利用分子標記建立臺灣黑熊（Ursus thibetanus formosanus）個體鑑別流程與應用

Establish the Procedure and Application for Individual Identification of Formosan Black Bears (Ursus Thibetanus Formosanus) Based on Molecular Markers

陳昇衛(Shengwei Chen)

指導教授 ： 黃美秀 ； 朱有田

國立屏東科技大學/農學院/野生動物保育研究所/碩士(2014年)

https://doi.org/10.6346/NPUST.2014.00023

摘要

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。
將建立好的個體鑑別流程應用於玉山國家公園大分地區自2010年10至隔年2月共採集251個臺灣黑熊排遺樣本，從中萃取出176個排遺DNA樣本，其中83個排遺DNA樣本的控制區域序列成功被增幅出來。藉由8組微衛星標記之基因型判定，又從這83個排遺DNA樣本共鑑別出35個個體，其期望個體鑑別率（P(ID)）以及手足個體鑑別率（P(ID)sib）皆低於0.01。還有此8組微衛星基因座的多型性資訊含量達0.5以上。個鑑別出的這35個個體，共鑑定出24個雄性及11個雌性個體。最後本研究建議臺灣黑熊個體鑑別流程：（1）先以D-loop序列擴增，再以性別鑑定，來控管樣本DNA品質，可節省實驗成本及時間。（2）結合螢光標記引子與毛細管電泳設備，增進性別鑑定的準確度及靈敏度。（3）UT3及UT31微衛星基因座可能不適合利用臺灣黑熊在排遺樣本上，另外參考適合的微衛星基因座，並加以測試。

關鍵字：臺灣黑熊、非侵入性採樣、個體鑑別、控制區域、微衛星標記、
性別鑑定

關鍵字

臺灣黑熊 ； 非侵入性採樣 ； 個體鑑別 ； 控制區域 ； 微衛星標記 ； 性別鑑定

並列摘要

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.

Keywords: Ursus thibetanus formosanus, non-invasive genetic sampling, individual identification, control region, microsatellite loci, gender determination

並列關鍵字

Ursus thibetanus formosanus ； non-invasive genetic sampling ； individual identification ； control region ； microsatellite loci ； gender determination

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