蝴蝶蘭是世界上最有價值的觀賞植物之一，其新品種的開發對全球的花卉產業具有重要的經濟意義。台灣原生種的姬蝴蝶蘭(Phalaenopsis equestris)，具有顏色變化大和花期長等迷人特性，因此常作為商業育種的重要親本，進行其遺傳和基因組研究將有助於促進新品種的開發和產出。具有效益之大片段插入DNA的細菌人工(人造細菌)染色體(bacterial artificial chromosome, BAC)基因庫，對於蝴蝶蘭的基因實質定位(physical mapping)、基因選殖、和分析基因結構與功能至關重要。
本研究以BamHI和HindIII限制酶酵素建構二套姬蝴蝶蘭BAC基因庫，共134,348個BAC選殖株，平均插入片段大小為103kb，大約覆蓋蝴蝶蘭單套基因組12倍。以12個專一性基因探針進行選殖測試，證實所有在蝴蝶蘭基因組中均勻分佈的基因探針，皆可在此基因庫中錨定獲得BAC選殖株。此外，以阿拉伯芥基因AtERS1(ethylene response sensor 1)和AtSOC1(SUPPRESSOR OF OVEREXPRESSION OF CO1)相關的蝴蝶蘭cDNA探針，從這兩套BAC基因庫，篩選出在姬蝴蝶蘭的乙烯受器基因PeERS1和調節開花時間的MADS-box基因PeSOC1 對應的BAC選殖株，經過次選殖並解序，得到此二基因的基因序列全長及構造。PeERS1包含5個外顯子(exon)和4個內含子(intron)，而PeSOC1包含7個外顯子和6個內含子，其中PeERS1和PeSOC1的第一內含子(intron 1)分別長達18,413 bp和37,171 bp，其長度遠超過已發表的ERS和SOC1。目前雖然姬蝴蝶蘭全基因組已被解序，但由於缺乏完整的蝴蝶蘭序列文獻，仍然有許多片段支架(scaffold)無法被組裝，於姬蝴蝶蘭基因組中仍存有7%，佔86 Mb的未知序列尚待釐清。在蘭花基因資料庫中的PeSOC1-1基因之第一內含子，存在五段未知序列，總長度為 4,351 bp，可由BAC基因庫選殖出的 PeSOC1，解序後的基因組序列比對而成功修正為4,669 bp。本研究結果表明這兩套姬蝴蝶蘭BAC基因庫是有用的資源，可應用於基因選殖，發展分子標誌，修正與完成次世代定序(next-generation sequencing, NGS)序列中高度複雜的未知序列。

Phalaenopsis orchid is one of the most valuable ornamentals in the world, and the development of new cultivars is economically vital to floricultural industries worldwide. P. equestris, one of the native species orchid in Taiwan, which has many attractive traits such as colorful, long-lasting flowers and is one of the important parents commonly used in commercial breeding programs. The genetics and genome researches on orchids will aid in the development of new cultivars to improve production. The availability of a large-insert bacterial artificial chromosome (BAC) genomic library is crucial for physical mapping, gene cloning, and analysis of gene structure and function in Phalaenopsis orchids.
In this study, two BAC libraries of the P. equestris were constructed using the BamHI and HindIII restriction enzymes. In total, the two libraries contain 134,348 clones with an average insert size of 103 kb and represent approximately 12 x haploid genome equivalents. Twelve gene-specific probes were used to screen the orchid BAC libraries. Positive clones were identified for all 12 probes, that are anchored to probes evenly distributed on the genomes of Phalaenopsis orchids. Moreover, the utility of both libraries was evaluated by screening for the presence of genes encoding ethylene receptor ERS1, and MADS-box that regulation of flowering time in orchids (PeSOC1) related to the Arabidopsis gene, AtERS1and AtSOC1, respectively. Subcloning and sequencing of BACs revealed that these BAC clones contain the corresponding genomic regions of PeERS1 and PeSOC1. The genomic sequence of the PeERS1 consists of 5 exons and 4 introns, the PeSOC1 comprises 7 exons and 6 introns, having the first intron of 18,413 bp and 37,171 bp, respectively, longer than those of ERS and SOC1 had ever been published. Although the whole-genome sequence of the P. equestris has been established, due to the lack of reference of Phalaenopsis orchid sequences, many scaffolds cannot be assembled. There are 86 Mb of the unknown bases corresponding to 7% of the Phalaenopsis genome. Five unknown fragments, a total of 4,351 bp in intron1 of PeSOC1 that is present in the OrchidBase, was successfully amended to 4,669 bp using PeSOC1 genomic sequences derived from BAC clones. These results indicated that the two BAC libraries are useful genomic resources for gene cloning, molecular marker development, and contribution to complete the unknown bases from the NGS (next-generation sequencing) sequences in P. equestris.

Table of contents
摘要 i
Abstract ii
致謝 iii
Table of contents iv
Table of tables vii
Table of figures viii
Abbreviation ix
1. Introduction 1
1.1 Development and prospect of Bacterial artificial chromosome (BAC) library 1
1.2 Application of BAC library 2
1.2.1 Construction of physical maps of whole genome 2
1.2.2 Map-based gene cloning 4
1.2.3. Combine BAC library and FISH technology in cytology and genomics study 4
1.2.4. Study of microsatellite sequence 5
1.3 The reason for constructing the Phalaenopsis BAC library 6
1.3.1 Phalaenopsis orchid is an important plant for Taiwan's export flower 6
1.3.2 The researches of Phalaenopsis orchids 7
1.3.3 Construction and application of BAC libraries 7
1.4. The impact of next-generation sequencing on genomics 8
1.4.1 Most commonly used high-throughput sequencing platforms 8
1.4.2 Overview of orchid genome sequencing 12
2. Materials and Methods 14
2.1 Plant material 14
2.2 High-molecular-weight DNA Preparation 14
2.3 Construction and characterization of BAC library 15
2.4 Preparation of specific probes 17
2.5 BAC library screening 18
2.6 Cloning of cDNAs coding for ethylene receptor (ERS1) or SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) 19
2.7 Real-time RT-PCR analysis of transcripts of PeERS genes 19
2.8 Sequence and Phylogenetic analysis 20
3. Results 22
3.1 Construction of two BAC library of Phalaenopsis equestris 22
3.1.1 Construction of two BAC library 22
3.1.2 Insert size estimation 22
3.1.3 Characterization of the BAC library 23
3.2 Application of BAC libraries of Phalaenopsis equestris 25
3.2.1 Isolation and screening of PeSOC1 from Phalaenopsis equestris and two BAC libraries 25
3.2.2 Isolation and screening of PeERS1 from Phalaenopsis equestris and two BAC libraries 30
3.2.3 Comparison of DNA sequences of PeSOC1 from BAC library and different versions of the OrchidBase data. 33
3.2.4 Application of the two BAC libraries of P. equestris for genomic analysis 36
4. Discussion 39
4.1 BAC library construction and characterization 39
4.1.1 High quality with large amounts of large-insert DNA is a critica factor in the successful construction of BAC library 39
4.1.2 The two BAC libraries are suitable for gene isolation and screening 41
4.2 Application of BAC libraries of Phalaenopsis equestris 42
4.2.1 cloning and characterization of PeSOC1 from P. equestris BAC library 42
4.2.2 BAC library as a tool for correct an fill the draft genome of P. equestris 44
References 48
 

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