Application of CHD1 Gene and EE0.6 Sequences to Identify Sexes of Several Protected Bird Species in Taiwan

Abstract

Many bird species, for example: Crested Serpent Eagle (Spilornis cheela hoya), Collared Scops (Owl Otus bakkamoena), Tawny Fish Owl (Ketupa flavipes), Crested Goshawk (Accipiter trivirgatus), and Grass Owl (Tyto longimembris)... etc, are monomorphic, which is difficult to identify their sex simply by their outward appearance. Especially for those monomorphic endangered species, finding an effective tool to identify their sex beside outward appearance is needed for further captive breeding programs or other conservation plans. In this study, we collected samples of Black Swan (Cygmus atratus) and Nicobar Pigeon (Caloenas nicobarica), two aviaries introduced monomorphic species served as control group, and Crested Serpent Eagle, Collared Scops Owl, Tawny Fish Owl, Crested Goshawk, and Grass Owl, five protected monomorphic species in Taiwan. We used sex-specific primers of avian CHD1 (chromo-helicase-DNA-binding) gene and EE0.6 (EcoRI 0.6-kb fragment) sequences to identify the sex of these birds. The results showed that CHD1 gene primers could be used to correctly identify the sex of Black Swans, Nicobar Pigeons and Crested Serpent Eagles, but it could not be used to correctly identify sex in Collared Scops Owls, Tawny Fish Owls, and Crested Goshawks. In the sex identification using EE0.6 sequence fragment, A, C, D and E primer sets could be used for sexing Black Swans; A, B, C, and D primer sets could be used for sexing Crested Serpent Eagles; and E primer set could be used for sexing Nicobar Pigeons and the two owl species. Correct determination of sex is the first step if a captive breeding measure is required. We have demonstrated that several of the existing primer sets can be used for sex determination of several captive breeding and indigenous bird species.

在臺灣許多保育鳥類，例如：大冠鷲（Crested Serpent Eagle, Spilornis cheela hoya）、領角鴞（Collared Scops Owl, Otus bakkamoena）、黃魚鴞（Tawny Fish Owl, Ketupa flavipes）、鳳頭蒼鷹（Crested Goshawk, Accipiter trivirgatus）和草鴞（Grass Owl, Tyto longimembris）等，由於屬於單外表型（monomorphic），公母外表相似，因此常無法由其外貌型態的不同，而判定其公母性別，故除了以傳統外貌分辨公母的方法，發展有效率的方法鑑別這些保育鳥類的公母，對於其保育及繁殖計畫極為重要。本研究以黑天鵝（Black Swan, Cygmus atratus）及綠簑鴿（Nicobar Pigeon, Caloenas nicobarica）兩種鳥類作為對照組，設計針對鳥類CHD1基因及EE0.6 DNA 序列，利用聚合酵素連鎖反應法可產生不同片段大小之特異性引子，進行大冠鷲、領角鴞、黃魚鴞、鳳頭蒼鷹和草鴞公母性別鑑定。試驗結果顯示，利用所設計CHD1 基因特異性引子，針對黑天鵝、綠簑鴿、大冠鷲及草鴞可準確進行公母鑑別，但是領角鴞、黃魚鴞及鳳頭蒼鷹等則無法利用此基因檢測進行性別鑑定。另在EE0.6 DNA序列特異性引子檢測結果顯示，利用A、C、D及E引子組可進行黑天鵝公母鑑別；A、B、C及D引子組可進行大冠鷲公母鑑別；E引子組可進行綠簑鴿、領角鴞和黃魚鴞公母鑑別；C、D及F引子組可進行草鴞公母鑑別。正確的性別鑑定，為進行保育類鳥類保育繁殖計畫最重要的第一步，本研究結果確立可利用CHD1 基因及 EE0.6 DNA序列特異性引子，進行上述保育鳥類之公母鑑別，而準確性高的保育鳥類公母鑑別分子檢測技術的建立，可提高人工配對繁殖效率，進而提昇對於上述臺灣稀有的保育鳥類的保育繁殖效率。