Cloning Of Sex-linked Gene Of The Tufted Deer And Identification Of Its Sex Chromosomes

There is extremely polymorphic karyotype in tufted deer.So far,six karyotypes have been reported.Firstly, two karyotypes, 2n=48 3 and 2n=47 , were reported by Zhang Xiran et al.and the cell strain was established (designated KIZ-81A).After that, another two karyotypes were reported, one was 2n=46? by Wang Zongren et al., the other was 2n=47 , by Shi Liming et al. . Recently, two new karyotypes were observed in our lab, one was 2n=48 (1998), the other was 2n=48 (2002). Analysis of the polymorphism,different chromosome number, morphism and length were showed. These changes focus in a few chromosomes, which are just the unpaired heteromorphic chromosomes with abundant heterochromatin.According to the human sex differentiation related ZFY and ZFX genes, a pair of primers was designed, and DNA fragments were amplified from the genomic DNA of male or female tufted deer. Subsequently the amplified fragments were cloned into the vector pMD18T and were sequenced. It is found that the sequences of ZFY gene and ZFX gene have 91% homology. Based on the different nucleotides, restriction site of Avail was found to be specific to ZFX gene. The results show that the combination of PCR with Avail digestion is a simple and sensitive way to identify the tufted deer sex.Using cloning of gene, chromosomal micromanuipulation, DOP-PCR, FISH and so on, some sex-linked gene of the tufted deer were cloned.For example, ZFX/ZFY, SRY, G6PD and DYZ repeat sequence. Subsequently, X chromosome and Y chromosome were identified by DOP-PCR and normal PCR. In addition, the human X-specific and Y-specific whole chromosome painting probes were hybridized onto metaphase spreads obtained from male of Elaphodus cephalophus.No signals were detected, which indicates the phylogenetic relationship between human and the tufted deer is not close.