臺灣博碩士論文加值系統

蝴蝶蘭(Phalaenopsis spp.)為全世界高經濟價值的花卉作物。然而香味蝴蝶蘭的育種有其困難性，因此較少有育種者將香味蘭花當成育種目標。為了節省育種時間，開發能偵測蝴蝶蘭香味的分子標誌(molecular marker)是非常需要的。大葉蝴蝶蘭 (P. bellina)因為具有甜美香氣，其香味為單萜類(monoterpene)，常被當作香花育種的親本。於本實驗室先前的研究發現，geranyl diphosphate synthase (PbGDPS) 是大葉蝴蝶蘭中香味合成之關鍵酵素。且利用10種不同香味的原生種蝴蝶蘭研究其GDPS啟動子的序列，發現它們的啟動子序列均非常相似，唯一不同的是重複序列之有無。經前人研究結果得知，在香味品種之中，完整的重複序列會伴隨著GDPS的表現，然而在不具香味的品種之中，除了具有不完整的重複序列且GDPS亦沒有表現。本論文研究利用14種原生種蝴蝶蘭以瞭解造成不同蝴蝶蘭香味的差異。經鏈鎖聚合反應 (PCR)擴增此重複片段，發現其具有300及400個核苷酸大小的片段。序列分析結果發現，除了重複片段(tandem repeats)具有較大的差異，不同原生種蝴蝶蘭的GDPS啟動子序列均非常相似。在400 bp的片段中，其具有兩個重複的片段，依據序列相似程度可分為repeat 1 及 repeat 2，並可再細分為ABCD及A’BC’D’各四個區域。而在300 bp的片段中，大片段的序列刪除存在repeat 1，因此只留下repeat 2的序列。然而在某些原生種蝴蝶蘭中，重複序列的刪除並不會影響GDPS基因的表現。進一步，利用蝴蝶蘭雜交族群所得之25個F1子代及其可能的父母本以發展香味的分子標誌。其中在400 bp片段的D區域，發現單一核苷酸多型性(single nucleotide polymorphisms, SNPs)的出現可能會造成單萜類香味物質的釋放量降低。然而在原生種蝴蝶蘭之中，大部分的SNPs及較長的刪除片段均存在於repeat 1之中。綜合上述現象，GDPS啟動子之重複序列中，repeat 2序列較repeat 1 保守，且大部分的突變均存於repeat 1，顯示repeat 2在蝴蝶蘭香味演化過程中較為重要，因此被保存下來。此重複序列具潛力發展為分子標誌，未來可能應用於早期偵測花香產生與否以協助香味蝴蝶蘭育種。然而由前述實驗得知，P. schilleriana, P. aphrodite subsp. formosana 及P. amboinensis var. common較不符合實驗所得之結果，因此具有這些血統的商業品種應避免使用之。

Phalaenopsis orchids are important crops with high economic value worldwide. However, breeding of scented Phalaenopsis orchids are with difficulties and thus the breeders usually have little successful targets on breeding orchids with floral scent. For time saving, molecular markers detecting the scent production is needed. Phalaenopsis bellina has sweet floral fragrance consisted of monoterpenes, and it usually is a seed or pod parent for breeding scent Phalaenopsis orchids. Previously, P. bellina geranyl diphosphate synthase (PbGDPS) is identified as the key enzyme for monoterpenes biosynthesis. In addition, the GDPS promoter from 10 native Phalaenopsis species were isolated and analyzed. They were very similar except the presence or absence of a tandem repeat sequences. In previous study, in scented Phalaenopsis species, the tandem repeat sequences were complete, and the GDPS was expressed. However, in scentless species, the incomplete tandem repeat sequences were detected and without GDPS expression. In this study, for developing the molecular marker from the tandem repeat sequences of GDPS promoter to detect the scent of monoterpene production, GDPS promoters of 14 native species Phalaenopsis spp. were amplified and analyzed. The PCR-amplified tandem repeat fragments in the promoter region showed 2 fragments of 400 bp and 300 bp. Sequence analysis showed that they were very similar except the presence or absence of two complete sets of tandem repeats. The 400-bp fragment contains the complete tandem repeats of repeat 1 and repeat 2, and each repeat can be subdivided as ABCD and A’B’C’D’ regions. The 300-bp fragment has a deletion of the repeat 1 and contains only the A’B’C’D’ regions. However, in some native species, the deletion of tandem repeat sequences did not affect the expression of GDPS. Furthermore, a population with 25 F1 progenies and their putative parents were analyzed for the development of molecular markers for assessing floral scent. Intriguingly, the single nucleotide polymorphisms (SNPs) occurred in the D region of tandem repeat in GDPS promoter might cause low production of monoterpene. However, no mutations were detected in the A’ region of the 400-bp fragment and the D’ region of the 300-bp fragment for all 14 native species regardless with or without scent. Most SNPs and large deletion existed in the repeat 1 rather than repeat 2. These results suggest that the repeat 2 is more conserved than the repeat 1 and may play an important role in orchid evolution for scent traits. In addition, the tandem repeat sequences of GDPS promoter may have potential to develop as molecular markers and apply for the early detection of the floral scent and facilitates scent orchid breeding in the future. However, the commercial cultivars which contain the parenthood of P. schilleriana, P. aphrodite subsp. formosana and P. amboinensis var. common were not suitable because their results were not so consistent with those in this study.

中文摘要 I
Abstract III
誌謝 V
List of Table IX
List of Figure X
List of Appendix Table XII
List of Appendix Figure XIII

1. Introduction 1
1.1 The importance of Phalaenopsis orchids 1
1.1.1 The unique of orchids 1
1.1.2 Phalaenopsis orchids in Taiwan 1
1.1.2.1 The economic aspect in Phalaenopsis 1
1.1.2.2 Breeding of Phalaenopsis orchids 2
1.2 Scent compounds and their biosynthesis pathway in plants 3
1.2.1 The characteristic of volatile organic compounds (VOCs) 3
1.2.2 Scent in Phalaenopsis bellina 3
1.2.3 Biosynthesis pathways of terpenoids 4
1.2.4 Critical enzyme for monoterpene biosynthesis in P. bellina 4
1.3 Previous study on GDPS promoter 5
1.3.1 GDPS is not expressed in the scentless P. equestris 5
1.3.2 Comparison of GDPS promoter in various native species of Phalaenopsis
orchids 6
1.3.3 The importance of repeat sequences in GDPS promoter 7
1.4 Molecular breeding in plants 7
1.4.1 Molecular marker and maker-assisted selection (MAS) in plants 7
1.4.2 PCR-based markers in plant 8
1.4.3 Development a molecular marker that detected the scented species of
Phalaenopsis 9
1.4.3.1 Examples of molecular markers developed from crops with fragrance 9
1.4.3.2 Development of a molecular marker from GDPS promoter sequence 9

2. Purpose 10

3. Materials and methods 11
3.1 Plant materials and growth conditions 11
3.2 Analysis of repeat sequence in GDPS promoter 11
3.2.1 Genomic DNA preparation 11
3.2.2 Detection of GDPS gene, promoter and repeats in various orchid genomes 12
3.2.3 Tandem repeat sequences isolation 12
3.2.4 Sequences analysis 14
3.3 Volatile compounds collection and analysis 14
3.3.1 Volatile compounds collection 14
3.3.2 Analysis of volatile compounds 14
3.4 Expression pattern of GDPS, LinS-like1, GerS-like1 and ERF9 in various
Phalaenopsis orchids 15
3.4.1 RNA preparation 15
3.4.2 Complementary DNA (cDNA) synthesis 16
3.4.3 Real time RT-PCR 17
3.4.4 Detection of the expression pattern of GDPS 17
3.5 Statistical analysis and the calculation of SNP polymorphism rate in the tandem
repeat sequences region 18

4. Results 19
4.1 Analysis of volatile compounds in 12 native species of Phalaenopsis orchids 19
4.2 Expression pattern of GDPS in 12 native species of Phalaenopsis orchids 19
4.3 Relative expression level of GDPS 20
4.4 Detection of GDPS gene, promoter and tandem repeat in genomic DNA of native
species of Phalaenopsis orchids 20
4.5 Sequences comparison and analysis of tandem repeats in the GDPS promoter
region 21
4.6 Analysis of volatile compounds in F1 hybrid (P. (bellina x Corona) ‘M734-1~25’) progenies and their putative parents 22
4.7 Relative expression level of GDPS 22
4.8 Detection of GDPS gene, promoter and tandem repeats in genomic DNA of
F1 progenies and putative parents 23
4.9 Comparison and analysis of tandem repeats in the GDPS promoter sequences in
hybrid P. M734 progenies and putative parents 24
4.10 The SNPs and the scent production of monoterpenes 24
4.11 The SNPs in the tandem repeats of GDPS promoter in the native Phalaenopsis species 25

5. Discussion 26
5.1 The scent production in Phalaenopsis orchids 26
5.2 The exception among the GDPS expression pattern, tandem repeat sequences and
terpenoids production 26
5.3 Specific primers for detection of tandem repeats of GDPS promoter 27
5.4 Molecular markers in Phalaenopsis spp. 28
5.5 SNPs in the tandem repeat sequences 28

6. Conclusions and perspectives 30

7. References 31

康有德、馬溯軒、洪立、方祖達、黃涵、鄭爕、凌德麟、李哖、鄭正勇、張喜寧、許圳塗、王自存 園藝概論 啟英文化 民國86年509-511 頁
Chen W.-H. 陳文輝 (2007) 台灣如何成為蝴蝶蘭王國？科學人雜誌 第69期: 76-82 頁
Chuang Y.-C. 莊育禎 (2009) Regulation mechanism of fragrance biosynthesis in Phalaenopsis orchids: the role of MYB transcription factors. 探討蝴蝶蘭香味合成之調控機制: MYB轉錄因子的角色，國立成功大學生命科學研究所碩士論文
Hsiao Y.-Y. 蕭郁芸 (2008) Studies on the biosynthesis pathway and its related genes of Phalaenopsis bellina floral scent. 大葉蝴蝶蘭香味 (Phalaenopsi. bellina) 生合成及其相關基因之研究，國立成功大學生命科學研究所博士論文
Hsu C.-Y. 許綺育 (2011) Analysis of the concomitance between repeat variation on GDPS promoter and scent production in Phalaenopsis orchids. 探討蝴蝶蘭GDPS啟動子上重複序列的差異與香味生成之相關性，國立成功大學生命科學研究所碩士論文
Acquaah G. (2012) Principles of Plant Genetics and Breeding. Wiley-Blackwell 2nd Edition. pp. 385-401.
Bronstein J.-L., Armbruster W.-S., and Thompson J.-N. (2014) Understanding evolution and the complexity of species interactions using orchids as a model system. New Phytologist 202: 373–375.
Collard B.-C.-Y., Jahufer M.-Z.-Z., Brouwer J.-B., and Pang E.-C.-K. (2005) An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic concepts. Euphytica 142: 169-196.
Chase M.-W. (2005) Classification of Orchidaceae in the age of DNA data. Curtis's Botanical Magazine 22: 2-7.
Christenson E.-A. (2001) Ecology and distribution. Phalaenopsis: A monograph. Portland: Timber Press. pp. 39-248.
Dudareva N., Klempien A., MuhlemannJ.-K., and Kaplan I. (2013) Biosynthesis, function and metabolic engineering of plant volatile organic compounds. New Phytologist 198: 16-32.
Dudareva N., Martin D., Kish C.-M., Kolosova N., Gorenstein N., Fäldt J., Miller B., and Bohlmann J. (2003) (E)-β-ocimene and myrcene synthase genes of floral scent biosynthesis in snapdragon: function and expression of three terpene synthase genes of a new terpene synthase subfamily. Plant Cell 15: 1227-1241.
Dudareva N., Negre F., Nagegowda D.-A., and Orlova I. (2006) Plant volatiles: recent advances and future perspectives. Critical Reviews in Plant Sciences 25: 417-440.
Guterman I., Shalit M., Menda N., Piestun D., Dafny-Yelin M., Shalev G., Bar E., Davydov O., Ovadis M., Emanuel M., Wang J., Adam Z., Pichersky E., Lewinsohn E., Zamir D., Vainstein A., and Weiss D. (2002) Rose scent: genomics approach to discovering novel floral fragrance–related genes. Plant Cell 14: 2325-2338.
Leeper, F.-J., and Vederas J.-C. (Eds.) (2000) Topics in current chemistry: biosynthesis: aromatic polyketides, isoprenoids, alkaloids. Springer, pp. 53-95.
Lin S., Lee H.-C., Chen W.-H., Chen C.-C., Kao Y.-Y., Fu Y.-M., Chen Y.-H., and Lin T.-Y. (2001) Nuclear DNA contents of Phalaenopsis sp. and Doritis pulcherrima. Journal of the American Society of Horticultural Science 126: 195-199.
Hsiao Y.-Y., Jeng M.-F., Tsai W.-C., Chuang Y.-C., Li C.-Y., Wu T.-S.,Kuoh C.-S., Chen W.-H. and Chen H.-H. (2008) A novel homodimeric geranyl diphosphate synthase from the orchid Phalaenopsis bellina lacking a DD(X)2-4D motif. Plant Journal 55: 719-733.
Hsiao Y.-Y., Pan Z.-J., Hsu C.-C., Yang P.-P., Hsu Y.-C., Chuang Y.-C., Shih S.-H., Chen W.-H., Tsai W.-C., and Chen H.-H. (2011) Research on orchid biology and biotechnology. Plant and Cell Physiology 52: 1467-1486.
Hsaio Y.-Y., Tsai W.-C., Kuoh C.-S., Huang T.-H., Wang H.-C., Wu T.-S., Leu
Y.-L., Chen W.-H. and Chen H.-H. (2006) Comparison of transcripts in Phalaenopsis bellina and Phalaenopsis equestris (Orchidaceae) flowers to deduce monoterpene biosynthesis pathway. BMC Plant Biology 6:14.
Jiang, G.-L. (2013) Molecular markers and marker-assisted breeding in plants. In: Andersen, S.-B. (Ed.). Plant Breeding from Laboratories to Fields. InTech, pp. 45-84.
Juwattanasomran R., Somta P., Chankaew S., Shimizu T., Wongpornchai S., Kaga A., and Srinives P. (2011) A SNP in GmBADH2 gene associates with fragrance in vegetable soybean variety ‘‘Kaori’’ and SNAP marker development for the fragrance. Theoretical and Applied Genetics 122: 533-541.
Kishimoto K., Nakayama M., Yagi M., Onozaki T., and Oyama-Okubo N. (2011) Evaluation of wild Dianthus species as genetic resources for fragrant carnation breeding based on their floral scent composition. Journal of the Japanese Society for Horticultural Science 80: 175-181.
Kovacha M.-J., Calingacionb M.-N., Fitzgeraldb M.-A., and McCoucha S.-R. (2009) The origin and evolution of fragrance in rice (Oryza sativa L.). Proceedings of the National Academy of Sciences of the United States of America 106: 14444-14449.
Knudsen J.-T., and Tollsten L. (1993) Trends in floral chemistry in pollination syndromes: floral scent composition in moth-pollinated taxa. Botanical Journal of the Linnean Society 113: 263-284.
Myint K.-M., Arikit S., Wanchana S., Yoshihashi T., Choowongkomon K., and Vanavichit A. (2012) A PCR-based marker for a locus conferring the aroma in Myanmar rice (Oryza sativa L.). Theoretical and Applied Genetics 125: 887-896.
Pichersky E., Noel T.-P., and Dudareva N. (2006) Biosynthesis of plant volatiles: nature’s diversity and ingenuity. Science 311: 808-811.
Raguso R.-A. (2008) Wake up and smell the roses: the ecology and evolution of floral scent. Annual Review of Ecology, Evolution, and Systematics 39: 549-569.
Taiz L., and Zeiger E. (2006) Plant Physiology. Sinauer Associates Publisher Inc., 4th edition, pp. 315-344.
Winter P. and Kahl G. (1995) Molecular marker and technologies for plant improvement. World Journal of Microbiology and Biochemistry 11: 438-448.
Yundaeng C., Somta P., Tangphatsornruang S., Wongpornchai S., and Srinives P. (2013) Gene discovery and functional marker development for fragrance in sorghum (Sorghum bicolor (L.) Moench). Theoretical and Applied Genetics 126:2897-2906.