臺灣博碩士論文加值系統

台灣的水稻品種有遺傳基礎狹隘之現象，研究水稻品種間親緣關係有助於日後在育種上選擇親本時的考量。本研究是利用Inter-Simple Sequence Repeats(ISSR) DNA分子標幟來（1）分析13個不同來源的越光品種之遺傳歧異度；（2）觀察兩個世代中八個水稻品種內的變異性，以瞭解ISSR分子標幟的穩定性；以及（3）探討水稻品種間遺傳歧異情形，並在使用的引子中篩選出核心引子。
從80個ISSR引子中，篩選出16個能清楚地產生多型性標幟之引子，以分析品種內（間）的差異。在分析不同來源的越光品種中，發現引子UBC811及UBC848分別在越光品種KHK11及KHK8各增幅出一多型性片段，而此二片段的序列分析結果，並未比對到任何水稻基因序列。將八個稻種經過兩個世代的栽培後做分析，結果每個稻種內均呈現單型性，世代間並無差異。將上述之材料，以11組Simple Sequence Repeats（SSR）引子進行分析。發現有三組引子OSR20、OSR22、RM249，在三個越光品種KHK6、KHK10及KHK11中，共產生五個多型性條帶；以引子OSR22、RM204及RM206分析稻種IR8及台南5號，發現在品種內呈現多型性，但世代間仍無差異。比較這兩種分子標幟，可得SSR的變異性大於ISSR。在兩個世代中，ISSR分子標幟是穩定的。
在探討品種間變異性的分析中，共選取76個原始祖先及栽培品種為材料，包含25個原始祖先與51個栽培品種（25個稉稻品種及26個秈稻品種）。16個ISSR引子共增幅出288個條帶，其中208個條帶具有多型性。經數據處理後得到所有品種的平均相似度為0.29，原始祖先為0.27，稉稻品種為0.66，秈稻品種為0.54。以樹狀圖分析造成多型性的核心引子，除了UBC810，其它15個引子都是核心引子。前人利用相同11組SSR引子，測得76個品種的平均相似度為0.40，原始祖先為0.42，稉稻品種為0.55，秈稻品種為0.63。比較ISSR與SSR所得之結果，對於親源關係較近之台灣水稻品種，ISSR分子標幟具有較佳之區分效果。

Rice (Oryza sativa L.) cultivars of Taiwan were developed on a narrow genetic base. Therefore, the knowledge of relationship among rice varieties is helpful in selecting mates for hybridization breeding program. In this study, we employed Inter-Simple Sequence Repeats (ISSR) DNA markers 1) to analyze the genetic variation of Koshihikri from thirteen seed sources; 2) to detect the stability of ISSR markers from eight rice varieties in two generations of seed propagation; 3) to evaluate the genetic diversity among rice varieties, and to identify the core primers that are informative in estimating diversity.
Sixteen of 80 ISSR primers were sufficient to produce polymorphic banding patterns. We have found specific fragments for KHK11 and KHK8 amplified the by primer UBC811 and UBC848, respectively. The eight rice varieties were propagated for two generations and showed monomorphic banding patterns. Three Simple Sequences Repeats (SSR) primers, OSR20, OSR22, and RM249, amplified five polymorphic banding patterns from eleven primers for SSR analysis. The three SSR primers, OSR22, RM204, and RM206 showed polymorphism within IR8 and Tainan 5, but no variation between two generations. Based on these results, SSR markers revealed more genetic variation than that by ISSR markers, and the ISSR marker is stable in two generations.
The genetic diversity among 76 rice varieties including 25 ancestors and 51 cultivars (25 Japonica and 26 Indica cultivars) were used for ISSR analysis. 16 ISSR primers amplified 288 banding patterns with 208 polymorphic. The averaged similarity estimates were 0.27 among ancestors, 0.66 among Japonica cultivars, 0.54 among Indica cultivars, and 0.29 among all rice varieties. Based on the clustering dendrograph, 16 ISSR primers were the core primer to differentiate varieties tested, except primer UBC810. The previous SSR data from the same material showed that the average similarity estimates among ancestors, Japonica cultivars, Indica cultivars, and all rice varieties were 0.42, 0.55, 0.63, and 0.40, respectively. ISSR showed lower averaged similarity estimates than SSR did. Therefore, ISSR analysis proved to be an efficient method for determining genetic diversity among Taiwanese rice varieties.

中文摘要 I
英文摘要 III
目錄 V
壹、 前言 1
貳、 前人研究 3
一、 作物種原遺傳變異性的流失 3
二、 台灣栽培稻品種之變遷與研究 4
三、 DNA分子標誌的建立及ISSR的應用 7
四、 稻種分類之研究 12
五、 ISSR在水稻方面的研究 15
參、 材料與方法 18
一、 試驗材料 18
二、 DNA的萃取與定量 19
三、 ISSR分析 21
四、 SSR分析 22
五、 歸群分析 23
六、 ISSR核心引子的篩選 24
七、 ISSR片段的選殖與定序 24
肆、 結果 26
一、 不同來源的越光品種之變異性 26
二、 品種內的變異性 28
三、 品種間變異性 29
伍、 討論 36
一、 不同來源越光品種之探討 36
二、 ISSR分子標幟穩定度之探討 37
三、 品種間變異性之探討 38
四、 篩選核心引子之探討 44
陸、 參考文獻 47
柒、 表與圖 57
捌、 附錄 91

行政院農業委員會、台灣省政府農林廳及亞太糧食肥料科技中心。
1987。台灣稻作品種圖誌。行政院農業委員會、台灣省政府農林廳及亞太糧食肥料科技中心。
林順福、陳成。 2002。作物育種觀念與技術之發展。科學農業 50： 110-121。
卓俊憲。 2003。利用AFLP及SSLP DNA分子標記探討台灣水稻品種和其原始祖先間之遺傳歧異性。國立中興大學生命科學研究所碩士論文。
張德慈。 1997。植物遺傳資源─未來植物生產的關鍵。台灣省農業試驗所。
黃真生。 1982。臺灣秈稻的展望。臺灣農業 18：38-47。
陳彥綾。 2000。台灣水稻品種核心引子之建立及分析。國立中興大學植物學研究所碩士論文。
楊小珍。 1997。台灣稻作品種及其親本遺傳變異之DNA多型性分析。國立中興大學植物學研究所碩士論文。
Ahbert, D., M. Lee, D. F. Austin, C. Livini, W. L. Woodman, S. J. Openshaw, J. S. Smith, K. Porter, and G. Dalton. 1996. Genetic diversity among elite sorghum inbred line assessed with DNA markers and pedigree information. Crop Sci. 36: 1385-1392.
Akagi, H., Y. Yolozeki, A. Inagaki, A. Nakamura, and T. Fujimura. 1996. A co-dominant DNA markers closely linked to the rice nuclear restore gene, Rf-1, identified with inter-SSR fingerprinting. Genome 39: 1205-1209.
Archak, S., A. B. Gaikwad, D. Gautam, E. V. Rao, K. R. Swamy, and J. L. Karihaloo. 2003. Comparative assessment of DNA fingerprinting techniques (RAPD, ISSR and AFLP) for genetic diversity of cashew (Anacardium occidentale L.) accessions of India. Genome 46: 362-369.
Bachmann, K. 1994. Molecular markers in plant ecology. New Phytol. 126: 403-418.
Becker, J. and M. Heun. 1995. Mapping of digested and undigested random amplified microsatellite polymorphisms in barley. Genome 38: 991-998.
Bhattacharya, R. C., S. R. Bhat, and V. L. Chopra. 1999. Applied Biotechnology. Science Publishers, Inc., New Hampshire, USA.
Blair, M. W., O. Panaud, and S. R. McCouch. 1999. Inter-simple sequence repeat (ISSR) amplification for analysis of microsatellite motif frequency and fingerprinting in rice (Oryza sativa L.). Theor. Appl. Genet. 98: 780-792.
Bornet, B., F. Goraguer, G. Joly, and M. Branchard. 2002. Genetic diversity in European and Argentinian cultivated potatoes (Solanum tuberosum subsp. tuberosum) detected by inter-simple sequence repeats (ISSRs). Genome 45: 481-484.
Bostein, D., R. L. White, M. Skolnick, and R. W. Davis. 1980. Construction of genetic linkage map in man using restriction fragment length polymorphisms. Am. J. Hum. Genet. 32: 314-331.
Chang, T. T. 1976. The origin, evolution, cultivation, dissemination, and diversification of Asian and Africa rice. Euphytica 25: 425-441.
Chang, T. T. 1984. Conservation of rice genetic resources: luxury or necessity? Science 224: 251-256.
Dos-Santos, J. B., J. Niehuis, P. Skroch, J. Tivang, and M. K. Slocum. 1994. Comparison of RAPD and RFLP genetic markers in determining genetic similarity among Brassica oleracea L. genotypes. Theor. Appl. Genet. 87: 909-915.
Doyle, J. J. and J. L. Doyle. 1990. Isolation of plant DNA from fresh tissues. Focus 12: 13-15.
Fang, D. Q., M. L. Roose, R. R. Krueger, and C. T. Federici. 1997. Fingerprinting trifoliate orange germplasm accessions with isozymes, RFLPs, and inter-simple sequence repeat markers. Theor. Appl. Genet. 95: 211-219.
Fernandez, M. E., A. M. Figueiras, and C. Benito. 2002. The use of ISSR and RAPD markers for detecting DNA polymorphism, genotype identification and genetic diversity among barley cultivars with known origin. Theor. Appl. Genet. 104: 845-851.
Glaszmann, J. C. 1987. Isozymes and classification of Asian rice varieties. Theor. Appl. Genet. 74: 21-30.
Gouião, L. and C. M. Oliveira. 2001. Molecular characterization of apple (Malus ×domestica Borkh.) using microsatellite (SSR and ISSR) markers. Euphytica 122: 81-89.
Goulden, C. H. 1941. Problems in plant selection. Proc. 7th International Genetic Congress, Edinburgh, pp. 132-133.
Hamada H. and T. Kakunaga. 1982. Potential Z-DNA forming sequences are highly dispersed in human genome. Nature 298: 396-398.
Harlan, J. R. 1976. Genetic resource in wild relatives of crops. Crop Sci. 16: 329-333.
Holcomb, J., D. M. Tolbert, and S. K. Jain. 1977. A diversity of genetic resource in rice. Euphytica 26: 441-450.
Huang, J. and S. M. Sun. 2000. Genetic diversity and relationships of sweet potato and its wild relatives in Ipomoea series Batatas (Convolvulaceae) as revealed by inter-simple sequence repeat (ISSR) and restriction analysis of chloroplast DNA. Theor. Appl. Genet. 100: 1050-1060.
Joshi, S. P., V. S. Gupta, R. K. Aggarwal, P. K. Ranjekar, and D. S. Brar. 2000. Genetic diversity and phylogenetic relationship as revealed by inter simple sequence repeat (ISSR) polymorphism in the genus Oryza. Theor. Appl. Genet. 100: 1311-1320.
Kato, S., H. Kosaka, and S. Hara. 1928. On the affinity of rice varieties as shown by the fertility of rice plants. Centr. Agric. Inst. Kyushu. Imp. Univ. 2: 241-276.
Kojima, T., T. Nagaoka, K. Noda, and Y. Ogihara. 1998. Genetic linkage map of ISSR and RAPD markers in Einkorn wheat in relation to that of RFLP markers. Theor. Appl. Genet. 96: 37-45.
Levin, I. N., E. Golboa, S. Yeselson, A. Shen, and A. Schaffer. 2000. Fgr, a major locus that modulates the fructose to glucose ratio in mature tomato fruits. Theor. Appl. Genet. 100: 256-262.
Lin, M. S. 1991a. Genetic base of Japonica rice varieties released in Taiwan. Euphytica 56: 43-46.
Lin, M. S. 1991b. Field uniformity of the Japonica rice region of Taiwan as estimated by relative genetic contribution. Theor. Appl. Genet. 83: 115-118.
Lin, M. S. 1992. Ancestral contribution to IRRI rice varieties (IR5-IR62). Jpn. J. Breed. 42: 437-442.
Lin, M. S. 1993. Changes in genetic diversity in the Japonica rice region of Taiwan. Bot. Bull. Acad. Sin. 34: 307-312.
Lin, M. S. and P. C. Hung. 1996. Ancestry of rice varieties of Taiwan. Agri. Asso. China N. S. 176: 88-100.
Lin, M. S. 2000. Cluster analysis of rice varieties of Taiwan based on coefficient of parentage. Agri. Asso. China 1: 89-98.
Litt, M. and J. A. Luty. 1989. A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. Am. J. Hum. Genet. 4: 397-401.
Mackill, D. J. 1995. Classifying japonica rice cultivars with RAPD markers. Crop Sci. 35: 889-894.
Mackill, D. J., Z. Zhang, E. D. Redona, and P. M. Colowit. 1996. Level of polymorphism and genetic mapping of AFLP markers in rice. Genome 39: 969-977.
Mackill, D. and X. Lei. 1997. Genetic variation for traits relates to temperature adaptation of rice cultivars. Crop Sci. 37: 1340-1346.
Martin, J. M., T. K. Blake, and E. A. Hockett. 1991. Diversity among north American spring barley cultivars based on coefficients of parentage. Crop Sci. 31: 1131-1137.
Morishima, H. and H. I. Oka. 1981. Phylogenetic differentiation of cultivated rice. 22 numerical evaluation of the indica-japonica differentiation. Jpn. J. Breed. 31: 402-413.
Mullis, K. B. and F. A. Faloona. 1987. Specific synthesis of DNA in vitro via a polymerase chain reaction. Methods Enzymol. 155: 335-350.
Nagaraju, J., M. Kathirvel, R. R. Kumar, E. A. Siddiq, and S. E. Hasnain. 2002. Genetic analysis of traditional and evolved Basmati and non-Basmati rice varieties by using fluorescence-based ISSR-PCR and SSR markers. Proc. Natl. Acad. Sci. U.S.A. 99: 5836-5841.
Nakagahra, M. 1978. The differentiation, classification and center of genetic diversity of cultivated rice (Oryza sativa L.) by isozyme analysis. Trop. Agr. Res. Ser. 11: 72-82.
Nei, M. and W. H. Li. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. U.S.A. 76: 5269-5273.
Ni, J., P. M. Colowit, and D. J. Mackill. 2002. Evaluation of genetic diversity in rice subspecies using microsatellite markers. Crop Sci. 42: 601-607.
Oka, H. I. 1958. Intervarietal variation and classification of cultivated rice. Ind. J. Genet. Plant Breed. 18: 79-89.
Paris, H. S., N. Yonash, V. Mozes-Daube, G. Tzuri, and N. Katzir. 2003. Assessment of genetic relationships in Cucurbita pepo (Cucurbitaceae) using DNA markers. Theor. Appl. Genet. 106: 971-978.
Prevost, A. and M. J. Wilkinson. 1999. A new system of comparing PCR primers applied to ISSR fingerprinting of potato cultivars. Theor. Appl. Genet. 98: 107-112.
Qian, W., S. Ge, and D. Y. Hong. 2001. Genetic variation within and among populations of wild rice Oryza granulata from China detected by RAPD and ISSR markers. Theor. Appl. Genet 102: 440-449.
Reddy, P. M., N. Sarla, and E. A. Siddiq. 2002. Inter simple sequence repeat (ISSR) polymorphism and its application in plant breeding. Euphytica 128: 9-17.
Rus-Kortekaas, W., M. J. M. Smulders, P. Arens, and B. Vosman. 1994. Direct comparison of levels of genetic variation in tomato detected by GACA — containing microsatellite probe and by random amplified polymorphic DNA. Genome 37: 375-381.
Sankar, A. A. and G. A. Moore. 2001. Evaluation of inter-simple sequence repeat analysis for mapping in Citrus and extension of genetic linkage map. Theor. Appl. Genet. 102: 206-214.
Second, G. 1982. Origin of the genic diversity of cultivated rice (Oryza spp.): study of the polymorphism scored at 40 isozyme loci. Jpn. J. Genet. 57: 25-57.
Staub, J. E., F. C. Serquen, and M. Gupta. 1996. Genetic markers, map construction, and their application in plant breeding. HortScience 31: 729-739.
Tanksley, S. D. 1983. Molecular marker in plant breeding. Plant Mol. Biol. Rep. 1: 3-8.
Tanksley, S. D., N. D. Young, A. H. Paterson, and M. W. Bonierbale. 1989. RFLP mapping in plant breeding: new tools for an old science. Biotechnology. 7: 257-264.
Tautz, D. and M. Renz. 1984. Simple sequences are ubiquitous repetitive components of eukaryotic genomes. Nucleic Acids Res. 12: 4127-4138.
Tautz, D. 1989. Hypervariability of simple sequences as a general source of polymorphic DNA markers. Nucleic Acids Res. 17: 6463-6471.
Tsumura, Y., K. Ohba, S. H. Strauss. 1996. Diversity and inheritance of inter-simple sequence repeat polymorphisms in Douglas-fir (Pseudostsuga menziesii) and sugi (Cryptomeria japonica). Theor. Appl. Genet. 92: 40-45.
Virk, P. S., B. V. Ford-Lloyd, M. T. Jackson, and H. J. Newbury. 1995. Use of RAPD for the study of diversity within plant germplasm collections. Heredity 74: 170-179.
Virk, P. S., J. Zhu, H. J. Newbury, G. J. Bryan, M. T. Jackson, and B. V. Ford-Lloyd. 2000. Effectiveness of different classes of molecular
makers for classifying and revealing variation in rice (Oryza sativa) germplasm. Euphytica 112: 275-284.
Vos, P., R. Hogers, M. Bleeker, M. Reijans, T. van de Lee, M. Hornes, A. Frijters, J. Pot, J. Peleman, M. Kuiper, and M. Zabeau. 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acid Res. 23: 4407-4414.
Wang, Z. Y., G. Second, and S. D. Tanksley. 1992. Polymorphism and phylogenetic relationships among species in the genus Oryza as determined by analysis of nuclear RFLPs. Theor. Appl. Genet. 83: 565-581.
Wang, G., S. Castiglione, J. Zhang, R. Fu, J. Ma, W. Li, Y. Sun, and F. Sala. 1994. Hybrid rice (Oryza sativa L.): identification and parentage determination by RAPD fingerprinting. Plant Cell Report 14: 112-115.
Weber, J. L. and P. E. May. 1989. Abundant class of human DNA polymorphism which can be typed using the polymerase chain reaction. Am. J. Hum. Genet. 44: 388-396.
Williams, J. G. K., A. R. Kubelik, K. J. Livak, J. A. Rafalski, and S. V. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18: 6531-6535.
Wu, K. S., and S. D. Tanksley. 1993. Abundance, polymorphism and genetic mapping of microsatellite in rice. Mol. Gen. Genet. 241: 225-235.
Yang, X. and C. Quiros. 1993. Identification and classification of celery cultivars with RAPD markers. Theor. Appl. Genet. 86: 205-212.
Yang, G. P., M. A. S. Maroof, C. G. Xu, Q. Zhang, and R. M. Biyashev. 1994. Comparative analysis of microsatellite DNA polymorphism in landraces and cultivars of rice. Mol. Gen. Genet. 245: 187-194.
Yu, L. X. and H. T. Nguyen. 1994. Genetic variation detected with RAPD markers among upland and lowland rice cultivars (Oryza sativa L.). Theor. Appl. Genet. 87: 668-672.
Zhang, Q., M. A. Saghai Maroof, T. Y. Lu, and B. Z. Shen. 1992. Genetic diversity and differentiation of Indica and Japonica rice detected by RFLP analysis. Theor. Appl. Genet. 83: 495-499.
Zhu, J., M. D. Gale, S. Quarrie, M. T. Jackson, and G. J. Bryan. 1998. AFLP markers for the study of rice biodiversity. Theor. Appl. Genet. 96: 602-611.
Zietkiewicz, E., A. Rafalski, and D. Labuda. 1994. Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification. Genomics 20: 176-183.