尿冰在梅花鹿种群研究中的价值

Usability of Urine Ice Samples for Population Research on Sika Deer

冬季的野外调查可以收集到动物排在雪地或地面上的尿冰。为评价尿冰在种群生态学研究中的价值，本研究从野外收集15份梅花鹿（CervusNippon）尿冰、从动物园收集2份已知性别的梅花鹿尿冰，以实验室保存的2份已知性别的梅花鹿肌肉样品为对照，采用凝胶回收试剂盒提取DNA，通过cytb和12SrRNA两个线粒体基因片段和AMEL、SRY两个核基因片段的PCR扩增，评价了尿冰DNA的可用性。结果表明：从1mL融化的尿冰中可提取出650ng的DNA。线粒体DNA分别成功扩增了0．472～1．2kb的片段，核基因成功扩增了225q25bp的片段。利用AMEL、SRY、ZFX3个基因片段进行性别鉴定时，已知性别样品的鉴定结果均完全正确。野外尿冰样品在AMEL基因片段（大小为282bp／225bp）的扩增成功率为88．2％，在MTI／DSRYl-H和ZFX1-L／ZFX1-H进行复合扩增时的成功率为94．1％。上述结果说明，尿冰DNA完全可以满足基于mtDNA和核DNA的遗传学分析，是有价值的DNA来源。

Urine can be rapidly frozen after urination in winter and collectedconveniently during field survey of population. In order to evaluate the usability of urine ice for population ecological research, we collected 15 urine ice samples left behind the sika deer （ Cervus nippon） in the wild, and 2 such samples of a female and a male sika deer collected in a zoo, too. 2 muscle tissue samples of a female and a male sika deer were used as standard reference. DNA was extracted using a commercial Agar- ose gel recovery kit. Fragments of cyt b gene and 12S rRNA gene on mtDNA and AMEL gene, SRY gene and ZFX gene were amplified. Results showed that up to 650 ng DNA could be extracted from lmL thawed urine ice. Fragment lengths of cyt b and 12S rRNA genes were 0.472 kb to 1.2 kb, re- spectively. The amplified fragments of AMEL gene, SRY gene and ZFX gene were from 225 bp to 425 bp in length. Sexing of urine ice samples of known sexes agreed to tissue samples. Successful rate of sexing of wild urine ice samples was 88.2% for AMEL gene and 94.1% for multiplex amplifi- cation of SRY gene. The results suggested urine ice could provide sufficient DNA for genetic analysis based on both mtDNA and nuclear DNA, and was usable for ecological studies of wild populations.