TSPY CN relative to the single copy AR gene, as determined by qPCR.

<p>CN was measured at three loci (Exon 1, 3, 5) in four breeds. Columns represent the average value (±SD) of 5 animals from the same breed. Different letters within efficiency calculation methods (STD or LinRegPCR) represent statistical significance according to two-way ANOVA (p<0.05).</p

The Porcine TSPY Gene Is Tricopy but Not a Copy Number Variant

<div><p>The testis-specific protein Y-encoded (TSPY) gene is situated on the mammalian Y-chromosome and exhibits some remarkable biological characteristics. It has the highest known copy number (CN) of all protein coding genes in the human and bovine genomes (up to 74 and 200, respectively) and also shows high individual variability. Although the biological function of TSPY has not yet been elucidated, its specific expression in the testis and several identified binding domains within the protein suggests roles in male reproduction. Here we describe the porcine TSPY, as a multicopy gene with three copies located on the short arm of the Y-chromosome with no variation at three exon loci among 20 animals of normal reproductive health from four breeds of domestic pigs (Piétrain, Landrace, Duroc and Yorkshire). To further investigate the speculation that porcine TSPY is not a copy number variant, we have included five Low-fertility boars and five boars with exceptional High-fertility records. Interestingly, there was no difference between the High- and Low-fertile groups, but we detected slightly lower TSPY CN at all three exons (2.56-2.85) in both groups, as compared to normal animals, which could be attributed to technical variability or somatic mosaicism. The results are based on both relative quantitative real-time PCR (qPCR) and droplet digital PCR (ddPCR). Chromosomal localization of the porcine TSPY was done using fluorescence in situ hybridization (FISH) with gene specific PCR probes.</p></div

Y-chromosome specific fluorescence in situ hybridization signal of FISH probe#1–5 with Tyramide signal detection on a boar metaphase.

<p>DAPI stained metaphase chromosomes are converted grayscale and AF594 hybridization signal on the short arm of the Y-chromosome is red.</p

Schematic view of the porcine TSPY gene (exons 1–5 in grey) and location of primers (green), TaqMan probes (red), PflMI enzyme cut positions (blue) and FISH probes (purple).

<p>Schematic view of the porcine TSPY gene (exons 1–5 in grey) and location of primers (green), TaqMan probes (red), PflMI enzyme cut positions (blue) and FISH probes (purple).</p

TSPY CN by ddPCR at exons 1, 3 and 5 in normal animals from four breeds, High-fertile and Low-fertile animals.

<p>Columns represent the average value (±SD) of 5 animals. Letters mark values that are statistically different (p<0.05).</p