Gene function & annotation

Unlocking the Bovine Y Chromosome Using a Hybrid Sequencing Approach

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Abstract

The Y chromosome is essential for sex determination and male fertility. However, in cattle, genomic characterisation of the Y chromosome is highly incomplete due to its repetitive nature, haploid structure, and extensive sequence homology with the X chromosome. All current bovine reference assemblies, including ARS-UCD1.3 derived from a female Hereford individual, lack any representation of the Y chromosome. Consequently, Y-linked sequences are either missing or inconsistently annotated across public databases, and reads in the Sequence Read Archive (SRA) often align ambiguously between X- and Y-linked regions. This limits the identification of male-specific variants and makes it challenging to incorporate them into genomic selection pipelines.To fill this gap, we created a high-quality assembly of the bovine Y chromosome using a hybrid sequencing approach combining long reads from Oxford Nanopore Technologies (ONT) and short, high-accuracy Illumina reads. Genomic DNA was isolated from Y-sorted semen of Holstein bulls, yielding material enriched with these sequences. Whole-genome Nanopore sequencing produced approximately 77 Gb of long-read data (mean read length 4.3-4.5 kb; N50 ≈ 6.2 kb; mean quality ≈ Q15), corresponding to 25x coverage per bull. A similar mean coverage was obtained for the Illumina short-read data used for polishing, ensuring balanced hybrid correction of the assembled contigs.The bioinformatics workflow included four main steps. First, we collected available bovine Y-chromosome reference sequences from public databases. Next, we aligned ONT long reads to these references and pulled out confidently mapped Y-linked reads, converting them back to FASTQ format. Then, we assembled these enriched ONT reads from scratch. Finally, we polished and refined the consensus sequence using high-accuracy Illumina data, taking advantage of Nanopore-specific tools. This streamlined pipeline enabled robust reconstruction of repetitive and ampliconic regions of the bovine Y chromosome.The resulting assembly achieved high contiguity (N50 > 1 Mb) and strong completeness (BUSCO > 95%), which is a significant improvement over existing Y-chromosome-related bovine resources. Comparative analysis indicated that the assembled Y sequence was highly similar to Y-linked contigs derived from public SRA datasets, validating assembly accuracy. Surprisingly, in Holstein bulls, there was an SRY region which was almost identical to the Hereford reference, but the rest of the Y-chromosomal regions were highly divergent between individuals, indicating substantial structural and sequence variability.This work presents a comprehensive and high-quality representation of the Holstein Y chromosome, establishing a practical pipeline for subsequent Y-chromosome studies in livestock. Beyond this, this assembly will be highly useful for the improvement of bovine reference genomes and the inclusion of Y-linked markers in genomic selection programs, especially those targeting male fertility and the evolution of sex chromosomes.

Keywords: 2026

How to Cite:

Żukowski, K., Strzała, T., Grzegorczyk, J., Słomian, D. & Skarwecka, M., (2026) “Unlocking the Bovine Y Chromosome Using a Hybrid Sequencing Approach”, World Congress on Genetics Applied to Livestock Production Digital Archive 2026(1): 2286373. doi: https://doi.org/10.31274/wcgalp.23966

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