利用逢機增殖多態性 DNA 進行大冠鷲和領角鴞性別鑑定

Abstract

大冠鷲、領角鴞、鳳頭蒼鷹、黃魚鴞和草鴞為台灣保育類的鳥類，由於公母外表相似，在保育繁殖上常會因無法分辨性別而降低成功配對機會。因此本試驗目的為利用逢機增殖多態性 DNA (random amplified polymorphism DNA, RAPD) 開發新的大冠鷲與領角鴞性別鑑定分子標誌，並期能應用在鳳頭蒼鷹、黃魚鴞和草鴞等其他物種。 本試驗先分別以禽類 CHD1 (chromo-helicase-DNA-binding 1) 基因和 EE0.6 (EcoRI 0.6-kb fragment) 序列的性別特異基因引子對上述五種鳥類進行性別鑑定。另外，再以 RAPD 技術對大冠鷲和領角鴞進行指紋分析，尋找新的大冠鷲和領角鴞性別鑑別特異片段。 試驗結果顯示，大冠鷲和草鴞可利用 CHD1 基因正確鑑定性別。而 EE0.6 序列皆可對上述五種鳥類進行性別鑑定，其分別有效的引子組為：大冠鷲：A、B、C 及 D 組引子；鳳頭蒼鷹：A、C 及 D 組引子；領角鴞和黃魚鴞：E組引子；草鴞：C、D 和 F 組引子。在開發新的大冠鷲和領角鴞之性別特異片段方面，其中二隨機引子 OPBB08 及 OPAT15 分別能於雌大冠鷲增殖出約 1.2 和 1.3 kb 的性別特異片段，但於雄大冠鷲則否；一隨機引子 OPAN15 能於雌領角鴞增殖出約 1.3 kb 的性別特異片段，但於雄領角鴞則否。 根據選殖的雌性特異片段，分別設計性別鑑定引子組檢測 41 隻大冠鷲與 16 隻領角鴞。其中大冠鷲性別鑑定引子組 CseBB08-5a (Crested Serpent Eagle BB08-5a) 、CseBB08-5b、CseBB08-7 和 CseAT15 皆可於雌大冠鷲擴增一性別特異片段 (分別為553、895、194和734 bp)，可準確分辨大冠鷲性別；且其中引子組 CseBB08-7 和 CseAT15 亦會在雌雄大冠鷲擴增一內在控制片段 (分別為 543 和 734 bp)，以確定 PCR 反應操作正確性。而領角鴞性別鑑定引子組 CsoAN15-1 (Collared Scops Owl AN15-1) 和 CsoAN15-6 分別可於雌領角鴞擴增一性別特異片段 (分別為 789 和 349 bp)，亦會在雌雄領角鴞擴增一內在控制片段 (分別為 543 和 734 bp)，可有效作為領角鴞性別鑑定之引子組。大冠鷲性別鑑定引子組 CseBB08-5b 亦可跨物種限制在雌鳳頭蒼鷹擴增一條約 900 bp 片段，準確鑑定鳳頭蒼鷹性別。 本研究確立 EE0.6 序列在大冠鷲、領角鴞和黃魚鴞性別鑑定上的應用，並找到新的鑑定大冠鷲和領角鴞性別特異片段，發展出新的性別鑑定引子組可快速且準確地鑑定大冠鷲和領角鴞的性別。故利用上述引子組比起 CHD1 基因和 EE0.6 序列更能夠提供作為大冠鷲、領角鴞和鳳頭蒼鷹性別鑑定之用，以提高人工配對繁殖效率，進而達到對於這些台灣稀有的保育類野生動物的保育繁殖。此外，亦能夠對於研究這些鳥類行為、族群等研究有所幫助，提供更快速準確的性別鑑定方法。

Crested Serpent Eagle (Spilornis cheela hoya), Collared Scops Owl (Otus bakkamoena), Crested Goshawk (Accipiter trivirgatus) Tawny Fish Owl (Ketupa flavipes) and Grass Owl (Tyto longimembris) are sexually monomorphic birds. To identify their sexes by their apparent morphology is difficult. Such characteristic complicates the research on establishing breeding program and conservation for these animals. Therefore, the objective of this study was to investigate sex-specific genetic markers of Crested Serpent Eagle and Collared Scops Owl using random amplified polymorphic DNA (RAPD) fingerprinting. we used sex-specific primers of avian CHD1 (chromo-helicase-DNA-binding 1) gene and EE0.6 (EcoRI 0.6-kb fragment) sequence to identify these five birds sex. Besides, 120 random primer sequences were from RAPD 10 mers kits (Operon Biotechnologies) and used for RAPD fingerprinting to search for sex-specific fragments of Crested Serpent Eagle and Collared Scops Owl. The results showed that CHD1 gene could correctly identify sex in Crested Serpent Eagle and Grass Owl only. Furthermore, EE0.6 sequence could also correctly distinguish sex of these birds, the useful primers sets are A, C and D for Crested Goshawk; E. primers sets for Collared Scops Owl and Tawny Fish Owl; C、D and F for Grass Owl. Moreover, The RAPD results showed that OPBB08 and OPAT15 random primers generated approximately 1.2 and 1.3 kb sex-specific fragments in female Crested Serpent Eagle and OPAN15 random primer generated a 1.3 kb sex-specific fragment in female Collared Scops Owl. The sex-specific fragments were constructed into the vectors for sequencing. According to the sex-specific fragments sequences, several primers were designed for sex identification in 41 Crested Serpent Eagle and 16 Collared Scops Owl respectively. The CseBB08-5a (Crested serpent eagle BB08-5a) and CseBB08-5b primers amplified 553 and 895 bp fragment respectivily in all female Crested Serpent Eagle but not in the male; the CseBB08-7 primers amplified a 194 bp fragment in all female Crested Serpent Eagles and a 543 bp fragment in all females and males; the CseAT15 primers amplified a 734 bp fragment in all female Crested Serpent Eagle and a 147 bp fragment in all females and males. The CsoAN15-6 (Collared scops owl AN15-6) primers from Collared Scops Owl genomic sequences amplified a 349 bp fragment in all female Collared Scops Owl and a 796 bp fragment in all females and males. The common amplified fragment in both male and female birds can be used as an internal control. In addition, we tested those primers on other species and found that CseBB08-5b of Crested Serpent Eagle could also be used for sex identification in Crested Goshawk. In this study, we have identified novel sex-specific DNA markers of Crested Serpent Eagle, Collared Scops Owl and Crested Goshawk. The use of these sex specific DNA markers would help in correctly identifying sexes before matching pair in a captive environment.