臺灣博碩士論文加值系統

摘 要

學號：M9211005
總頁數：71

論文名稱：分子標記在番石榴基因型遺傳相似性分析
與性狀鑑定上的應用

學校名稱：國立屏東科技大學
系所： 農園生產系 研究所

畢業時間及摘要別：九十三學年度第二學期碩士學位論文摘要

研究生：陳袖真
指導教授：陳幼光 博士

論文摘要內容：


番石榴(Psidium guajava L.)為桃金孃科(Myrtaceae)番石榴屬(Psidium)之熱帶及亞熱帶果樹，為台灣重要經濟果樹之一。番石榴的品種種類繁多，外表性狀極為相似，品種間不易區別，常造成育種上的困擾。分子標記的發展及在植物遺傳育種上的廣泛應用，亦為植物的品系鑑定提供一有效工具。本實驗係利用逢機擴增多型性DNA (random amplified polymorphic DNA，RAPD)、簡單序列重複間序列 (inter-simple sequence repeats，ISSR)及擴增片段長度多型性 (amplified fragment length polymorphism，AFLP) 三種以PCR (polymerase chain reaction)為基礎的DNA指紋技術作為番石榴品系鑑定的依據。分析84個RAPD引子，從中篩選出37個具多型性的引子(佔44 %)，平均每引子可產生6.68個多型性條帶；分析100個ISSR引子，發現46個引子可擴增出條帶，然只有11個引子能產生較多的多型性條帶(佔23.9%)，平均每引子可產生6個多型性條帶；而以AFLP而言，於11組引子對中有3組的引子可產生較多的多型性條帶(佔27.7 %)，平均每引子可產生10個多型性條帶。以NT-SYS軟體依據Nei與Li之UPGMA (unweighted pair-group method with arithmetic mean)法分析番石榴品系間的遺傳相似性，RAPD、ISSR與AFLP估算所測試番石榴品系間的遺傳相似性分別介於0.30-0.99、0.39-0.99與0.53-0.99之間；於親緣關係圖中顯示此三種分子標記皆能區分不同的番石榴並將其分群出來，如草莓番石榴；而對於果實有或無籽、果皮與葉片呈紅或綠色及果實是否具更年性等性狀，則以RAPD與AFLP較能將其作明顯的分群。在利用標記進行番石榴性狀的鑑定上，已找到可以區別番石榴紅色與綠色葉片顏色與更年性與非更年性果實兩種特性之RAPD標記。此外，‘水晶拔’被認為是‘泰國拔’芽條變異而來，從本試驗發現兩者的遺傳相似性在0.95以上，且亦找到可區別‘水晶拔’與‘泰國拔’的條帶，其是否與‘水晶拔’無籽化相關，此極値得進一步探討。

Abstract

Student ID: M9211005
Title of thesis: Application of Molecular Markers in Genotype Similarity
Analysis and Characteristics Identification of Guava
(Psidium guajava L.)
Total page: 70
Name of institute: Department of Plant Industry
Graduate date: July 18, 2005 Degree Conferred: Master
Name of student: Hsiu-Chen Chen Adviser: Dr. Yu-Kuang Chen
The contents of abstract in this thesis:
Guava (Psidum guajava L.) belongs to the family Myrtaceae and is distributed throughout the tropics. It is one of the economically important fruits in Taiwan. Due to the similarity in their appearance, different cultivars cannot be easily distinguished from each other, which causes confusion in breeding. Molecular markers have been developed and applied broadly in plant genetics and breeding. This technology provides an effective tool in cultivar identification. In this study, three PCR (polymerase chain reaction)-based DNA fingerprinting techniques, including RAPD (random amplified polymorphic DNA), ISSR (inter-simple sequence repeats), and AFLP (amplified fragment length polymorphism) were used to determine genetic similarity among 63 guava genotypes. Based on the screening of 84 RAPD primers, 46 ISSR primers found were 44%, 23.9%, and 27.7%, respectively. The average number of polymorphic bands generated by each polymorphic primer was 6.68, 6, and 10, respectively. Using UPGMA (unweighted pair-group method with arithmetic mean) for dendrogram construction, the genetic similarity of distances derived from RAPD, ISSR, and AFLP data were between 0.30-0.99, 0.39-0.99 and 0.53-0.99, respectively. Each type of the markers used can distinguish the difference among guava genotypes and group them according to the dendrogram constructed. RAPD and AFLP were better in grouping characteristics of seedlessness, fruit skin and leaf colors, and climacteric trait. RAPD, ISSR, and AFLP markers are able to distinguish between red-and green-leaf color and between acclimacteric and non-acclimacteric fruit, respectively. In addition, ‘Shuei-Jing Bar’, considered as a sport of ‘Tai-Kuo Bar’, had a similarity with ‘Tai-Kuo Bar’ around 0.95 in this study. The bands showing a difference between ‘Shuei-Jing Bar’ and ‘Tai-Kuo Bar’ were also revealed. It will be worthwhile to further explore whether these DNA fragments have anything to do with seedlessness in ‘Shuei-Jing Bar’.

目 錄
頁次
中文摘要……………….…………………………………..………………..I
英文摘要……………….…………………………………………………..III
誌謝……………………………………………………..…………………..V
目錄……………………….………………………………………………..VI
圖表索引…………………………………………….……………………..IX
壹、前言……………………………………………...………………………1
貳、前人研究………………………………………...………………………2
一、番石榴簡介……………………………...…………………………2
二、番石榴引種與選種…………………………...……………………2
三、番石榴的性狀…………………………….………………………..5
(一) 番石榴葉片及果實顏色……………..………………………5
(二) 番石榴果實的更年性…………………..……………………6
(三) 其他性狀…………………..…………………………………7
四、DNA指紋技術……..………………………………………………7
(一) RFLP..…………………………………..……………………..8
(二) RAPD………………………………..………………………..8
(三) SSR與ISSR………….………………………………..………9
(四) AFLP….……………………………………………………..10
(五) 分子標記在植物上的應用…………………………………11
1. 遺傳相似性分析與分類…………….…………………….11
2. 體細胞變異性的分析…………….……………………….13
3. 連鎖圖譜的建立…………………………………………..13
頁次
4. 其他應用…………………………………………………..14
參、材料與方法…………………………………………………………….15
一、植物材料與來源……………………………….…………………15
二、試驗方法……………………………………….…………………15
(一) 基因組DNA的萃取與純化…………………………………15
(二) RAPD-PCR分析……………………………………………...16
(三) ISSR 分析…………………………………………………….17
(四) AFLP 分析……………………………………………………17
(五) 遺傳相似性的分析…………………………………………..20
(六) RAPD在番石榴性狀鑑定上的應用…………………………20
肆、結果…………………………………………………………………….26
一、RAPD分析番石榴品系間的遺傳相似性…..………….……….26
二、ISSR分析番石榴品系間的遺傳相似性.……….…..…………33
三、AFLP分析番石榴品系間的遺傳相似性……...………………40
四、RAPD+ISSR分析番石榴品系間的遺傳相似性………..…….45
五、RAPD+ISSR+AFLP分析番石榴品系間的遺傳相似性……...48
六、RAPD在番石榴性狀鑑定上的應用…….…………………….51
(一) 可區別番石榴葉片顏色之分子標記的篩選………………..51
(二) 可區別番石榴果實後熟特性之分子標記的篩選…………..51
伍、討論……………………………………………………………….……57
一、番石榴品系間之遺傳相似性……………………….……………57
二、與番石榴特殊性狀相關的標記…………………….……………60
三、‘泰國拔’及其芽條變異株遺傳組成的異同………...…………...60
頁次
陸、結論……………………………………………..……………………….63
參考文獻…………………………………………………………………….64
作者簡介…………………………………………………………………….71

參 考 文 獻

丁慶成 (2002) 運用AFLP於香魚片有毒河魨之檢測。國立屏東科技大學食品科學系碩士論文 p.36-44。
王昭月、莊耿彰、范明仁 (2001) 以ISSR與RAPD分子標記分析火鶴花栽培種間遺傳相似性。中華農業研究 50：54-67。
王武彰 (1995) 番石榴。台灣農家要覽第二冊 第二版，第51~58頁。豐年社。台北市。
行政院農業委員會 (2003) 農業統計年報，第102~103頁。
林慧玲 (1998) 番石榴果實後熟生理之研究。國立台灣大學園藝學系博士論文。
柯立祥 (1996) 臺灣番石榴產業之經營及展望。台灣熱帶地區果園經營管理研討會專刊，第109~117頁。台灣省高雄區農業改良場。
柯見螢、楊堯文、陳福旗 (2003) 利用逢機擴大多型性DNA（Random Amplified Polymorphic DNA, RAPD）標記探討白鶴芋品種之遺傳變異性。中國園藝49：45-53。
范明仁、王昭月、羅舜芳、許庭榮、L. M. Engle (2001) 應用多型性分子標記進行番椒屬(Capsicum)種原種內與種間遺傳歧異度之分析。中華農業研究 50：29-42。
洪聖峰、張祖亮、陳右人 (2001) 應用逢機增殖多型性去氧核糖核酸(RAPD)標誌於筊白(Zizania latifolia Turcz.)品種與其黑穗病(Ustilago esculenta P. Henn.)系之鑑別。中國園藝 47：331-340。
莊淑貞、王小華、胡澤寬 (2002) 單雜交青割玉米品種胚部SSR (simple sequence repeat)標誌在種子純度鑑定的應用。農林學報 51：9-18。
曹儀植 (2002) 植物分子生物學，第142~159頁。高等教育出版社。北京。
溫佳思、蕭如英 (1999) 應用同功酶、逢機擴大多型性核糖核酸及型態特徵研究粗莖鐵炮百合與台灣百合之分類地位。中國園藝 45：293-302。
郭純德 (1986) 乙烯生化合成之機制及調節作用。科學農業 34: 173-183。
郭純德、蔡平里 (1986) 果實之更年性。科學農業 34: 240-247。
郭純德、蔡平里 (1987) 乙烯與果實後熟。科學農業 35: 200-210。
張哲嘉、林宗賢 (1998) 台灣番石榴生產之現況與改進。中國園藝 44：116-124.
陳敏祥 (1985) 台灣番石榴之栽培管理與產期調節。果樹產期調節研討會專集，第87~92頁。台中區農業改良場特刊第1號。
劉邦基 (1993) RFLP標誌在果樹育種與品系鑑別的應用。果樹育種研習會專刊，第139~146頁。台灣省農業試驗所特刊第37號。
劉邦基、張有明、許華欣 (1995) RAPD標誌在蔬菜育種上的應用。蔬菜育種研討會專刊，第269~274頁。台灣省桃園區農業改良場特刊第7號。
劉玠吟 (2004) 無籽番石榴之倍體數、花粉活力及雜交稔實率。國立屏東科技大學農園生產系碩士論文。
劉祖惠、林弘茂、張德淵、吳瑞鈺 (2003) 應用增殖限制酵素DAN片段多型性分析蝴蝶蘭花色性狀與變異鑑別的研究。中國園藝49：221-231。
謝鴻業、王武彰、李梨瑜 (1997) 番石榴品種改良-雜交育種。提昇果樹產業競爭力研討會專集Ⅲ，第21~26頁。台灣省台中區農業改良場。
謝鴻業 (2000) 珍珠拔與水晶拔之特性與栽培管理。高雄區農業專訊 31：16-17。
謝鴻業 (2002) 栽培番石榴應有的正確觀念。高雄區農業專訊 40：24-26。
顏秀芬、林宗賢 (1986) 番石榴兩栽培品種果實採收後呼吸型式及自動催化產生乙烯能力之比較。中國園藝 32: 224-232。
Boritzki, M., J. Plieske, and D. Struss (2000) Cultivar identification in sweet cherry (Prunus avium L.) using AFLP and microsatellite markers. Acta Hort. 538: 505-510.
Botstein, D., R. L. White, M. Skolnick, and R. W. Davis (1980) Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am. J. Hum. Genet. 32: 314-331.
Brown, P. T. H., F. D. Lange, E. Kranz, and H. Lorz (1993) Analysis of single protoplasts and regenerated plants by PCR and RAPD technology. Mol. Gen. Genet. 237: 311- 317.
Castrillo, L. A., J. D. Vandenberg, and S. P. Wraight (2003) Strain-specific detection of introduced Beauveria bassiana in agricultural fields by use of sequence-characterized amplified region markers. J. Invertebr. Pathol. 82: 75-83.
Degani, C., J. Deng, A. Beiles, R. Ei-Batsri, M. Goren, and S. Gazit (2003) Identifying lychee (Litchi chinensis Sonn.) cultivars and their genetic relationships using intersimple sequence repeat (ISSR) markers. J. Amer. Soc. Sci. 128: 838-845.
Erlich, H. A. (1992) Genetic analysis using the polymerase chain reaction. Annu. Rev. Genet. 26: 479- 506.
Giese, H., A.G. Holm-Jensen, H. Mathiassen, B. Kjaer, S. K. Rasmussen, H. Bay, and J. Jensen (1994) Distribution of RAPD markers on a linkage map of barley. Hereditas 120: 267-273.
Guo, W. W., Y. J. Cheng, C. L. Chen, C. H. Fu, and X. X. Deng (2004) Molecular characterization of several intergeneric somatic hybrids between Citrus and its related genera. Acta Hort. 632: 259-264.
Huaracha, E., M. Xu, and S. S. Korban (2004) Narrowing down the region of the Vf locus for scab resistance in apple using AFLP-derived SCARs. Theor. Appl. Genet. 108: 274-279.
Kriegner, A., J. C. Cervantes, K. Burg, R. O. M. Mwanga, and D. Zhang (2003) A genetic linkage map of sweerpotato ( Ipomoea batatas (L.) Lam) based on AFLP markers. Mol. Breed. 11: 169-185.
Lagercrantz, U., H. Ellegren, and L. Andersson (1993) The abundance of various polymorphic microsatellite motif differs between plants and vertebrates. Nucleic Acids Res. 21: 1111-1115.
Lau, O. L., and K. H. Yung (1974) Synergistic effect of kinetin on IAA-induced ethylene production. Plant & Cell Physiol. 15: 29-35.
Lau, O. L., and S. F. Yang (1973) Mechanism of a synergistic effect of kinetin on auxin-induced ethylene production. Plant Physiol. 51: 1011-1014.
Lau, O. L., and S. F. Yang (1975) Interaction of kinetin and calcium in relation to their effect on stimulation of ethylene production. Plant Physiol. 55: 738-740.
Le Clerc, V., M. Briard, and P. Revollon (2002) Influence of number and map distribution of AFLP markers on similarity estimates in carrot. Theor. Appl. Genet. 106: 157-162.
Lee, G. P., C. H. Lee, and C. S. Kim (2004) Molecular markers derived from RAPD, SCAR, and the conserved 18S rDNA sequences for classification and identification in Pyrus pyrifolia and P. communis. Theor. Appl. Genet. 108: 1487-1491.
Li, A., and S. Ge (2001) Genetic variation and clonal diversity of Psammochloa villosa (Poaceae) detected by ISSR markers. Ann. Bot. 87: 585-590.
Mokrani, L., L. Gentzbittel, F. Azanza, L. Fitamant, G. Al-Chaarani, and A. Sarrafi (2002) Mapping and analysis of quantitative trait loci for grain oil content and agronomic traits using AFLP and SSR in sunflower (Helianthus annuus L.). Theor. Appl. Genet. 106: 149-156.
Mueller, U. G., and L. L. Wofenbarger (1999) AFLP genotyping and fingerprinting. Tree 14: 389-394.
Mukhlesur, R. M., and Y. Hirata (2004) Homology of seed coat specific marker of B. juncea with brown seeded cultivar of B. rapa. J. Biol. Sci. 4: 731-734.
Murray, M. G., and W. F. Thompson (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8: 4321-4325.
Nakasone, H. Y., and R. E. Paull (1998) Guava. Tropical Fruits. p.149-172. In: CAB International, New York, USA.
Nei, M. (1973) Analysis of gene diversity in subdivided populations. Proc. Nat. Acad. Sci. USA 70: 3321-3323.
Nei, M. (1987) Phylogenetic trees. Molecular Evolutionary Genetics. p.287-326. In: Columbia University Press New York Guilford, Surrey.
Nei, M., and W. Li (1979) Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Nat. Acad. Sci. USA 76: 5269-5273.
Novelli, V. M., M. Cristofanl, and M. A. Machado (2000) Evaluation of microsatellite markers in cultivars of sweet orange (Citrus sinensis L. Osbeck). Acta Hort. 535: 47-50.
Park, S. O., and K. M. Crosby (2004) Identification and confirmation of RAPD and SCAR marker linked to the ms-3 gene controlling male sterility in melon (Cucumis melo L.). J. Amer. Soc. Hort. Sci. 129: 819-825.
Polanco, C., and M. L. Ruiz (2002) AFLP analysis of somaclonal variation in Arabidopsis thaliana regenerated plants. Plant Sci. 162: 817-824.
Prakash, D. P., P. Narayanaswamy, and S. N. Sondur (2002) Analysis of molecular diversity in guava using RAPD markers. J. Hort. Sci. Biotechnol. 77: 287-293.
Ratnaparkhe, M. B., M. Tekeoglu, and F. J. Muehlbauer (1998) Inter-simple- sequence-repeat (ISSR) polymorphisms are useful for finding markers associated with disease resistance gene clusters. Theor. Appl. Genet. 97: 515-519.
Ray, P. K. (2002) Guava. Breeding Tropical and Subtropical Fruits. p.143-155. In: Narosa Publishing House, New Delhi, India.
Reamon-Buttner, S. M., J. Schondelmaier, and C. Jung (1998) AFLP markers tightly linked to the sex locus in Asparagus officinalis L. Mol. Breed. 4: 91-98.
Saghai-Maooof, M. A., K. M. Soliman, R. A. Jorgenson, and R. W. Allard (1984) Ribosomal DNA spacer-length polymorphisms in barloy: Mendelian inheritance chromosomal location and population dynamics. Proc. Natl. Acad. Sci. USA 81: 8014-8018.
Severson, D. W., S. E. Brown, and D. L. Knudson (2001) Genetic and physical mapping in mosquitoes: molecular approaches. Annu. Rev. Entomol. 46: 183-219.
Sharifani, M. M., and J. F. Jackson (2000) Characterization of pear species and cultivars using RAPD primers. Acta Hort. 538: 499-504.
Simioniuc, D., R. Uptmoor, W. Friedt, and F. Ordon (2002) Genetic diversity and relationships among pea cultivars revealed by RAPDs and AFLPs. Plant Breed. 121: 429-435.
Simone, M. D., M. Morgante, M. Lucchin, P. Parrini, and A. Marocco (1997) A first linkage map of Cichorium intybus L. using a one-way pseudo-testcross and PCR-derived markers. Mol. Breed. 3: 415-425.
Sneath, P. H. A., and R. R. Sokal (1973) Numerical Taxonomy. The principles and practice of numberical classification. p.230-234. In: W. H. Freeman and Company, San Francisco, California, USA.
Struss, D., R. Ahmad, and S. M. Southwick (2003) Analysis of sweet cherry (Prunus avium L.) cultivars using SSR and AFLP markers. J. Amer. Soc. Hort. Sci. 128: 904-909.
Vos, P., R. Hogers, M. Bleeker, M. Reijans, T. V. D. Lee, M. Hornes, A. Frijters, J. Pot, J. Peleman, M. Kuiper, and M. Zabeau (1995) AFLP: a new technique for DNA fingerprinting. Nucl. Acids Res. 23: 4407-4414.
Welsh, J., and M. McClelland (1990) Fingerprinting genomes using PCR with arbitrary primer. Nucl. Acids Res. 18: 7213-7218.
Williams, J. G. K., A. R. Kubelik, K. J. Livak, J. A. Rafalski, and S. T. Tingey (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl. Acids Res. 18: 6531-6535.
Yakubov, B., O. Barazani, and A. Golan-Goldhirsh (2005) Combination of SCAR primers and touchdown-PCR for sex identification in Pistacia vera L. Sci. Hort. 103: 473-478.
Zhebentyateva, T. N., and Y. M. Sivolap (2000) Genetic diversity of apricot by isozyme and RAPD analyses. Acta Hort. 538: 525-529.
Zietkiewicz, E., A. Rafalski, and D. Labuda (1994) Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification. Genomics 20: 176-183.