臺灣博碩士論文加值系統

鸚鵡在鳥類分類學上屬鸚形目（Psittaciformes），主要分布於南北緯度之間，目前已知約有374種鸚鵡分佈於世界各地，而全球其他許多國家雖然非鸚鵡原棲息地，但由於其美麗的羽色以及高智商能力，因此鸚鵡在世界各國仍為極受歡迎之伴侶動物之一。由於除了桃面情侶鸚鵡、虎皮鸚鵡、玄鳳鸚鵡以及玫瑰環頸鸚鵡外，其餘300多種鸚鵡皆屬於華盛頓公約（Convention on International Trade in Endangered Species of Wild Fauna and Flora, CITES）附錄一或附錄二之列管動物，因此，為了對應走私案例，開發能在走私鳥蛋或少量跡證的情況下作出精確結果的分子種別鑑定法有其必要性，而應用聚合酶連鎖反應（Polymerase chain reaction; PCR）為基礎之分子鑑定技術，為合理之第一選擇，其中，逢機增殖多型性DNA標誌法（random amplified polymorphic DNA, RAPD）是以逢機序列之短核苷酸引子（primers），於低煉合溫度下進行PCR反應，使其盡量產生多型性環帶（bands），並且不需任何樣品資訊。因此，本試驗共使用了6組，120種逢機引子以 RAPD方式對8-15種鸚鵡進行種別分析，共從中取得52組可能是種別特異序列環帶。分析以逢機引子OPD15自綠頰錐尾鸚鵡（Pyrrhura molinae）增殖之437 bp序列中，分別選取首尾序列26 bp與27 bp設計序列專一增殖區域（Sequence characterized amplified region, SCAR）引子，稱之為GCC-F/R，而以逢機引子OPG7自和尚鸚鵡（Myiopsitta monachus）增殖之529 bp序列中，分別選取首尾序列20 bp設計SCAR引子，稱之為Monk-F/R，再以引子GCC-F/R對包含綠頰錐尾鸚鵡在內之15種鸚鵡以及不同個體之16隻綠頰錐尾鸚鵡進行PCR增殖，而以引子Monk-F/R對包含和尚鸚鵡在內的15種鸚鵡以及不同個體的12隻和尚鸚鵡進行PCR增殖，其結果皆顯示引子GCC-F/R與Monk-F/R之PCR產物具有種別專一片段，而其他種鸚鵡則均無。因此，本試驗設計之此二對新引子可分別對綠頰錐尾鸚鵡以及和尚鸚鵡進行種別鑑定。

In taxonomy, parrots belong to gegnus Psittaciformes and distribute mainly between north and sough latitudes. Until now, it is known that there are about 374 kinds of parrots around the world. Although Taiwan is not the original habitat of parrots, parrots are one of the most popular companion animals in Taiwan due to their beautiful plumage and high intelligence. However, all of the parrot species, except for Agapornis roseicollis, Melopsittacus undulates, Nymphicus holandicus and Psittacula krameri, are endangered and protected by CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) under different degrees of protection by listed on Appendix I or Appendix II. Therefore, it is necessary to develop molecular diagnosis methods with smuggled eggs or trace of material evidence to identify species, and the polymerase chain reaction (PCR)-based methods would be the first reasonable choice. Among various methods, the PCR-based random amplified polymorphic DNA (RAPD) analysis is conducted by short random primer sequences at low annealing temperature to produce multiple-band PCR products without knowing the sample information. Hence, RAPD was applied for identification of parrot species in this study. A total of 120 random primers from 6 series were used to identify the species-specific fragment in genomic DNA from 8-15 parrot species, and 52 possible bands were obtained after RAPD. After further analysis, two pairs of sequence characterized amplified region (SCAR) primers, called GCC-F/R and Monk-F/R, were designed according to sequencing results of the 437 bp RAPD product obtained from Pyrrhura molinae by random primer OPD15 and 529 bp RAPD product obtained from Myiopsitta monachus by random primer OPD7, respectively. GCC-F/R primer sequences were identical to the first 26 bp and the last 27 bp from the 437 bp RAPD product of Pyrrhura molinae, and Monk-F/R primer sequences were from the 529 bp RAPD product of Myiopsitta monachus. The GCC-F/-R and Monk-F/-R were applied to evaluate their species specificity. The electrophoresis result revealed that only genomic DNA from green-cheeked conure could be amplified with GCC-F/-R primers, and only monk parakeets genomic DNA could be amplified with Monk-F/-R primers after validation among 16/ 12 intra- and 15 inter species, indicating that these two pairs of SCAR primers could be applied for identification of monk parakeets and green-cheeked conure.

誌謝辭.i
摘要.ii
Abstract.iii
目次.v
圖次.vii
表次.x
前言.1
文獻檢討.2
一、鸚鵡簡介.2
（一）鸚鵡之演化與分類.2
（二）鸚鵡之羽色.6
二、鳥類之性別鑑定.9
三、物種之種別鑑定.11
（一）粒線體DNA之分子鑑定法.12
（二）增幅片段長度多型性（Amplified fragment length polymorphism; AFLP）.13
（三）限制酶片段長度多形性（Restriction Fragment Length Polymorphisms）, RFLP.14
（四）微衛星序列（Microsatellite Sequence.15
（五）簡單序列重複間序列（inter-simple sequence repeat; ISSR）.16
（六）即時聚合酶連鎖反應（Real-time PCR）.17
（七）單鍊構形多型性（Single-strand conformation polymorphism; SSCP）.17
（八）逢機增幅多型性DNA（Random amplified Polymorphic DNA; RAPD）.18
材料方法.22
結果.32
一、RAPD種別鑑定.32
（一）逢機引子OPC系列.32
（二）逢機引子OPD系列.32
（三）逢機引子OPE系列.33
（四）逢機引子OPG系列.33
（五）逢機引子OPH系列.34
（六）逢機引子OPI系列.34
二、以SCAR引子進行種別鑑定.48
（一）逢機引子OPC系列48
（二）逢機引子OPD系列49
（三）逢機引子OPE系列.57
（四）逢機引子OPG系列.57
（五）逢機引子OPH系列.69
（六）逢機引子OPI系列.70
討論.72
結論.88
參考文獻.89
附錄.92

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