Isolation and Sequencing of the Y Chromosome in Mediterranean River Buffalo Using Laser Microdissection-Based NGS

Background/Objectives: The Y chromosome plays a crucial role in male fertility, sex determination, and spermatogenesis, yet it remains poorly characterized in Mediterranean river buffalo (Bubalus bubalis, 2n = 50) because of its high repeat content, extensive heterochromatin, and complex palindromic structures. Although a chromosome-level Y assembly is available for swamp buffalo (2n = 48), no equivalent reference exists for the river type. Methods: To address this gap, Y chromosomes from 10 Mediterranean buffalo bulls were isolated by laser microdissection following peripheral blood culture and whole-chromosome amplification. Probe specificity was verified by FISH, and amplified Y chromosomes were sequenced using Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA). Sequencing data were assembled and analysed through de novo assembly, repeat identification, sequence alignment, and variant detection. Comparative analyses included alignment to the swamp buffalo Y chromosome and annotation of Y-linked genes using the Bos taurus reference genome. Results: FISH confirmed the specificity of the isolated material, showing strong signals on the Y chromosome and on X/Y PAR and heterochromatic regions. Sequencing generated over 240 million paired-end reads, and de novo assembly produced 566,815 contigs. Repeat analysis identified 3.91% repetitive elements, mainly SINEs, while variant calling detected more than 23,000 variants. Comparative analyses mapped several contigs to the swamp buffalo Y chromosome and Y-linked genes. Annotation against the B. taurus genome identified 26 unique genes, including homologs shared with the X chromosome, and revealed MSY gene duplications, including 10 copies of TSPY and 3 of HSFY. Conclusions: These findings show that laser microdissection with NGS enables effective access to the buffalo Y chromosome, representing a milestone in the characterization of the river type genome, and providing a basis for studies on buffalo male fertility and breeding programs.