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研究生：	蔡雅閔 Tsai, Ya-Min
論文名稱：	台灣白花蝴蝶蘭的第一與第十七遺傳連鎖群在蘭嶼姬蝴蝶蘭染色體上的共線性 Colinearity of genetic linkage groups 1 and 17 of Phalaenopsis aphrodite subsp. formosana on chromosomes of Phalaenopsis equestris
指導教授：	張松彬 Chang, Song-Bin
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 生命科學系 Department of Life Sciences
論文出版年：	2016
畢業學年度：	104
語文別：	英文
論文頁數：	65
中文關鍵詞：	台灣白花蝴蝶蘭、蘭嶼姬蝴蝶蘭、螢光原位雜合、共線性、粗絲期染色體、染色體圖譜
外文關鍵詞：	P. aphrodite, P. equestris, Fluorescent in situ hybridization (FISH), Colinearity, Pachytene chromosome, chromosome map
相關次數：	點閱：121 下載：3
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蘭科(Orchidaceae)為開花植物中最大的一科，包含25,000個以上的物種遍佈全球。其中蝴蝶蘭屬(Phalaenopsis)為全球園藝市場中非常受歡迎且具高經濟價值的觀賞植物，而系統性育種是有效率與永續地發展蘭花產業最重要的基礎。蝴蝶蘭基因組研究能有助於系統性育種，其中遺傳連鎖圖譜和染色體核型分析等細胞遺傳研究在育種中扮演相當重要的角色，但目前蝴蝶蘭的細胞遺傳相關研究依然相當缺乏。本研究使用高解析度的粗絲期(pachytene)染色體為材料，利用螢光原位雜合(fluorescent in situ hybridization, FISH)技術分析台灣白花蝴蝶蘭(Phalaenopsis aphrodite subsp. formosana)染色體圖譜的第一與第十七遺傳連鎖群(genetic linkage groups)與基因序列在蘭嶼姬蝴蝶蘭(Phalaenopsis equestris)染色體上的分布，並將兩種原生蝴蝶蘭的染色體圖譜做比較性核型分析。根據定位結果顯示，第十七遺傳連鎖群與PEPC基因位於台灣白花蝴蝶蘭第十二對染色體，而位於蘭嶼姬蝴蝶蘭第七對染體上；第一遺傳連鎖群與45S rDNA位於台灣白花蝴蝶蘭第十六對染色體，位於蘭嶼姬蝴蝶蘭第十三對染色體上。經比較性核型分析結果顯示PEPC基因、45S rDNA與兩個台灣白花蝴蝶蘭的遺傳連鎖群在蘭嶼姬蝴蝶蘭上皆具共線性(Colinearity)。經由兩種台灣原生種蝴蝶蘭的染色體圖譜比較性核型分析，除了可以了解染色體的重組與演化，增進蝴蝶蘭的基因體細胞遺傳學研究，且可加速以這二種蝴蝶蘭為親本的新雜交品種分子育種。

Orchidaceae is one of the largest families in angiosperms, and more than 25,000 species have been found around the world. Phalaenopsis hybrids are the popular and high-value commodity in the global horticulture market. Systematic breeding is the essential base for efficient and sustainable development of Phalaenopsis hybrids. Consequently, the developments of genome studies will facilitate systematic breeding for Phalaenopsis and genetic linkage map and chromosome karyotyping are the crucial part for their breeding program. To date, the cytogenetic characterization of Phalaenopsis genome is still limited. Here, we integrate two genetic linkage groups of P. aphrodite and genes to pachytene chromosomes of P. equestris by fluorescent in situ hybridization (FISH) mapping. We also analyzed comparative karyotype based on the results of FISH mapping of P. aphrodite and P. equestris. Our results showed D_L17 and phosphoenolpyruvate carboxylase gene (PEPC) are located on chromosome 12 of P. aphrodite and chromosome 7 of P. equestris. Also, we discovered 45S rDNA and D_L1 were mapped on chromosome 16 of P. aphrodite and chromosome 13 of P. equestris. Overall, we found the consistent orientation and arrangement of our selected markers on these chromosomes between P. aphrodite and P. equestris. Our data provided a benefit for future cytogenetic and chromosomal rearrangement and evolutionary studies in orchids and facilitate in systematic breeding for the new hybrids using these two Phalaenopsis parents.

摘要............................	................I
Abstract.......................................II
致謝...........................................III
Index..........................................IV
List of Table..................................VI
List of Figure................................VII
Chapter 1 Introduction..........................1
1.1 Phalaenopsis orchids........................1
1.2 Cytogenetics in plant genomics..............1
1.2.1 Application of Cytogenetics in plant......1
1.2.2 Fluorescence in situ hybridization (FISH).2
1.2.3 Comparative FISH mapping..................3
1.2.4 The resolution of FISH signal.............5
1.2.5 Previous cytogenetic studies in orchids...6
1.3 Previous cytogenetic and genetic studies in Phalaenopsis....................................6
1.4 Purpose.....................................8
Chapter 2 Materials and Methods................10
2.1 Plant materials............................10
2.2 Pachytene chromosome preparation...........10
2.2.1 Chromosome stage of pollen mother cells (PMCs) examination....................................10
2.2.2 Preparation of acid-clean slides.........10
2.2.3 Prepare chromosome spread by drop method.11
2.3 DNA of probe isolation.....................12
2.3.1 Plasmid DNA extraction...................12
2.3.2 Bacterial artificial chromosome (BAC) DNA extraction.....................................12
2.3.3 BAC DNA purification.....................13
2.3.4 Evaluation of BAC DNA quality............14
2.3.5 Probe labeling by nick translation.......14
2.3.6 Probe quantification (dot blotting)......15
2.4 Fluorescence in situ hybridization (FISH)..15
2.5 Cytological measurements and analysis......17
Chapter 3 Results..............................18
3.1 Comparative mapping of PEPC and D_L17 scaffolds between P. aphrodite and P. equestris..........18
3.1.1 FISH mapping of PEPC and D_L17 on chromosome 12 in P. aphrodite...................................18
3.1.2 FISH mapping of PEPC and D_L17 on chromosome 7 in P. equestris...................................19
3.1.3 Colinearity of PEPC and D_L17 between P. aphrodite and P. equestris...............................20
3.2 Comparative mapping of 45S rDNA and BACs of D_L01 between P. aphrodite and P. equestris..........21
3.2.1 FISH mapping of 45S and three BACs of D_L1 on chromosome 16 in P. aphrodite..................21
3.2.2 FISH mapping of 45S and BACs of D_L01 on chromosome 13 in P. equestris.............................22
3.2.3 Colinearity of 45S and BACs of D_L01 between P. aphrodite and P. equestris.....................23
Chapter 4 Discussion...........................25
4.1 The analysis of chromosome karyotyping between P. aphrodite and P. equesris......................25
4.2 Colinearity and rearrangements between P. aphrodite and P. equestris...............................25
4.3 The dispersed repeats of BAC-FISH results in P. equestris......................................26
4.4 Conclusion.................................27
Chapter 5 Reference............................29
Chapter 6 Tables...............................35
Chapter 7 Figures..............................46
Appendix 1 FISH mapping of PEPC, scaffold_545 and scaffold_959 on the chromosomes in P. aphrodite	60
Appendix 2 The cytogenetic order of PEPC and D_L17 in P. aphrodite......................................61
Appendix 3 The draft karyotype of P. aphrodite.62
Appendix 4 The published karyotype of P. equestris	...............................................63
Appendix 5 FISH mapping of 45S and BACs of D_L01 on the chromosomes in P. aphrodite....................64
Appendix 6 The cytogenetic order of PEPC and BACs of D_L01 in P. aphrodite..........................65

                                    

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