmDA1–3 as a promising candidate for subsequent functional studies.

To validate the expression patterns of CmDA1 genes, we performed quantitative real-time PCR analysis in the melon inbred line HN15 across various tissues and fruit developmental stages (Figure 8). The results revealed distinct expression profiles among the four genes, with CmDA1–3 consistently showing the highest expression levels across all examined samples.

In roots, all genes reached their maximum expression, with CmDA1–3 showing particularly strong accumulation (mean: 17.81). CmDA1–1 and CmDA1–2 displayed moderate root expression (means: 7.81 and 4.83), while CmDA1 showed the weakest expression (mean: 3.08). This pattern continued in stems and leaves, where CmDA1–3 maintained the highest levels (means: 7.39 and 12.16), followed by CmDA1-1 (means: 4.44 and 6.29).

During fruit development from 7 to 30 DAP, the genes displayed characteristic temporal patterns. CmDA1–3 maintained stable, high expression throughout this period (means: 5.75 at 7 DAP to 6.86 at 30 DAP). In comparison, CmDA1–1 showed moderate but gradually decreasing expression during fruit maturation (means: 2.75 at 7 DAP to 1.48 at 30 DAP). Both CmDA1 and CmDA1–2 maintained relatively low expression levels across all developmental stages, with CmDA1–2 reaching particularly low levels at later stages (mean: 0.55 at 22 DAP). These qRT-PCR results corroborate the expression trends observed in our transcriptome data and highlight CmDA1–3 as the most prominently expressed member in both vegetative tissues and developing fruits, suggesting its potential importance in melon growth and development.