Molecular Method Based on Hydrolysis Probe Assays to Identify the Sex Chromosomes of Iberian Desman (Galemys pyrenaicus) Using Non-Invasive Sampling.

Abstract

Desmans belong to the subfamily Desmaninae, which are members of the family Talpidae. Desmans and moles show limited sexual dimorphism, making unclear sex discrimination by phenotypic assessment. The Iberian desman (Galemys pyrenaicus) is an endangered species with a severe population decline. Knowledge of sex and sex ratio is essential for conservation and management. Based on these arguments and although previous conventional PCR studies amplifying DBX/DBY genes were relatively successful in sexing the desman, high-resolution sex-specific PCR has been requested. All these reasons encouraged us to develop new species-specific RT-qPCR assays by TaqMan probes to determine the sex in desman, especially with genetic material from non-invasive samples. Accordingly, efficiency, limit of detection (LOD), specificity, and DNA analysis from faeces were verified. The target genes DBX and DBY were amplified with gDNA from both sexes, with Y-chromosome consistently absent in the female. Despite the modest efficiency, regression analysis (R² > 0.999) indicated a linear range of the DBX and DBY assays extending from 20 to 0.2 ng/µL DNA. LOD analyses estimated that twice as much gDNA was needed in males as in females for DBX detection. Paradoxically, the Y-chromosome required three times as much gDNA as the X-chromosome using a male sample. Therefore, an unexpected dosage imbalance in the genome in favour of the X chromosome was discussed in light of an apparent multicopy nature of the DBX gene and with a sexing success rate of 49.9% of the non-invasive samples, supporting Fisher's principle for the mammalian XX/XY sex system, as expected.