臺灣博碩士論文加值系統

本研究利用逢機增幅多型性DNA（Random Amplified Polymorphic DNA，簡稱RAPD）分子標記對台灣咖啡種原初步篩選、鑑定，以利快速獲得台灣Coffea arabica咖啡間遺傳歧異度與遺傳結構資料，試驗材料為2006年台灣咖啡節收集11個台灣咖啡族群樣品，總數為110個DNA混合樣品。在RAPD標記分析結果中，使用11個逢機引子，總共產生170個條帶，選取其中清晰且具多型性107個條帶，計算遺傳相似性介於67％至97％。再根據（Unweighted pair-group method with arithmetic average，簡稱UPGMA）進行群聚分析，繪製分群樹狀圖，此與（Principal Coordinate Analysis，簡稱PCoA）結果相似，雖曾多次國外引種，然而所有參試的遺傳相似度高達75％，只有南投縣與雲林縣歧異度略高，而與其他族群分開，此顯現台灣咖啡族群確實有再次由原生地引種，提高本地栽培種原歧異度的必要性。
實驗結果配合歷史文獻記載，推測可能是因為普遍選擇單一種苗，以致族群內歧異度偏低，而族群間種原關係相近，也造成部分族群併入別群，依據群聚分析結果，顯現台灣咖啡受環境阻隔影響，停留日據時代規模，使分化程度不明顯，無論以各別樣品或不同層級樣區的角度進行分析，台灣地區咖啡生豆遺傳歧異度偏低，對於大規模病害發生應戒慎恐懼。

The RAPD (Random Amplified Polymorphic DNA) markers were used to analyze the Taiwan coffee germplasm for understanding the genetic diversity and population structure. Eleven coffee accessions from the participant of the 2006 Taiwan Coffee Festival were used in this research. One hundred and ten blended DNA samples from the eleven coffee accessions were able to produce 170 unambiguous bands by 11 RAPD primers. One hundred and seven polymorphic bands were chosen for estimating the genetic diversity. The estimated genetic similarities between accessions were ranging from 67% to 97%. The cluster analysis based on the polymorphic bands using UPGMA methodology reveal similar result as the data analyzed by principle coordinate analysis. The average similarity between accessions was estimated to be 75% and not able to distinguished from one another other than the Nantou and Yunlin accessions. The result presented an evidence of narrow genetic diversity of the current coffee germplasm. Input of foreign germplasm should be required to broaden the genetic base of local germplasm.
The records from local historical coffee introduction documentation provided an explanation to the great similarity estimated by this research. The similarity between the accessions could be due to the spreading the coffee germplasm during the Japanese occupation.

口試委員會審定書………………………………………………Ι
誌謝………………………………………………………………Ⅱ
目錄………………………………………………………………Ⅲ
圖目錄……………………………………………………………Ⅴ
表目錄……………………………………………………………Ⅵ
附圖目錄…………………………………………………………Ⅶ
附表目錄……………………………………………………Ⅶ
中文摘要……………………………………………………Ⅷ
Abstract…………………………………………………………Ⅸ
前言………………………………………………………………1
前人研究……………………………………………………………3
ㄧ、台灣咖啡歷史……………………………………………3
（一） 咖啡傳入台灣…………………………3
（二） 農業試驗階段……………………………………4
（三） 民間經營企業栽培………………………………6
二、咖啡傳播世界地圖……………………………………7
三、咖啡形態特性………………………………………9
四、咖啡在遺傳育種上的研究………………………………10
五、分子標誌在植物育種上的利用………………………16
六、分子標誌在咖啡上的研究…………………19
材料與方法………………………………………23
ㄧ、試驗材料………………………………………23
二、實驗方法...........................................23
（一） 樣品研磨……………………………………23
（二） 咖啡生豆樣品DNA抽取與純化…………24
（三） DNA品質檢定與定量……………………26
（四） DNA分析( RAPD analysis)……………………………26
（1）反應溶液總體積與試劑濃度……………26
（2）PCR反應……………………………………27
（3）電泳膠片製作及RAPD多型性條帶分析...27
（4）遺傳資料分析……………………28
結果…………………………………………………………31
ㄧ、110個生豆樣品RAPD分析結果………………31
二、11個咖啡族群RAPD分析結果………………38
三、六個地區RAPD分析結果…………………………42
討論…………………………………………………49
（一）混合樣品與單粒樣品之探討…………………49
（二）台灣咖啡族群歸群分析之探討…………………49
（三）C. arabica遺傳歧異度之探討………………………50
結論………………………………………………………………………51
參考文獻……………………………………………………52
附錄…………………………………………59

中央研究所林業部。1928。台灣咖啡の栽培史と其成績。台灣農事報（第22卷第8號）。台北市。p. 81。

王曉俐等作。2004。植物基因轉殖與分子檢查技術。植物生物技術教學資源中心主編。台中市。pp. 189-190。

田口 護。2004。咖啡大全。積木文化。台北市。p. 16。

田代安定。1916。恆春熱帶植物殖育場事業報告（1-6輯）。台灣總督府殖產局。台北市。pp. 196-213。

李淵百。2002。計量遺傳學入門。國立編譯館出版。台北市。pp. 49-62。

林均雅。2005。恆春山茶之族群內遺傳結構。國立台灣大學生態學與演化生物學研究所。台北市。pp. 27-28。

胡智益。2004。台灣茶樹種原葉部性狀及序列變異之探討。國立台灣大學農藝學研究所碩士論文。台北市。pp. 13-14。

財政部關稅總局。2006; 2007。我國進出口貨物數量與價值查詢表：烘制咖啡和咖啡及其製品三大分類的整體進口量。
http://web.customs.gov.tw

唐贊袞。1891。台陽見聞錄（下卷）。成文出版社。台北市。p. 169。

黃弼臣。1953。台灣之咖啡。台灣銀行季刊6:90-104。台北縣。

蔡啓�琚C1972。台灣之過去及現在。台灣研究叢刊第107種。台北市。p. 385。

郭華仁。2008。作物生產概論。台灣大學農藝學系種子研究室。台北市。p. 3, pp. 42-49。

陳述、胡凱康。1995。玉米核酸逢機增殖多型性（RAPD）遺傳歧異分析系統之建立。中華農業研究。44:251-257。

殖產局農務課。1935。熱帶產業調查會調查書：珈琲。台北市。pp. 1-9, p.24。

臺糖經濟研究室。1969。臺灣文獻史料叢刊第七輯：劉銘傳撫臺前後檔案。大通書局。台北市。p. 9。

臺灣總督府農業試驗所。1932。臺灣農家便覽增訂五版。台灣農友會發行。台北市。pp. 106-108。

臺灣總督府農業試驗所。1944。臺灣農家便覽改訂增補第六版。台灣農友會發行。台北市。pp. 14-15。

臺灣省政府農林廳。1946-1982。臺灣農業年報。南投縣。

劉建甫。2001。RAPD與AFLP分子標誌應用於培地茅種原遺傳歧異度分析。國立台灣大學農藝學研究所碩士論文。台北市。p. 7。

澤田兼吉。1933。台灣に於ける咖啡栽培歷史。臺灣農事報第321號。台南市。pp. 1-7。

蕭其來。1956。台灣的咖啡。海外文庫出版社。台北市。

蕭其來。1958。南洋的咖啡。海外文庫出版社。台北市。pp. 15-22。

顏采衣。2007。鑑別台灣主要稉稻品種的InDel標記之開發。國立台灣大學生物資源暨農學院農藝學研究所碩士論文。台北市。p. 13, p. 23。

櫻井芳次。1929。珈琲【東部台灣開發研究資料第一輯】。台灣總督府殖產局。台北市。pp. 1-23, pp. 152-156。

韓懷宗。2000。咖啡萬歲。聯經出版事業公司。台北市。pp. 3-10。

Aga, E., E. Bekele, and T. Bryngelsson. 2005. Inter-simple sequence repeat（ISSR）variation in forest coffee trees（Coffea arabica L.）populations from Ethiopia. Genetica 124:213-221.

Aga, E., T. Bryngelsson, E. Bekele, and B. Salomon. 2003. Genetic diversity of forest Arabica coffee (Coffea arabica L.) in Ethiopia as revealed by random amplified polymorphic DNA (RAPD) analysis. Hereditas 138:36-46.

Agwanda, C. O., P. Lashermes, P. Trouslot, M. C. Combes, and A. Charrier. 1997. Identification of RAPD markers for resistance to coffee berry disease, Colletotrichim kahawae, in arabica coffee. Euphytica 97:241-248.

Altintas, S., F. Toklu, S. Kafkas, B. Kilian, A. Brandolini, and H. özkan. Estimating genetic diversity in durum and bread wheat cultivars from Turkey using AFLP and SAMPL markers. Plant Breeding 127: 9-14.

Anthony, F., J. Berthaud, J. L. Guillaumet, and M. Lourd. 1987. Collecting wild Coffea species in Kenya and Tanzania. Plant Genet. Resources Newsl 69:23-29.

Anthony , F., B. Bertrand, O. Quiros, A. Wilches, P. Lashermes, and J. Berthaud. 2001. Genetic diversity of wild coffee (Coffee arabica L.) using molecular markers. Euphytica 118:53-65.

Anthony, F., M. C. Combes, C. Astorga, and B. Bertrand. 2002. The origin of cultivated Coffea arabica L. varieties revealed by AFLP and SSR markers. Theor. Appl. Genet. 104:894-900.
Berthou, F., C. Mathieu, and F. Vedel. 1983. Chloroplast and mitochondrial DNA variation as indicator of phylogenetic relationships in the genus Coffea L.. Theor. Appl. Genet. 65:77-84.

Bussell, J. D. 1999. The distribution of random amplified polymorphic DNA（RAPD）diversity amongst populations of Isotoma petraea（Lobeliaceae）. Molecular Ecology 8:775-789.

Carvalho, A. 1988. Principles and practices of coffee plant breeding for productivity and quality factors:Coffea arabica. In: R. J. Clarke and R. Macrae（Eds）Coffee vol 4: Agronomy. Elsevier Applied Science, London, pp. 129-165.

Chaparro, A. P., M. A. Cristancho, H. A. Cortina, and A. L. Gaitán. 2004. Genetic variability of Coffea arabica L. accessions from Ethiopia evaluated with RAPDs. Genetic Resources and Crop Evolution 51:291-297.

Cros, J., M. C. Combes, P. Trouslot. F. Anthony, S. Hamon, A. Charrier, and P. Lashermes. 1998. Phylogenetic analysis of chloroplast DNA variation in Coffea L.. Mol. Phylog. Evol. 9:109-117.

Crochemoro, M. L., L. M. Nunes, G. A. Andrade, H. B. C. Molinari, and M. E. Vasconcellos. 2004. Varietal identification of coffee seeds by RAPD technique. Brazilian archives of biology and technology 47:7-11.

Crow, J. F. 1986. Basic concepts in population, quantitative, and evolutionary genetis. W. H. Freeman and Company. New York. pp. 12-13, pp. 62-63.

Coste, R. 1992. Coffee: The plant and the product. The Macmillan Press Ltd. London. pp. 1-11.

Davidson, J. W. 1903. The island of Formosa, past and present. SMC Publishing Inc. Washington. pp. 554-555.

Dice, L. R. 1945. Measures of the amount of ecological association between species. Ecology 26:297-302.

Diniz, L. E. C., C. F. Ruas, V. P. Carvalho, F. M. Torres, E. A. Ruas, M. O. Santos, T. Sera, and P. M. Ruas. 2005. Genetic diversity among forty coffee varieties assessed by RAPD markers associated with restriction digestion. Brazilian archives of biology and technology 48:511-521.

Haarer, A. E. 1956. Modern coffee production. Leonard Hill, London.

He, G., C. S. Prakash, and R. L. Jarret. 1995. Analysis of genetic diversity in a sweetpotato（Ipomoea batatas）germplasm collection using DNA amplification fingerprinting. Genome 38:938-945.

Herrea, J. C., M. C. Comes, F. Anthony, A. Charrier, and P. Lashermes. 2002. Introgession into the allotetraploid coffee（Coffea arabica L.）: segregation and recombination of the C. canephora genome in the tetraploid interspecific hybrid（Coffea arabica × C. canephora）. Theor. Appl. Genet. 104:661-668.

Jaccard, P. 1908. Nouvelles recherches sur la distribution florale.-Bull. Soc. Vaud. Sci. Nai. 44:223-270.

Khattak, J. Z. K., J. L. Christiansen, A. M. Torp, and S. B. Andersen. 2007. Genetic microsatellite markers for discrimination of spinach cultivars. Plant Breeding 126:454-456.

Kunihisa, M., N. Fukino, and S. Matsumoto. 2006. Development of PCR-RFLP markers on strawberry and the indentificaiton of cultivers and their progeny. Acta Hort. 708:517-521.

Lashermes, P., E. Couturon, N. Moreau, M. Paillard, and J. Louarn. 1996a. Inheritance and genetic mapping of self-incompatibility in Coffea canephora Pierre. Theor. Appl. Genet. 93:458-462.

Lashermes, P., J. Cros, M. C. Combes, P. Trouslot, F. Anthony, S. Hamon, and A. Charrier. 1996b. Inheritance and restriction fragment length polymorphism of chloroplast DNA in the genus Coffea L. Theor. Appl. Genet. 93:626-632.

Lashermes, P., P. Trouslot, F. Anthony, M. C. Combes, and A. Charrier. 1996c. Genetic diversity for RAPD markers between cultivated and wild accessions of Coffea arabica. Euphytica 87:59-64.

Lashermes, P., M. C. Combes, P. Trouslot, and A. Charrier. 1997. Phylogenetic relationships of coffee-tree species（Coffea L.）as inferred from ITS sequences of nuclear ribosomal DNA. Theor. Appl. Genet. 94:947-955.

Lashermes, P., M. C. Combes, J. Robert, P. Trouslot, A. D. Hont, F. Anthony, and A. Charrier. 1999. Molecular characterization and origin of the Coffea arabica L. genome. Mol. Gen. Genet. 261:259-266.

Lashermes. P., S. Andrzejewski, B. Bertrand, M. C. Combes, S. Dussert, G. Graziosi, and P. Trouslot. 2000. Molecular analysis of introgressive breeding in coffee（Coffea arabica L.）. Theor. Appl. Genet. 100:139-146.

Lewontin, R. C. 1972. The apportionment of human diversity. Evol. Biol. 6:381-398.

Li, W., L. Jiang, S. zhou, C. Wang, L. Liu, L. Chen, H. Ikehashi, and J. Wan. 2007. Fine mapping of pss1, a pollen semi-sterile gene in rice（Oryza sativa L.）. Theor. Appl. Genet. 114:939-946.

Maluf, M. P., M. Silvestrini, L. M. C. Ruggiero, O. G. Filho, and C. A. Colombo. 2005. Genetic diversity of cultivated Coffea arabica inbred lines assessed by RAPD, AFLP, and SSR marker systems. Sci. Agric. 62:366-373.

Mammadov, J. A., W. S. Brooks, C. A. Griffey, and M. A. S. Maroof. 2007. Validating molecular markers for barley leaf rust resistance genes Rph5 and Rph7. Plant Breeding 126:458-463.

Martellossi, C., E. J. Taylor, D. Lee, G. Graziosi, and P. Donini. 2005. DNA extraction and analysis from processed coffee beans. J. Agric. Food Chem. 53:8432-8436.

Masumbuko, L. I., T. Bryngelsson, E. E. Mneney, and B. Salomon. 2003. Genetic diversity in Tanzaian Arabica coffee using random amplified polymorphic DNA (RAPD) markers. Hereditas 139:56-63.

Masumbuko, L. I, and T. Bryngelsson. 2006. Inter simple sequence repeat（ISSR）analysis of diploid coffee species and cultivated Coffea arabica L. from Tanzania. Genetic Resources and Crop Evolution 53:357-366.

Montagnon, C, and P. Bouharmont. 1996. Multivariate analysis of phenotypic diversity of Coffea arabica. Genetic Resources and Crop Evolution 43:221-227.

National Coffee Association of U.S.A., Inc（NCA）. 2006. Coffee Trade Statistics: ICO Exports to All Destinations.
http://www.ncausa.org

Nei, M. 1972. Genetic distance between populations. Am. Nat. 106:283-292.

Nei, M. 1973. Analysis of gene diversity in Subdivided populations. Proc. Nat. Acad. Sci. USA 70:3321-3323.

Nei, M, and W. H. Li. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. 76:5269-5273.

Orozco-Castillo, C., K. J. Chalmers, R. Waugh, and W. Powell. 1994 . Detection of genetic diversity and selective gene introgression in coffee using RAPD markers. Theor. Appl. Genet. 87:934-940.

Orozco-Castillo, C., K. J. Chalmers, W. Powell, and R. Waugh. 1996. RAPD and organelle specific PCR re-affirms taxonomic relationships with the genus Coffea. Plant Cell Reports 15:337-341.

Pendergrast, M. 1999. Uncommon grounds: history of coffee and how it transformed our world. Basic Book, A member of the Perseus Books Group. New York. pp. 4-9.

Poncet. V., P. Hamon, J. Minier, C. Carasco, S. Hamon, and M. Noirot. 2004. SSR cross-amplification and variation within coffee trees（Coffea spp.）. Genome 47:1071-1081.

Prakash, N. S., D. V. Marques, V. M. P. Varzea, M. C. Silva, M. C. Combes, and P. Lashermes. 2004. Introgression molecular analysis of a leaf resistance gene from Coffea Liberica into C. arabica L.. Theor. Appl. Genet. 109:1311-1317.

Prakash, N. S., M. C. Comes, S. Dussert, S. Naveen, and P. Lashermes. 2005. Analysis of genetic diversity in Indian robusta coffee genepool（Coffea canephora）in comparison with a representative core collection using SSRs and AFLPs. Genetic Resources and Crop Evolution 52:333-343.

Reddy, M. P., N. Sarla, and E. A. Siddiq. 2002. Inter simple sequence repeat（ISSR）polymerphism and its application in plant breeding. Euphytica 128:9-17.

Rohlf, F. J. 2005. NTSYSpc. Applied Biostatistics Inc. New York.

Shannon, C. E., and W. Weaver. 1949. The mathematical theory of communication. Urbana IL: University of Illinois Press.

Silvestrini, M., M. G. Junqueira, A. C. Favarin, O. G. Filho, M. P. Maluf, M. B. Silvarolla, and C. A. Colombo. 2007. Genetic diversity and structure of Ethiopian, Yemen and Brazilian Coffea arabica L. accessions using microsatellites markers. Genet. Resour. Crop Evol. 54:1367-1379.

Tautz, D, and M. Renz. 1984. Simple sequence are ubiquitous repetitive components of eukaryotic genomes. Nucleic acids Research 12:4127-4138.

Van, P. Y, and R. Wachter. 1994. TREECOM for Windows: A software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput. Appl. Biosci. 10: 569-570.

Vos, P., R. Hogers, M. Bleeker, M. Reijans, T. Lee, M. Hornes, A. Frijters, J. Peleman, M. Kuiper, and M. Zabeau. 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research 23:4407-4414.

Willame, H. U. 1935. All about coffee. SCAA, New York.

Williams, J. G. K., A. R. Kubelik, K. J. Livak, J. A. Rafalski, and S. V. Tingey. 1990. DNA polymorphism amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18: 6531-6535.

Wright, S. 1931. Evolution in Mendelian populations. Genetics 16:97-159.

Yamamoto, M., T. Tomita, M. Onjo, K. Ishihata, T. Kubo, and S. Tominaga. 2007. Genetic diversity of white sapote（Casimiroa edulis La Llave & Lex.）demonstrated by intersimple sequence repeat analysis. HortScience 42:1329-1331.

Yan, G., H. Long, W. song, and R. Chen. 2008. Genetic polymorphism of Malus sieversii populations in Xinjiang, China. Genet. Resour. Crop Evol. 57:171-181.

Yeh, F. C., R. C. Yang, T. J. Boyle, Z. H. Ye, and J. X. Mao. 1997. POPGENE 1.32. The User Friendly Shareware for Population Genetic Analysis. Molecular Biology and Biotechnology, Centre University of Alberta, Edmonton, Canada.

Zietkiewicz, E., A. Rafalski, and D. Labuda. 1994. Genome fingerprinting by simple sequence repeat（SSR）-anchored polymerase chain reaction amplification. Genomics 20:176-183.

Zimmermann A., J. Lüthy, and U. Pauli. 1998. Quantitative and qualitative evaluation of nine different extraction methods for nucleic acids on soya bean food samples. Zeitschrift für Lebensmittel-Untersuchung und -Forschung A 207:81-90.