Nicotiana sylvestris為栽培菸草的母本，共有十二對染色體，多為中位中節和近中位中節；其染色體長度和長短臂比例相似，且染色體上缺乏明顯的細胞遺傳標記，所以一直無法建立正確的核型。本實驗以N. sylvestris基因組DNA為探針，N. plumbaginifolia基因組DNA為覆蓋DNA，與N. sylvestris根尖細胞進行基因組螢光原位雜交 (genomic in situ hybridization, GISH)；結果顯示雜交訊號在 N. sylvestris染色體的分布有強弱的差異，且染色體上訊號的多寡和位置亦不同。根據基因組原位雜交結果，以及染色體長度和長短臂比例、衛星體的有無，並輔以本實驗室建立的N. plumbaginifolia-sylvestris異源單染色體添加系，我們建立一個正確的N. sylvestris的核型，未來能應用於栽培菸草的基因組演化的分析，添加系中變異型N. sylvestris染色體的分析，DNA序列的實質定位，以及N. sylvestris中串聯性重複序列的定位等研究上。

Nicotiana sylvestris (2n=24) is the maternal parent of cultivated tobacco (N. tabacum). The lengths and arm ratios of N. sylvestris chromosomes are similar, and there are no obvious cytological markers on the chromosomes. Therefore, it is difficult to identify the individual chromosomes by traditional cytogenetic techniques. In this study, we hybridized somatic metaphase chromosomes of N. sylvestris and N. plumbaginifolia-sylvestris monosomic addition lines using genomic DNA of N. sylvestris as a probe and unlabeled genomic DNA of N. plumbaginifolia as a block. The results show that patterns of hybridization signals in individual N. sylvestris chromosomes are distinctly different. Based on patterns of genomic in situ hybridization (GISH), chromosome lengths, arm rations, and positions and sizes of satellites, we construct a karyotype for N. sylvestris. The karyotype is useful for investigation of the evolution of cultivated tobacco, identification of chromosomes in aberrant addition lines, and localization of repetitive, single and low-copy sequences.

中文摘要…………………………………………………………………1
英文摘要…………………………………………………………………2
壹、前言…………………………………………………………………3
貳、材料與方法…………………………………………………………8
一、植物材料……………………………………………………….8
二、菸草基因組DNA的抽取…………………………………….. 8
三、基因組原位雜交……………………………………………….9
參、結果…………………………………………………………………13
一、N. sylvestris基因組原位雜交…………………………………13
二、N. plumbaginifolia-sylvestris異源單染色體添加系的基因組原位雜交………………………………………………………...14
三、N. sylvestris的核型……………………………………………15
肆、討論…………………………………………………………………20
伍、參考文獻…………………………………………………………....26

Anamthawat-Jónsson, K., and J. S. Heslop-Harrison. 1995. Establishing relationships between closely related species using total genomic DNA as a probe. Meth. Mol. Biol. 50: 209-225
Appels, R., C. Driscoll, and W. J. Peacock.1978. Heterochromatin and highly repeated DNA sequences in rye (Secale cereale). Chromosoma 70: 67-89
Banks, M. S., and P. K. Evans. 1977. Cytological markers in different tobacco species and their hybrids. J. Hered. 68: 395-399
Bland, M. M., D. F. Matzinger, and C. S. Leving. 1985. Comparison of the mitochondrial genome of Nicotiana tabacum with its progenitor species. Theor. Appl. Genet. 69: 535-541
Chang, Y. S.. 1996. The nucleolar organizer regions of five Nicotiana species. Master thesis, Departement of Botany, National Taiwan University, Taipei, Taiwan.
Chen, C. C., S. K. Chen, M. C. Liu, and Y. Y. Kao. 2002. Mapping of DNA markers to arms and sub-arm regions of Nicotiana sylvestris chromosomes using aberrant alien addition lines. Theor. Appl. Genet., in press.
Chen, C. C., C. R. Huang, and K. Y. To. 1985. Anther cultures of four diploid Nicotiana species and chromosome numbers of regenerated plants. Bot. Bull. Acad. Sin. 26: 147-153
Chen, C. C., Y. Y. Kao, F. M. Lee, and R. F. Lin. 2001. Somatic hybridization between Nicotiana sylvestris Speg. & Comes and N. plumbaginifolia Viv.. In: T. Nagata and Y. P. S. Bajaj (eds). Biotechnology in Agriculture and Forestry 49, Springer-Verlag, Berlin and Heidelberg, pp. 292-303
Cionini, P. G., P. Bassi, R. Cremonini, and A. Cavallini. 1985. Cytological localization of fast renaturing and satellite DNA sequences in Vicia faba. Protoplasma 124: 106-111
Endo, T. R., and B. S. Gill. 1996. The deletion stocks of common wheat. J. Hered. 87: 295-307
Galasso, I., T. Schimdt, and D. Pignone. 2001. Identification of Lens culinaris ssp. culinaris chromosomes by physical mapping of repetitive DNA sequences. Chromosome Res. 9: 199-209
Gawel, N. J., and R. I. Jarret. 1991. A modified CTAB DNA extraction procedure for Musa and Ipomoea. Plant Mol. Biol. Rep. 9: 262-266
Gerstel, D. U.. 1960. Segregation in new allopolyploids of Nicotiana. I. Comparison of 6x (N. tabacum x tomentosiformis) and 6x (N. tabacum x otophora). Genetics 45: 1723-1734
Gerstel, D. U.. 1963. Segregation in new allopolyploids of Nicotiana. II. Dicordant ratios from individual loci in 6X (N. tabacum X N. sylvestris) Genetics 48: 677-689
Goodspeed, T. H.. 1954. The genus Nicotiana. Chronica botanica, Waltham, Mass
Goodspeed, T. H., and P. Avery. 1939. Trisomic and other types in Nicotiana sylvestris. J. Genet. 38: 381-458
Goodspeed, T. H., and R. E. Clausen. 1928. Interspecific hybridization in Nicotiana. VIII. The sylvestris-tomentosa-tabacum hybrid triangle and its bearing on the origin of tabacum. Univ. Calif. Publ. Bot. 11: 245-256
Gray, J. C., S. D. Kung, and S. G. Wildman. 1974. Origin of Nicotiana tabacum L. detected by polypeptide composition of fraction I protein. Nature 252: 226-227.
Huang, H. C., and C. C. Chen. 1988. Genome multiplication in cultured protoplasts of two Nicotiana species. J. Hered. 79: 28-32
Jakowitsch, J., I. Papp, M. A. Matzke, and A. J. M. Matzke. 1998. Identification of a new family of highly repetitive DNA, NTS9, that is located predominantly on the S9 chromosome of tobacco. Chromosome Res. 6: 649-659
Jansen, M. A. K., R. E. van den Noort, M. Y. A. Tan, E. Proinsen, L. M. Lagrimini, and R. N. F. Thorneley. 2001. Phenol-oxidizing peroxidases contribute to the protection of plants from ultraviolet radiation stress. Plant Physiol. 126: 1012-1023
Kandra, L., and G. J. Wagner. 1998. Pathway for the biosynthesis of 4-methyl-1-hexanol volatilized from petal tissue of Nicotiana sylvestris. Phytochemistry 49: 1599-1604
Kenton, A., A. S. Parokonny, Y. Y. Gleba, and M. D. Bennett. 1993. Characterization of the Nicotiana tabacum L. genome by molecular cytogenetics. Mol. Gen. Genet. 240: 159-169
Kobayashi, Y., Y. Dokiya, M. Sugiura, Y. Niwa, and M. Sugita. 2001. Genomic organization and organ-specific expression of a nuclear gene encoding phage-type RNA polymerase in Nicotiana sylvestris. Gene 279:33-40
Koukalová, B., J. Reich, R. Matyášek, V. Kuhrová, and Bezdêk. 1989. A BamHI family of highly repeated DNA sequences of Nicotiana tabacum. Theor. Appl. Genet. 78: 77-80
Lee, F. M., and C. C. Chen. 1990. Somatic hybridization between Nicotiana sylvestris and N. otophora without the application of selection. J. Hered. 81: 313-317
Lee, F. M., and C. C. Chen. 1992. Preferential recovery of somatic hybrids from protoplast fusion of two Nicotiana species in the absence of artificial selection.Taiwania 37: 11-18
Leitch, A. R., T. Schwarzacher, D. Jackson, and I. J. Leitch. 1994. In situ hybridization: a practical guide. BIOS Scientific Publishers Limited, Oxford.
Lim, K. Y. R., Kovarik, R. Matyášek, M. Bezdêk, C. P. Lichtenstein, and A. R. Leitch. 2000a. Gene conversion of ribosomal DNA in Nicotiana tabacum is associated with undermethylated, decondensed and probably active gene units. Chromosoma 109: 161-172
Lim, K. Y., R. Matyášek, C. P. Lichtenstein, A. R. Leitch. 2000b. Molecular cytogenetic analyses and phylogenetic studies in the Nicotiana section Tomentosae. Chromosoma 109:245-258
Lin, R. F., and C. C. Chen. Cytological studies of interspecific somatic hybrids in Nicotiana. Bot. Bull. Acad. Sin. 31: 179-187
Matyášek, R., B. Gazdova, J. Fajkus, and M. Bezdêk. 1997. NTRS, a new family of highly repetitive DNAs specific for the T1 chromosome of tobacco. Chromosoma 106: 369-379
Matyášek, R., B. Koukalová, and J. Reich. 1989. Isolation and sequencing of HRS60dim1, a dimeric member of the HRS60-family of a tobacco DNA repeat. Nucleic Acids Res. 17: 4377
Murashige, T., and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497
Negrutiu, I., and J. F. Muller. 1981. Culture conditions of protoplast-derived cells of Nicotiana sylvestris for mutant selection. Plant Cell Reports 1:14-17
Negrutiu, I., M. Jacobs, and M.Caboche. 1984. Advances in somatic cell genetics of higher plants-the protoplast approach in basic studies on mutagenesis and isolation of biochemical mutants. Theor. Appl. Genet. 67: 289-304
Neumann, P., M. Nouvozá, and J. Macas. 2001. Molecular and cytogenetic analysis of repetitive DNA in pea (Pisum sativum L.). Genome 44:716-728
Okamuro, J. K., and R. B. Goldberg. 1985. Tobacco single-copy DNA is highly homologous to sequences present in the genomes of its diploid progenitors. Mol. Gen. Genet. 198: 290-298
Olmstead, R., and J. D. Palmer. 1991. Chloroplast DNA and systematics of the Solanaceae. In: Hawkes, J. G., R. N. Lester, M. Nee, and N. Estrada (eds). Solanaceae III. Taxonomy, chemistry, evolution. Royal Botanic Gardens, Kew, London, pp161-168
Parokonny, A. S., and A. Kenton. 1995. Comparative physical mapping and evolution of the Nicotiana tabacum L. karyptype. In: P. E. Brandham and M. D. Bennett (eds). Kew Chromosome Conference IV., Royal Botanic Garens, Kew, pp. 301-320
Parokonny, A. S., A. Y. Kenton, Y. Y. Gleba, and M. D. Bennett. 1992. Genome reorganization in Nicotiana asymmetric somatic hybrids analysed by in situ hybridization. Plant J. 2: 863-874
Raina, S. N., and V. Rani. 2001. GISH technology in plant genome research. Meth. Cell Sci. 23: 83-104
Sabar, M., R. D. Paepe, and Y. Kouchkovsky. 2000. Complex I impairment, respiratory compensations, and photosynthetic decrease in nuclear and mitochondrial male sterile mutants of Nicotiana sylvestris. Plant Physiol. 124: 1239-1249
Schwarzacher, T., K. Anamthawat-Jónsson, G. E. Harrison, A. K. M. R. Islam, J. Z. Jia, I. P. King, A. R. Leitch, T. E. Miller, S. M. Reader, W. J. Rogers, M. Shi, and J. S. Heslop-Harrison. 1992. Genomic in situ hybridization to identify alien chromosomes and chromosome segments in wheat. Theor. Appl. Genet. 84: 778-786
Sheen, S. J.. Isozyme evidence bearing on the origin of Nicotiana tabacum L.. Evolution 26: 143-154
Shibata, F., M. Hizume, and Y. Kuroki. 2000. Molecular cytogenetic analysis of supernumerary heterochromatic segments in Rumex acetosa. Genome 43: 391-397
Suen, D. F., C. K. Wang, R. F. Lin, Y. Y. Kao, F. M. Lee, and C. C. Chen. 1997. Assignment of DNA markers to Nicotiana sylvestris chromosomes using monosomic alien addtion lines. Theor. Appl. Genet. 94: 331-337.
Sumner, A. T.. 1990. Chromosome banding. Unwin Hyman Ltd, Lodon.
Wang, C. K.. 2000. Characterization of 4R repeated sequences in the genus Nicotiana and investigation of their evolution in cultivated tobacco, N. tabacum, and its progenitors. Doctoral dissertation, Department of Botany, National Taiwan University, Taipei, Taiwan.
Zoller, J. F., Y. Yang, R. G. Hermann and U. Hohmann. 2001. Comparative genomic in situ hybridization (cGISH) analysis on plant chromosomes revealed by labeled Arabidopsis DNA. Chromosome Res. 9: 357-375