Regulator of Microtubule Dynamics 2 (RMDN2) encodes a key regulator of microtubule dynamics, which is essential for cell division and cytoskeletal homeostasis. It is broadly expressed across various tissues and belongs to the microtubule-associated protein family. Current research has linked RMDN2 to human diseases, particularly cancer, suggesting that it may function as a cancer-promoting driver with potential diagnostic, prognostic, and therapeutic value [1]. Moreover, a genome-wide association study identified a significant association between the single nucleotide polymorphism (SNP) rs2113389 at the RMDN2-CYP1B1 locus and Alzheimer’s disease [2]. Despite these findings, the genetic variation and biological function of RMDN2 remain poorly characterized, especially in animals.

RMDN2 shares homology with Regulator of Microtubule Dynamics 3 (RMDN3, PTPIP51), a mitochondrial protein that mediates lipid radical transfer from mitochondria to the endoplasmic reticulum to mitigate oxidative stress [3]. While RMDN3 has been relatively well studied, the structural characteristics, specific roles, and gene structure of RMDN2 in chickens remain unexplored. Our previous study identified RMDN2 as a candidate gene influencing chicken follicle development, with expression levels increasing during follicular development and peaking in F1 follicles [4], suggesting a potential role in ovarian function and egg-laying performance.

Molecular marker-assisted selection is an effective strategy for enhancing the efficiency of egg production breeding. SNPs are ideal molecular markers due to their high density, low mutation rates, stable inheritance biallelic polymorphism, and strong representativeness [5]. Numerous SNPs associated with laying traits have been identified in poultry, including variants in the RFamide-related peptide (RFRP) gene in Zhenning Yellow chickens [6], prolactin (PRL) gene in chickens [7], and tyrosine aminotransferase (TAT) gene in Muscovy ducks [8]. These findings support the potential of SNPs as effective molecular markers for improving egg production. Given this, the continued discovery of novel SNPs promises to provide valuable tools for breeding. In our preliminary study, we identified four mutations in the coding sequence region of the RMDN2 gene through pooled sequencing. However, the relationship between these RMDN2 gene variants and laying performance remains unclear and warrants further investigation.

Mutations in key genes can markedly influence important phenotypic traits, and bioinformatics approaches offer powerful tools to investigate these relationships. For instance, polymorphism analyses of Cytochrome P450 Family 11 Subfamily A Member 1 (CYP11A1) in sheep have linked specific variants to litter size [9]. Similarly, a missense mutation in growth differentiation factor 9 (GDF9) is predicted to alter the protein’s tertiary structure and affect follicle-stimulating factor activity, highlighting its potential as a genetic marker for improving sheep litter size [10]. In Nigerian sheep, computational assessment of Prion Protein (PRNP) polymorphisms using PolyPhen-2 and PROVEAN suggested a deleterious effect for p.Arg154His, in contrast to the benign p.His171Gln [11]. Further, structural and functional predictions for the Melatonin Receptor 1A (MTNR1A) gene in Indonesian fine-tailed ewes indicated that the p.Val127Ile SNP may severely disrupt protein structure and stability, correlating with litter size variation [12]. Collectively, these studies demonstrate how bioinformatics tools can effectively prioritize putative causal SNPs and interpret their functional implications for complex traits.

In this study, we examined the association between polymorphisms in the RMDN2 gene and reproductive performance in an F₂ population generated from a cross between Rhode Island White and White Leghorn chickens. Furthermore, bioinformatics analyses were performed to preliminarily investigate the potential functional and regulatory roles of RMDN2. The results of this work will contribute to a theoretical framework that can guide molecular-assisted breeding strategies for enhancing egg production performance.

In poultry production, reproductive traits like egg production, egg weight, and AFE are crucial economic indicators. This study examined F₂ resource populations derived from two high-yielding egg-laying chicken breeds subjected to long-term intensive selection for improved laying performance. Four novel SNPs within the RMDN2 gene were identified, with genetic diversity at these loci being moderate to low. Prolonged selective breeding typically leads to the reduction of genetic diversity [14], this mechanism is primarily achieved through selective sweeps, whereby the fixation of a beneficial mutation is accompanied by a reduction in genetic diversity in the surrounding genomic region [15,16]. Interestingly, the wide-type GG and GG genotypes of SNP1 and SNP3 showed lower frequencies and were associated with the lower EN66 compared to other genotypes. As a result of long-term selection, the population likely experienced genetic bottlenecks due to the elimination of individuals with lower egg production. It is hypothesized that RMDN2 may have been indirectly selected during breeding programs, with its genetic variations linked to the enhancement of egg production. However, the current findings suggest that the utility of this marker for future breeding efforts is limited in this population, as the EN66 values of the alternative genotypes are close to each other. Combined genotypic effects of multiple SNPs, particularly those involving heterozygous configurations, can significantly influence egg production traits through cooperative interactions [17]. The individual effects of SNPs are often small compared with their combined effects, highlighting the importance of haplotype-level analysis in molecular marker-assisted selection [18]. The common heterogeneity observed in this quantitative trait supports the potential of hybrid breeding to enhance egg production [19]. Future studies should increase the sample size and employ whole-genome analysis to incorporate multiple genetic loci, alongside environmental factors, to gain a deeper understanding of their roles in egg production traits. Such research will provide a more comprehensive reference for the genetic improvement of other chicken populations.

Given that the functional relationship between genetic markers and trait phenotypes cannot be effectively captured by a single SNP, LD and haplotype analysis were employed to investigate the association between these loci and reproductive traits [20]. The H1H1 haplotype combination, derived from the three SNPs on the PRKCA gene, was advantageous for enhancing eggshell strength and thickness in female ducks [21]. Similarly, the H1H1 combination on the MC1R gene was effective in increasing the black plumage proportion in Guangxi Yao chickens [22]. In the present study, the H1H1 combination was associated with low values of BWFE, BW105, and FEW, whereas the H2H2 and H1H4 combinations were associated with elevated FEW values. Selection for a lighter body weight in chicken breeding lines may enhance reproductive performance, as individuals with earlier AFE and lighter FEW tend to have higher total egg number [23]. Notably, the H1H4 combination, formed by heterozygous genotypes of SNP1, SNP2, SNP3 and the GG genotype of SNP4, showed the highest EN66, despite having the lowest allele frequency. This observation may be explained by heterosis, which is common in quantitative traits and supports the potential of hybrid breeding for improving egg production [24]. However, future studies should explore the beneficial effects of heterozygous haplotypes on reproductive traits by increasing sample sizes, in order to fully assess and leverage the breeding potential of the H1H4 combination.

The analysis of physicochemical properties analysis indicates that the missense mutation induces subtle but potentially meaningful changes in the biochemical characteristics of RMDN2, which may collectively influence its structure and function. Alteration in amino acid composition can modify charge distribution and hydrophobicity, both of which are critical determinants of protein folding and molecular interactions [25]. These effects may modulate interaction efficiency without causing complete loss of function. Hydrophobicity plays a central role in stabilizing protein structure and directing secondary structure formations [26]. Previous studies have demonstrated that increased hydrophobic character can enhance core packing and thermal stability [27], but it may also predispose proteins to adopt β-sheet–rich conformations under specific conditions [28]. These shifts can influence functional dynamics, especially for proteins requiring structural flexibility. Protein stability represents a balance between maintaining structural integrity and preserving conformational adaptability [29]. Excessive stabilization may restrict the dynamic motions required for catalysis or regulation [30]. Therefore, even minor stability changes may have disproportionate functional consequences. Overall, the mutation may act as a fine-tuning factor, subtly influencing folding behavior, interaction networks, and functional regulation.

Further in silico analysis revealed that RMDN2 lacks signal peptide and glycosylation sites, but it may function as a transmembrane protein, consistent with its role within the RMDN family [3]. Transmembrane regions are typically composed of α-helices. Although the hydrophobic distribution within this region (amino acids 9–27) remains largely unchanged, the introduction of β-sheets may influence helical continuity, potentially affecting the transmembrane insertion process [31]. Therefore, the presence of β-sheets in the RMDN2 transmembrane region might similarly expose hidden phosphorylation sites or ligand-binding motifs, potentially impacting signal transduction. A decrease in α-helix content can affect the hydrophobic interface, inhibiting protein oligomerization and affecting functions like signal transduction or cell adhesion [32]. Disordered regions often have post-translational modification sites and are involved in dynamic protein interactions [33]. The primary function of the coiled coil is to mediate protein oligomerization and ligand binding [34]. SNP1 and SNP2 mutations located in the coiled-coil region (79–106 residues), while SNP3 mutation located the disordered region (residues 112–181), lead to a reduction in α-helix and an increase random coil and β-sheet, implying that these mutations may influence the post-translational function of RMDN2 and its role in signal transduction. RMDN2, as a member of RMDN family, is involved in microtubule dynamics, which are critical for chromosome pairing and cell division [35]. Hence, mutations in RMDN2 could potentially influence the post-translational function and signal transduction involved in microtubule dynamic process. However, the precise advantages and disadvantages of RMDN2 need to be explored through experiment validation in future studies.

Additionally, we observed that the mutant allele frequency was higher than that of the wild type, and the EN66 of the homozygous mutant was close to that of the heterozygous genotype. This suggests that the SNP mutations in RMDN2 are unlikely to impair its function. On the contrary, mutations may enable RMDN2 to more actively affect ovarian function, thus contributing to increased egg production.

Phylogenetic analysis of RMDN2 demonstrated complete sequence homology between chicken and quail, with notable conservation observed among species within the same order. However, homology significantly decreased across different taxonomic orders, indicating that species from distinct orders may exhibit similar evolutionary responses to environmental changes [36]. This phenomenon is also observed universally when employing marker genes in studies of species evolution [37,38]. Our findings suggest that RMDN2 could serve as a valuable tool for comparative evolutionary studies across species.

This study investigated genetic polymorphisms within the CDS region of the RMDN2 gene in an F₂ chicken resource population, identifying four novel SNPs. The heterozygous genotypes of SNP1, SNP3, and SNP4 were associated with increased EN66, while the heterozygous genotype of SNP2 was associated with elevated BWFE. The H1H1 haplotype combination was associated with lower BWFE, BW105, and FEW. Missense mutations were predicted to influence the function of RMDN2 protein. Phylogenetic analysis demonstrated high conservation within species of the same order and limited sequence conservation across species of different orders. These findings contribute to the understanding of genetic factors influencing laying traits and provide a foundation for further exploration of the RMDN2 gene’s role in poultry. However, the limitations of the current study, including the small sample size and the lack of experimental validation, must be acknowledged, as they may affect the robustness and generalizability of the conclusions. Future studies, including larger sample sizes and experimental validation of the predicted functional effects, are necessary to confirm the role of RMDN2 in poultry breeding.