High-impact variants on the X chromosome in Italian Holstein cattle: Implications for male cattle health

Introduction

The Holstein Friesian (HF) is one of the most widely distributed and economically important dairy cattle breeds worldwide. The breed originated in the Netherlands through the crossing of black Batavian and white Friesian cattle, resulting in animals with superior milkproducing ability even under limited feeding conditions (Elischer, 2014) [2] . Among HF populations, the Italian Holstein has gained particular importance due to its exceptional milk yield and major contribution to the dairy industry, especially in the production of high-value dairy products such as Parmigiano-Reggiano and Mozzarella cheese. Consequently, genomic studies in Holstein cattle are highly relevant for improving productivity, fertility, disease resistance and overall breeding efficiency. Fertility and production traits in dairy cattle are strongly influenced by genetic factors. Earlier studies demonstrated that variation in fertility traits possesses a substantial genetic component, making genomic evaluation an important tool for livestock improvement programmes (Jansen, 1985) [7] . With the advancement of next-generation sequencing technologies, identification of functional genomic variants associated with economically important traits has become increasingly feasible. Whole exome sequencing (WES) is a targeted sequencing approach that focuses specifically on protein-coding regions of the genome, where the majority of functional mutations are located (Ng et al., 2009; Mamanova et al., 2010) [9,8] .

Quality Control and Read Preprocessing

The quality of raw sequencing reads was assessed using FastQC to evaluate sequence quality scores, GC content, duplication levels and adapter contamination. Low-quality bases and adapter sequences were removed using Trimmomatic. Reads containing ambiguous bases and short reads were filtered to improve downstream alignment accuracy. Following preprocessing, high-quality reads were retained for subsequent analysis. Quality-filtered reads exhibited improved Q20 and Q30 scores, indicating high sequencing accuracy.

Read Alignment

Filtered paired-end reads were aligned to the Bos taurus reference genome (ARS-UCD2.0/bosTau9.fa) using the Burrows-Wheeler Aligner MEM (BWA-MEM) algorithm.

The generated Sequence Alignment Map (SAM) file was converted into Binary Alignment Map (BAM) format and subsequently sorted using SAM tools for downstream analysis.

BAM File Processing and Coverage Analysis

The sorted BAM file was indexed using SAM tools to facilitate efficient data access during variant analysis.

Sequencing depth and genome coverage statistics were assessed to evaluate the quality and reliability of exome coverage across the reference genome.

Variant Calling

Variant calling was performed using BCF tools to identify single nucleotide polymorphisms (SNPs) and insertiondeletion mutations (INDELs) from the aligned sequencing data. The identified variants were stored in Variant Call Format (VCF) for downstream functional annotation and analysis.

Variant Annotation

Functional annotation of identified variants was performed using SnpEff implemented through the Galaxy platform. The X chromosome (NC_037357.1) was extracted from the annotated dataset for further analysis. High-impact X-linked variants were filtered using the vcfR package in R based on the following criteria: mapping quality (MQ) > 30, read depth (DP) > 10, quality score (QUAL) > 20 and highimpact functional annotation. 1. Since male cattle possess only one X chromosome, deleterious mutations affecting X-linked genes may exert stronger phenotypic effects due to the absence of a second compensatory allele. Therefore, identification of high-impact X-linked variants is important for understanding reproductive performance, growth, metabolism and developmental traits in cattle. Fertility is one of the most economically important traits in dairy cattle and previous studies have shown that reproductive efficiency can be improved through better management practices and hormonal interventions (Darwash et al., 2001) [4] . However, a substantial proportion of variation in fertility traits is also influenced by genetic factors (Jansen, 1985) [7] . In this context, identification of high-impact genomic variants associated with reproductive and physiological functions is important for understanding the molecular basis of fertility and productivity in cattle. In the present study, exploratory analysis identified highimpact variants on the X chromosome affecting genes including POLA1, GPR119, IGSF1, CCNB3, OFD1 and HUWE1. The biological significance of these variants may be particularly important in male cattle because bulls possess only one X chromosome, increasing the likelihood of phenotypic expression of deleterious mutations.

Results and Discussion

Frameshift variants identified in POLA1 may have important implications for cellular proliferation and genomic stability. The POLA1 gene encodes the catalytic subunit of DNA polymerase alpha, which is essential for DNA replication and cell division. Previous studies have shown that mutations in POLA1 can alter gene splicing and reduce transcript and protein levels, resulting in immune dysfunction and developmental abnormalities (Starokadomskyy et al., 2016) [11] . In affected males, POLA1-associated disorders have been linked to growth retardation, recurrent infections and systemic developmental defects (Pezzani et al., 2013) [10] . Therefore, disruption of POLA1 in cattle may potentially influence growth, immunity and reproductive performance. The identified variant in GPR119 may also possess functional importance due to the gene's role in metabolic regulation. GPR119 belongs to the G-protein-coupled receptor superfamily and is involved in glucose-dependent insulin secretion as well as incretin hormone release (Yang et al., 2018) [13] . Alterations in this gene could impair glucose homeostasis, feed efficiency and metabolic adaptation in cattle, potentially affecting growth and production traits. Similarly, variants identified in IGSF1 may influence growth and reproductive physiology. Previous studies in mice have highlighted IGSF1 as a candidate gene associated with skeletal muscle development and muscle mass regulation (Verbrugge et al., 2018) [12] . Since the gene is also involved in endocrine signalling pathways, functional disruption may affect developmental processes and reproductive efficiency in cattle. A frameshift mutation identified in CCNB3 may have reproductive significance due to its role in cell cycle progression and meiotic regulation. Earlier reports demonstrated that CCNB3 is indispensable for female fertility in mice and is associated with successful meiotic progression (Chotiner et al., 2022) [1] . Although the present study focused on male cattle, disruption of this gene may still affect germ cell development and spermatogenesis, potentially influencing bull fertility. In addition to the identified frameshift INDELs, SNP variants detected in OFD1 and HUWE1 may also possess important biological significance in cattle development and reproduction. The OFD1 gene, also known as CXORF5, has been associated with several developmental syndromes in humans, including the X-linked dominant Oral-Facial-Digital type 1 syndrome (Ferrante et al., 2001) [5] . Previous studies demonstrated that OFD1 exhibits stage-specific expression during embryonic development, particularly in genital ridges, nervous tissues and craniofacial structures. The gene is also highly expressed in epithelial tissues lining the oral and nasal cavities (Ferrante et al., 2001) [5] . Since OFD1 plays a major role in ciliary function and organ development, mutations affecting this gene may potentially contribute to developmental abnormalities, reproductive defects and impaired organogenesis in cattle. Similarly, variants identified in HUWE1 may influence important cellular regulatory mechanisms. The HUWE1 gene encodes an E3 ubiquitin protein ligase involved in protein degradation, apoptosis, DNA damage response and cell cycle regulation. Previous reports in humans demonstrated that increased copy number variations in HUWE1 are associated with cognitive impairment and neurological abnormalities in males (Froyen et al., 2012) [6] . Since HUWE1 participates in multiple cellular homeostasis pathways, functional alterations in this gene may affect cellular proliferation, reproductive physiology and developmental stability in cattle.

Conclusion

The present exploratory whole exome analysis identified high-impact variants on the X chromosome of an Italian Holstein bull, including frameshift INDELs and spliceassociated SNPs. The genes associated with the variants are associated with metabolism, reproductive function, DNA replication, cellular regulation and developmental processes. The findings provide preliminary insights into candidate functional variants that may influence fertility, growth and overall health in cattle, highlighting the importance of whole exome sequencing for screening breeding animals and identifying potentially important genomic variants.

References

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