Length difference between equine ZFX and ZFY genes and its application for molecular sex determination

Abstract

Purpose

We analyzed the sex chromosome-encoding ZFX-ZFY genes and tested molecular sexing using the amplification patterns of intron 9 of ZFX-ZFY in the horse.

Methods and results

The amplification of the ZFX-ZFY produced two distinct patterns, reflecting sexual dimorphism based on a length difference between the X and Y chromosomes. The amplification products from foals showed two distinct bands: one was common to all foals and mares, indicating that this band was amplified from ZFX, while the other was specific to some foals, indicating that it was from ZFY. The result based on the PCR assay was identical to the results of amplification of the Y chromosome-specific SRY gene and those of investigations of the phenotypic gender in three different horse populations.

Conclusion

We suggest that this PCR strategy for determining sexes by comparing the amplification patterns of ZFX-ZFY genes is a convenient and precise method for discriminating sexes in horses.

Most mammals have an X-Y chromosome sex determination system. Sexual differentiation is determined primarily by the presence or absence of the Y chromosome, and the sex determining region Y (SRY) gene plays a key role in inducing male development, while inhibiting female differentiation [1–3].

Control of the sex ratio of animals is desirable in the livestock industry. In mammals, the X-Y homologous sequences for the X- and Y-linked genes zinc finger, amelogenin-X and -Y (AMELX-AMELY), and SMCX/SMCY have been compared [4–6]. PCR assays for molecular sexing are often more useful because they provide sensitive, precise, rapid, and reliable results. To date, PCR-based sex typing has been used for embryonic sexing in cattle, mice, and pigs [7–12]. To establish a rapid and precise molecular sexing method using PCR amplification for horse breeding, we analyzed the sequence characteristics and sexual dimorphism of the ZFX and ZFY genes and compared the results to those obtained from Y chromosome-specific SRY gene amplification and phenotypic investigation.

Total of 128 genomic DNA samples were collected from three horse populations (61 Thoroughbreds, 20 Tsushima native horses from Japan, and 47 Jeju native horses from South Korea). To amplify the intron 9 of ZFX and ZFY genes, the primer pair was designed from exon 8 and 10 of the sequences of human and cattle [13]. The primer sequences for the amplification of ZFX and ZFY genes were as follows: eqZF9iF, 5′-AAA TCA AAA CCT TCA TGC CAA T-3′ and eqZF9iR, 5′- TTC CGG TTT TCA ATT CCA TC-3′. To compare the ZFX-ZFY amplification patterns to those from the Y chromosome specific SRY, which was amplified with SRY-specific primers [14] eqSRYf, 5′- TGC TAT GTC CAG AGT ATC CAA CA -3′ and eqSRYr 5′-TGA GAA AGT CCG GAG GGT AA-3′. PCR reaction was carried out in 25-μl volumes, containing distilled water, 2.5 μl of reaction buffer, 200 μM of dNTPs, 2.0 units of Taq DNA polymerase (Promega, USA), 10 mM of each primer, and about 100 ng of total DNA. Amplification was performed using the PTC-200 thermal cycler (MJ Research, USA) under the following conditions: an initial 2-min denaturation at 94°C was followed by 35 cycles of 45 s at 94°C, 60 s at 58°C, and 60 s at 72°C. The PCR products for ZFX-ZFY and SRY gene were separated on 2.0% agarose gels containing ethidium bromide, and purified each band using a QIAEX II Gel Extraction Kit (Qiagen, USA). After purification of PCR products, for each homozygous genotype the PCR amplified fragments from three individuals were selected and sequenced using ET Dye-Terminator Sequencing kit (Amersham Pharmacia, USA). To compare the intron 9 sequences of equine ZFX and ZFY with those from cattle, we also determined the intron 9 sequences of ZFX and ZFY from cattle. The nucleotide sequences for intron 9 and flanking regions obtained in this study were deposited in GenBank database under accession numbers DQ179227 (bovine ZFY), DQ179228 (bovine ZFX), DQ179229 (equine ZFY), and DQ179230 (equine ZFX). To scan the repeat DNA elements, all sequences were analyzed using web programs CENSOR (http://www.girinst.org/censor) and RepeatMasker (http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker).

The amplified PCR product for ZFX-ZFY genes showed two distinct band patterns. Those from some foals had heteroduplex bands (553- and 604-bp), while those from mares had a single 604-bp band, respectively (Fig. 1a). The band that foals shared with mares is likely that amplified from the X chromosome, while the foal-specific band could be amplified from the Y chromosome. The results of amplifications of the intron 9 flanking region of the ZFX-ZFY genes were identical to those based on investigations of phenotypic gender and a PCR assay of the Y chromosome-specific SRY gene. Figure 1b shows that the foals had an SRY band, while the mares did not. Since the SRY gene is specific to the Y chromosome, the presence of an SRY gene means that the DNA was isolated from animals possessing a Y chromosome. In other words, without exception, all individuals in three horse populations (Thoroughbred, Tsushina native horse, and Jeju native horse), that possessed the 553-bp ZFY PCR product also had the SRY PCR product. The DNA sequences of the purified PCR products, which included the complete intron 9 and flanking exons 9 and 10, were determined. Two nucleotide substitutions were found on exon 9 that did not involve an amino acid substitution or length difference. No mutations were found on the exon 10 partial sequences in ZFX and ZFY. However, the intron 9 sequence of ZFX was 51-bp longer than that of ZFY, making it readily distinguishable on agarose gels, and various nucleotide substitutions were also identified. The nucleotide alignment shows the nucleotide substitutions and indels of the sequences of the two partial exons and intron 9 of ZFX and ZFY (Fig. 2). The sequence characteristics of the ZFX and ZFY genes in cattle and pigs, while the horse sequence of intron 9 of ZFX has not been reported [13]. To our knowledge, ours is the first study to determine the sequence of intron 9 of ZFX and compare it to that of ZFY. The length variation is due to multiple indels between the two sequences. The presence of interspersed repeat elements on the intronic regions of the ZFX-ZFY genes causes marked length variation among mammalian species [13, 15]. Using the programs RepeatMasker and CENSOR, we identified fragments of the interspersed repeat sequence. A short interspersed nuclear element (SINE)-like sequence was found in cattle ZFY intron 9 and two Alu-like sequences are present in the human ZFY intron 9. However, no retroelement-derived sequence was found in intron 9 flanking regions of the equine ZFX-ZFY genes. This result is similar to previous reports documenting the absence of a SINE-like sequence on intron 7 of the equine ZFX and ZFY gene, in contrast to the human and bovine ZFY intron 7 [13].

Fig. 1.

PCR amplification patterns of intron 9 flanking region of equine ZFX and ZFY genes a and SRY gene b. Arrows indicate the lengths of each PCR product

Fig. 2.

Comparison of the sequences of intron 9 flanking region of equine ZFX and ZFY genes with those of bovine genome. Asterisks and dots indicate the identical sequence comparing to that of bovine ZFY. Dashes indicate nucleotide deletion. Rectangles in exon 9 are nucleotide substitution mutations between equine ZFX and ZFY. Underlined sequences are SINE-like element Bov-tA found in intron 9 of bovine ZFY using RepeatMasker and CENSOR program comparing to that previously reported (GenBank Accession no. of X46124)

The molecular sexing of horses had been described using the SRY and AMELX-AMELY genes [16]. Stallions have a Y-specific SRY band and AMELX-AMELY heteroduplex bands, while mares only have homoduplex AMELX. The detectable dimorphic patterns had been also documented in horses in the ZFX and ZFY genes using HaeIII PCR-RFLP and subsequent DNA sequencing [5]. However in this study, we found a length difference, despite the relationship of the insertion/deletion of the SINE element, more rapidly and easily using a simple amplification and electrophoresis. Moreover, the result using this molecular approach for sex determination of horses was identical to the results of phenotypic investigations and another PCR assay based on the amplification of the Y chromosome-specific SRY gene (Fig. 1b). All horse populations tested had no XY females; therefore, phenotypic males and females were distinguished using both PCR amplification tests using the SRY and ZFX-ZFY genes. Our analysis of the intronic organization showing sexual dimorphism on the ZFX and ZFY genes will contribute to molecular sexing as a useful method for selective breeding and for understanding the evolution of the horse genome.