臺灣博碩士論文加值系統

蕨類配子體型態矮小相似，無足夠外型特徵以成功鑑別物種，但使用DNA分子條碼可提供較足夠的分子特徵，解決外部型態特徵鑑別蕨類配子體與未鑑別物種之困難。然而目前蕨類植物無一個通用所有物種的分子條碼，本研究以蕨類研究常用的matK、rbcL及trnL-L-F三個分子條碼區段，依據引子通用性(PCR成功率高低)、定序序列品質(扣除因重複鹼基造成讀序失敗後之成功率)、與物種區別效力(最小種間遺傳距離是否大於最大種內距離、種內個體能否形成單系群)三項標準加以評估效力，選取最有潛力發展為通用的蕨類植物分子條碼。
本研究樣囊括分類階層範圍為台灣蕨類植物28科77屬217種。第一項評估PCR成功率結果，trnL-L-F可成功放大99.33%物種、rbcL達88.55%、matK為67.34%
，若採取通用性加類群專一性兩階段引子放大matK，PCR成功率提高為85.86%。第二項定序品質成功率，在rbcL為100%、matK為98.01%、trnL-L-F為86.69%。第三項物種區別效力中，種間大於種內遺傳距離差距(Barcoding gap物種對)比例trnL-L-F為95.61%、rbcL為95.42%、matK為87.70%。種內個體形成單系群比例trnL-L-F為90%、rbcL為85%、matK為82%。綜合三項標準，trnL-L-F具最高分子條碼效力。本研究因廣泛取樣跨類群之台灣蕨類植物，對比過去其他研究大多僅採用一項標準的結果更為可信，由於trnL-L-F物種區別效力之種間種內遺傳距離差距比例與單系群比例，均超過90%，單一條碼trnL-L-F即可在蕨類通用性鑑種。

Identification of fern species, and particularly fern gametophyte is a difficult task because there are not many morphological characters available. DNA barcoding helps to identify morphologically related gametophytes and unidentified specimens with molecular characters. However, there is only a few works on developing universal DNA-barcode in ferns. In this study, I evaluated discriminatory powers of three chloroplast barcodes, matK, rbcL and trnL-L-F, by proposed criteria such as primer universality (PCR success rate), sequence quality (mononucleotide repeats disrupting individual sequencing reads) and discrimination success. The discrimination success of species is accessed by two different methods for comparison. A distance-based analysis checks if the minimum uncorrected interspecific P-distance involving a species was larger than its maximum intraspecific distance (indication of a barcoding gap); and a Bayesian phylogenetic analysis examines whether individuals of the same species can be clustered together (checking for porportions of well-supported monophyly of species).
28 fmailies 77 genera 217 species of the Taiwanese ferns were selected to assess DNA discriminatory powers of matK、rbcL and trnL-L-F. Primer universality assessment resulted in 99.33% PCR success rate for trnL-L-F, the highest among three; mean while, 88.55% for rbcL and 67.34% for matK. To improve the success rate of matK, two-stage PCR (universal primer pairs then lineage-specific primer pairs) were performed, the new matK success rate increased to 85.86%. Sequence quality checking resulted in 100% rbcL sequences no disrupting reads caused by mononucleotide repeat (tandem repeat), whereas 98.01% in matK and 86.69 % in trnL-L-F. Barcoding gaps can be found in 95.61% trnL-L-F pairwise distance, 95.42% by rbcL and 87.70% by matK. On the other hand, the discrimination success inferred by Basyien monophyly indicated 90% of within-species trnL-L-F sequences were successfully resolved when forming a monophyletic group with sufficient bootstrap support, in rbcL this was 85% and in matK this was 82%. To sum up, trnL-L-F has the highest discriminatory power to tell different fern species apart. Because the discrimination success of trnL-L-F in our well-sampled Taiwanese ferns is above 90%, higher enough to be the most universal DNA barcode for ferns.

目錄 Ⅰ
摘要 Ⅲ
Abstract Ⅳ
第一章、前言…………………………………………………......................................1
一、分子條碼對蕨類植物研究之重要性…………………………………............1
二、分子條碼法則與應用…………………….…………………………………...1
三、評估分子條碼之準則與策略…………………………………………………2
四、分子條碼研究現況…………………………………………………................4
五、具潛力候選分子條碼…………………………………………………............5
六、研究理論策略架構及目的………………………………………....................6
第二章、材料與實驗方法………………………………………...................................9
一、取樣材料………………………………………………….................................9
二、實驗方法流程………………………………………………….........................9
(一) DNA萃取…………………........................................................................9
(二) 聚合酶鏈鎖反應及定序……...................................................................11
(三) 序列排列與分析………….......................................................................11
第三章、分析方法.................................................................................................13
一、聚合酶連鎖反應擴增序列成功率...............................………......……….......13
二、重複鹼基序列中斷讀序比例…………………................................................13
三、物種鑑別成功率…………................................................................................13
(一) 種間及種內遺傳距離差距 (barcoding gap) 物種對比例….....…...….13
(二) 成功鑑別物種單系群比例.......................................................................14
第四章、結果…………………………………………………………......………........16
一、引子通用性之測試…………………………………………………………..16
二、定序品質之評估………………………………………………………….....16
三、物種區別效力之計算…………………………………………………….....17
(一)Barcoding gap物種對比例......................................................................17
(二)成功鑑別物種單系群之比例...................................................................19
第五章、討論........................................................................................................22
一、引子通用性trnL-L-F最高.............................................................................22
二、定序品質rbcL最佳........................................................................................23
三、物種區別效力trnL-L-F最佳..........................................................................24
四、trnL-L-F、rbcL和matK優缺點.....................................................................29
五、結合數段分子條碼以達最高蕨類物種區別效力評估..................................30
第六章、貢獻與展望...............................................................................................32
參考文獻…………….................................................................................................34
圖………………….....................................................................................................41
表………………......................................................…...............................................44
附錄…...........................………………………….….................................................53

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