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研究生:林仲毅
研究生(外文):Jhong-YiLin
論文名稱:利用葉綠體DNA作為分子標誌,以鑑別不同品系的蝴蝶蘭
論文名稱(外文):Develop the chloroplast DNA markers to distinguish Phalaenopsis species
指導教授:張清俊張清俊引用關係
指導教授(外文):Ching-Chung Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:英文
論文頁數:81
中文關鍵詞:葉綠體DNA分子標誌蝴蝶蘭屬
外文關鍵詞:cpDNA markermoth orchidsPhalaenopsis
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經由比較台灣原生種阿嬤蝴蝶蘭(P. aphrodite subsp. formosana) 以及姬蝴蝶蘭(P. equestris)的葉綠體基因組,已發現到兩種蝴蝶蘭在演化上產生的許多變異區域,特別是位於基因間隙(intergenic spacers) 及內插子(introns)。本研究目的為開發蝴蝶蘭葉綠體的DNA分子標誌,應用於品種鑑定、親本分析及蝴蝶蘭的親緣演化的研究。本實驗從容易變異的基因間隙區域及內插子設計出28組引子對,利用聚合酵素鏈鎖反應去擴增蝴蝶蘭的DNA,利用PCR產物長度的變異與(或)序列的不同,用以區別不同原生種的蝴蝶蘭與不同的姬蝴蝶蘭亞種。實驗結果發現rps16-trnQ(UUG)區域為具有最佳的區分效果,可以將15種原生種蝴蝶蘭區分成13組。而結合trnN-rpl32, rps19-psbA, petA-psbJ, rps16-trnQ(UUG), trnT(UGU)-trnL(UAA), rps15-ycf1, petN-psbM, trnE(UUC)- trnT(GGU)基因間隙和petD內插子,則可以區分姬蝴蝶蘭與其它兩個姬蝴蝶蘭亞種。並發現trnE(UUC)- trnT(GGU), trnN-rpl32, accD-psaI, rps15-ycf1, petN-psbM, petA-psbJ, trnF(GAA)-ndhJ, psbA-trnK(UUU)基因間隙和atpF內插子,可以分辨出易混淆的兩種蝴蝶蘭(P. aphrodite和P. amabilis)。此外,葉綠體分子標誌可以用來追朔最原始的母本,研究中發現雜交種的親本與英國皇家園藝協會(Royal Horticultural Society)註冊的栽培種蝴蝶蘭不同。在rps16-trnQ及trnR(UCA)-atpA區域定序後也發現到在蝴蝶蘭間存在許多變異,包括序列的插入/缺失(InDel)、點突變(point mutation)及一些簡單重複序列(simple sequence repeat, SSR)。

Previously, the comparative chloroplast genomic study between two native moth orchids, P. aphrodite and P. equestris in Taiwan have identified many evolutionary hot-spot regions, particularly in intergenic spacers and introns, which are potentially useful as molecular markers to distinguish Phalaenopsis species. To explore the possibility of developing cpDNA as molecular markers, the conserved 28 pairs of PCR primers are designed in the flanking regions of polymorphic sites such as insertion/deletion (InDel), and subsequently have been used to amplify the DNA among moth orchids. Our study showed that based on the length variation of rps16-trnQ(UUG) intergenic region, 15 native species of moth orchids could be successfully separated into 13 groups. In addition, the combination of length variation from any two of the following markers such as the intergenic spacers of trnN-rpl32, rps19-psbA, petA-psbJ, rps16-trnQ(UUG), trnT(UGU)-trnL(UAA), rps15-ycf1, petN-psbM, trnE(UUC)- trnT(GGU) and petD intron could be successfully used to identify the three subspecies of P. equestris. Furthermore, the length variation of the intergenic regions such as trnE(UUC)-trnT(GGU), trnN-rpl32, accD-psaI, rps15-ycf1, petN-psbM, petA-psbJ, trnF(GAA)-ndhJ, psbA-trnK(UUU) and using atpF intron could distinguish P. aphrodite and P. amabilis which their morphological traits are easily been confused in cultivation. Moreover, based on the mode of maternal inheritance for plastids in moth orchid, the female ancestor of hybrid species could be correctly traced back according to the cpDNA markers, but it is not consistent with their genealogy registered in Wildcatt database of Royal Horticulture Society. By sequencing the rps16-trnQ and trnR(UCA)-atpA intergenic regions, the location of many variable sites such as InDel, single nucleotide substitution, and simple sequence repeat (SSR) among moth orchids were found. In conclusion, the development of cpDNA molecular markers would be very useful for commercial breeding program and species identification for proprietary variety protection in orchid industry.

中文摘要 6
Abstract 7
Acknowledgements 8
1. Literature review 9
1.1 DNA marker technology 9
1.1.1 Non-PCR-based methods 9
1.1.2 PCR-based methods 10
1.1.3 Comparison of the common used molecular markers 11
1.2 Orchids and species identification 12
1.3 The properties of chloroplast genome 13
1.3.1 Chloroplast genome structure 14
1.3.2 Chloroplast genome application 15
1.3.3 Chloroplast DNA markers 16
1.3.4 Chloroplast DNA markers as a barcode in orchids 17
1.3.5 Comparative chloroplast genomes between P. aphrodite and P. equestris 18
1.4 Aims 19
2. Materials and methods 20
2.1 Materials 20
2.2 Methods 20
2.2.1 Extraction of the Plant genomic DNA 20
2.2.2 Designing of PCR primers 21
2.2.3 PCR amplification 21
2.2.4 Gel electrophoresis 21
2.2.5 DNA sequencing and phylogenetic analysis 23
3. Results 24
3.1 Develop cpDNA markers based on comparative chloroplast genomics 24
3.2 Distinguish the P. amabilis and P. aphrodite by cpDNA markers 27
3.3 Distinguish the subspecies of P. equestris by cpDNA markers 28
3.4 Maternally inherited mode of chloroplast DNA based on cpDNA markers 29
3.5 Trace the female ancestor of hybrids orchids by cpDNA marker 30
4. Discussion 32
References 37
Table 1 The Phalaenopsis species and their source used in this study 42
Table 2 Classification of 66 natural species of Phalaenopsis and 17 most common used parents in breeding 43
Table 3 The PCR primers used in this study. 44
Table 4 The successful rate of PCR amplification. 45
Table 5 The molecular identity of 15 native and 21 hybrid species on the basis of 8 cpDNA markers. 46
Table 6 The molecular identity of 15 native moth orchids based on seven most potential cpDNA markers. 47
Table 7 The molecular identity of 15 native moth orchids obtained from Kaohsiung district agricultural research and extension station based on seven most potential cpDNA markers. 48
Table 8 The percentage of pair-wise sequence identity or divergence in the trnR(UCA)-atpA intergenic spacers among 15 native and 10 hybrids of moth orchids. 49
Table 9 The percentage of pair-wise sequence identity or divergence in the rps16-trnQ intergenic spacers among 15 native species and 5 hybrids of moth orchids. 50
Table 10 The expected PCR size of trnL intron versus estimated length in gel. 51
Table 11 The percentage of pair-wise sequence identity or divergence in the trnL introns among 15 native species of moth orchids. 52
Table 12 The conditions of the polymerase chain reaction. 53
Table 13 The composition of 6% polyacrylamide gel. 53
Fig. 1 The regions were tested for the development of cpDNA markers in this study. 54
Fig. 2 Identification of 15 native species and 5 hybrid species of moth orchids by cpDNA markers. 55
Fig. 3 Identification of 14 species of moth orchids by cpDNA markers. 59
Fig. 4 Identification of 15 native species of moth orchids by cpDNA markers. 60
Fig. 5 The multiple sequence alignment of trnR(UCU)-atpA intergenic spacers among 15 native species and 10 hybrids of moth orchids. 62
Fig. 6 The phylogenetic tree of trnR(UCU)-atpA intergenic spacers among 25 moth orchids. 63
Fig. 8 The multiple sequence alignment of rps16-trnQ(UUG) intergenic spacers among 15 native species and 5 hybrids of moth orchids. 70
Fig. 9 The phylogenetic tree of rps16-trnQ intergenic spacers among moth orchids. 71
Fig. 10 The multiple sequence alignment of trnL introns from 15 native species. 73
Fig. 11 The phylogenetic tree of trnL introns among 15 moth orchids. 74
Fig. 12 Molecular identification between P. aphrodite and P. amabilis. 75
Fig. 13 Molecular identification of three subspecies of P. equestris. 76
Fig. 14 The multiple sequence alignments of trnN-rpl32 markers among 3 subspecies of P. equestris. 77
Fig.15 Maternal inheritance of chloroplast DNA in moth orchids. 78
Fig. 16 Identification of 20 hybrid species of moth orchids by cpDNA markers. 79
Appendix Fig. 1. The floral morphology of moth orchids used in this study. 80
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