Sex-determining region y(SRY) and amelogenin y (AMELY) genes amplification in pregnant mare maternal plasma and relation between these genes and sex

In this study, peripheral blood samples were taken from 24 mares (13 female fetuses, 11 male fetus pregnancies) at each month of pregnancy (1-11 months), and the amplification efficiencies of SRY and AMELY genes were compared. The mares included in the study were of Arabian racehorses and their ages ranged from 5-15 years. Body condition scores (BCS) ranged from 5-7. Study groups were classified according to newborn sex, which was confirmed postnatally. After cfDNA isolation, their densities were found to vary between 5.9-64 ng/µl. As a result of statistical analysis, it was observed that there was no significant difference between Ct values (amplification amount) of AMELY gene depending on gender in pregnancies carrying male and female fetuses (p>0.05). Amplification specificity was checked for each sample in the dissolution curve analyzes performed after the qRT-PCR method. In this analysis, the detectability of the investigated genes related to fetal sex was determined in the maternal cfDNA samples studied. There is high sequence similarity between the AMELY gene on the Y chromosome and the AMELX gene on the X chromosome. However, unlike AMELX, a 24-base deletion in the AMELY gene sequence enabled the two gene products to be separated from each other after PCR. Similarly, when the primer design specific to the SRY gene was designed, in-silico analyzes using different algorithms confirmed that only the SRY sequence was amplified, while amplification was observed in females after qRT-PCR. However, in the dissolution curve analyzes we performed, the amplification of the female homologous region and the amplification of the actually targeted SRY gene were clearly identified and confirmed. When the resolution curve analysis of the AMELY and SRY genes of male and female fetuses is considered at all gestational months, the detection power for both genes increases from the 5th gestational month and the highest detection rate is between 9-10. observed between months. In this project, the efficacy of AMELY and SRY genes in sex determination was compared using fetal cfDNAs from maternal circulation at all gestational months (1-11 months) in mares. We believe that the use of multiple sex-specific markers in the determination of fetal sex from maternal serum in pregnant mares will increase the accuracy of the test.