Male sex determination of most mammals is sry-dependent. However, there appears to be no sry gene in Microtus mandarinus mandarinus, one of a few rodents that have lost sry but retain fertile males and females (Chen et al., 2008). In 2006, it was found that mutation in the R-spondin1 gene (RSPO1) was linked to female-to-male sex reversal (Parma et al., 2006). We chose RSPO1 as a candidate gene for sex determination in M. mandarinus mandarinus because one allele of RSPO1 appeared to be linked to male sex. Then, we asked if the RSPO1 allele is localized on sex chromosomes using fluorescence in situ hybridization (FISH) techniques. Experimental animals included five males and four females. They belonged to two pedigrees with two generations (Fig. 1B). The following primers for PCR amplification of a RSPO1 fragment corresponding to a part of RSPO1 mRNA 3’UTR were used: forward 5’-CATCCACCCACCCATCCAACTATCC-3’; reverse 5’-ATCTCCGACAATCAAGTCTCACAGC-3’. Polymerase chain reactions were performed according to standard procedure. Ten microlitres of each amplified product was analysed by the polymerase chain reaction-single strand conformation polymorphism. The remaining portion of each amplified product was cloned into a vector and sequenced. After each amplified product was sequenced, it was found that all four female animals were homozygous while allele (CG) was associated with a heterozygous male (Fig. 1A, B).

M. mandarinus mandarinus chromosomes have no gene homologous to any gene on the Y chromosome of related species (Zhu et al., 2004). Thus, some genes on the Y chromosome of M. mandarinus mandarinus can be either homologous to genes on autosomal chromosomes, or divergent from any genes on the Y chromosome of sister species. We tested if one allele of RSPO1 is localized on the sex chromosomes of M. mandarinus mandarinus.