Exclusion of Sall 4 as the sex-determining gene in the Mandarin vole Microtus mandarinus mandarinus

In previous studies, we have shown that the Sry HMG-box is absent in Microtus mandarinus mandarinus (M. m. mandarinus), suggesting that sex determination of M. m. mandarinus is independent of the Sry gene. We amplified a 312 bp fragment within exon 2 of the Sall4 gene in the mouse and M. m. mandarinus using polymerase chain reaction (PCR) and detected Sall4 using fluorescence in situ hybridization (FISH) technique. The probes for the Sall4 gene were labeled with digoxigenin using PCR and hybridized to chromosomes and interphase nuclei of the mouse and M. m. mandarinus. Our results suggested that Sall4 exists in the genome of male and female M. m. mandarinus, and the sequence within exon 2 of the gene is the same in the mouse and M. m. mandarinus. The results also showed that Sall4 is localized on chromosome 6 in M. m. mandarinus. As they are the sex chromosomes in M. m. mandarinus, the results excluded the Sall4 gene from being the testis-determining factor in this species. We propose that in M. m. mandarinus, sex determination is controlled by another yet unknown gene on the sex chromosomes.