臺灣黑熊（Ursus thibetanus formosanus）屬亞洲黑熊，為臺灣特有亞種，於1989年列為瀕臨絕種保育類動物。對於一個瀕臨絕種的物種來說，長期野外族群監測是非常重要的，然有鑑於大型哺乳類動物之稀有性及廣泛活動範圍，以非侵入性採樣方法結合分子遺傳技術建立野外族群個體鑑別資料遂為必然之研究技術。但是相關技術運用在野外臺灣黑熊尚未成熟且標準化，本研究目的乃建立臺灣黑熊個體鑑別流程。其流程的技術包含：（1）利用粒線體DNA之控制區域（D-loop 序列）前段序列（685 bp）作為分子標記來進行排遺物種鑑定以及鑑定樣本DNA品質；（2）利用8組4個核苷酸重複序列之微衛星標記進行微衛星標記訊息多樣性評估與對排遺樣本的個體鑑別；（3）結合螢光標記引子及毛管電泳以性別決定區域（sex-determining region of Y- chromosome, SRY）、zinc finger（ZF）及amelogenin（AMEL）基因作為性別鑑定的分子標記。以上三項技術皆以圈養臺灣黑熊的血液及排遺DNA做過實驗條件測試，並針對個體鑑別技術方法進行單盲試驗。
Formosan black bears (Ursus thibetanus formosanus) are a subspecies of Asian black bears in Taiwan, which are locally endangered. Long-term population monitoring is very important for conserving endangered species. Due to the rarity and wide home range of large carnivores, it is necessary to establish the information of individual identification from wild populations through non-invasive genetic sampling and molecular genetic techniques. However, the related methods have been neither well-established for the study of Formosan black bears. Therefore, the objective was to establish procedures for individual identification of wild Formosan black bears. The procedures included several technical steps. (1) Partial control region (D-loop) sequence (685 bp) of mitochondrial DNA, a molecular marker, was used to identify the species and confirm the quality of fecal DNA from fecal samples. (2) 8 polymorphic tetranucleotide repeat (GAAA) microsatellite loci from Formosan black bears were used to evaluate the polymorphic information and identify individuals from fecal samples. (3) The sex-determining region of Y-chromosome (SRY), zinc finger (ZF), and amelogenin (AMEL) gene as markers of gender determination with fluorescent label primer and capillary electrophoresis was used to identify the sex of individuals. These experiment conditions of the three techniques mentioned above were also tested by using blood and fecal DNA extracted from captive bears as templates, and a single-blinded trial was taken to verify the accuracy of individual identification.
The established procedures for individual identification were then applied to examine 251 fecal samples of wild Formosan black bears collected at Dafen in Yushan National Park during the acorn season (from October 2010 to February 2011). 176 fecal DNA samples were extracted, and the D-loop sequence were successfully amplified in 83 fecal DNA samples. Through genotyping in all of the selected 8 microsatellite loci, 35 individuals were identified. The expected probability of identity (P(ID)) and sibling probability of identity (P(ID)sib) both were lower than 0.01, and the polymorphic information content (PIC) was above 0.5. There were 24 males and 11 females from 35 individuals identified. Regarding the procedures for individual identification of bears, we suggest that the D-loop sequence could be amplified in advance and followed by gender determination for controlling the quality of fecal DNA. Besides, the gender determination with fluorescent label primers and capillary electrophoresis equipment is more accurate and sensitive than the agarose gel electrophoresis. Finally, these 2 microsatellite loci, UT3 and UT31, seems unsuitable for individual identification of fecal samples of Formosan black bears, and other appropriate markers can be further developed.