Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/25578
標題: 以不同性別專一引子鑑定鸚鵡性別以及利用cytochrome b與12S ribosomal RNA序列鑑定鸚鵡種別
Sexing parrots by different sex-specific primers and application of cytochrome b and 12S ribosomal RNA sequences to identity the parrot species
作者: 廖秭妤
Liao, Tz-Yu
關鍵字: 鸚鵡;parrot;性別鑑定;種別鑑定;sex determination;species determination
出版社: 動物科學系所
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摘要: 
世界上所有鳥類中約有60%以上無法利用外表辨識雌雄,此稱為單型性(monomorphic),意即體型大小相當,羽色無明顯差異。其中鸚形目(Psittaciformes)鳥類約有350種左右,能以外表辨別雌雄的鸚鵡僅60幾種,故幾乎有80%以上的鸚鵡為單型性,因此使用目測觀察鸚鵡的性別為困難之事。利用聚合酶連鎖反應(polymerase chain reaction, PCR),針對chromodomain helicase DNA binding protein 1(CHD-1)基因設計特定引子如P1/P2/P3、P4/P5、P2/P8、1272H/1237L、2550F/2718R與primer1/2等,並利用這些引子進行PCR擴增以檢視雌雄相異之片段以做為判別。於本試驗中,利用引子P1/P2/P3配合限制酵素Dde I切割、引子2550F/2718R與引子primer1/2擴增性別特異性片段,成功檢驗19種鸚鵡性別。粒線體DNA(mitochondria DNA, mtDNA)常用於種別鑑定,本試驗挑選Cytb以及12S rRNA作為粒線體種別鑑定基因,並利用National Centre for Biotechnology Information(NCBI)中之BLASTn系統與現有之database做比對,依據比對結果,序列最大相似度高於98%可做為種別確認之指標。本試驗萃取26種鸚鵡之粒線體DNA進行種別鑑定,其中使用Cyt b粒線體DNA成功鑑定19種鸚鵡種別;而使用12S rRNA則成功鑑定其中13種鸚鵡種別。因此,若NCBI中之databse種別數量夠齊全時,使用Cyt b及12S rRNA鑑定種別,不失為容易且精準之方式。除了利用粒線體DNA外,本試驗中並嘗試random amplified polymorphic DNA (RAPD)方式鑑定17種鸚鵡種別,利用40支逢機引子逢機擴增17種鸚鵡之基因組DNA,並從中尋找種別特異性片段。於本試驗中,逢機引子OPH-17在紅腹金剛鸚鵡(Orthopsittaca manilata)中增殖出一片段與膠片上其他種別不相同,經純化、定序後,卻發現從同一個體所擴增之8段序列間,彼此沒有顯著相似度,反覆操作過後再現性不高。因此,本試驗並沒有找到能做為種別特異性標誌之序列,於穩定操作之技術仍需改進。

Approximately 60% of avian species are sexually monomorphic, and most of the parrots could not be sexed by feather colors or any other morphological characteristics in appearances. It has been reported that specific DNA sequences could be amplified by using polymerase chain reaction (PCR) with the specific primers of chromodomain helicase DNA binding protein 1 (CHD-1) gene. In birds, there are two genes related to CHD-1, CHD-W and CHD-Z, which are located in chromosome W and Z, respectively. Since these two genes are evolutionarily conserved, and their sequences vary sex specific manner, application of the differences between the nucleotide sequences could be used for sex identification. Multiple primers, including P1/P2/P3, P4/P5, P2/P8, 2550F/2718R, primer1/2 and 1272H/1237L have been designed according to the sequence of introns or exons in CHD-1 gene. By using different combinations of these specific primers for PCR, 19 species of examined parrots could be successfully determined in this study. Amplification and sequencing fragments of mitochondrial DNA (mtDNA) genes, such as cytochrome oxidese I (COI), cytochrome b (Cytb), 12S ribosomal RNA (12S) and 16S ribosomal RNA (16S) are widely used for species identification. In this study, Cytb and 12S were tried to identify different species of parrots. Comparisons of sequences were conducted by using the BLASTn portal within the National Centre for Biotechnology Information (NCBI) database. The threshold value for sequence similarities of Cytb or 12S rRNA genes between analyzed samples and database is 98%. Twenty-six species of parrots were analyzed in this study. Results showed that the sequences of Cytb gene in 19 species of examined parrots could be found in NCBI. Furthermore, 13 species of analyzed parrots have the high similarity in 12S gene published in database. However, 2 species were found neither the sequences of cyt b nor 12S rRNA published in NCBI. Hence, in addition by comparison of mtDNA to identify parrot species is an accurate method, uploading the unpublished sequences would help the integrity of database. In addition to use mitochondrial DNA for species identification, random amplified polymorphic DNA (RAPD) were used to identify 17 different parrot species. In this study, 40 random primers were employed. One of these primer, OPH-17, was successfully amplified a specific band in Orthopsittaca manilata. After analyzing 8 sequences amplified from the same individual, there are no significant correlations among these 8 sequences. Thus, it is not possible to find species specific sequence using the 40 random primers in this study. More stricted conditions way are further tried to inprove reproducibility.
URI: http://hdl.handle.net/11455/25578
其他識別: U0005-0502201316422000
Appears in Collections:動物科學系

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