番茄ps-2雄性不育基因的PCR分析与定位
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摘要
番茄雄性不育的研究对于番茄育种极其重要。本研究从保加利亚引入了含雄性不育基因ps-2的品系CMC，从中选出稳定品系383作材料进行了相关研究。结果表明该品系在北京地区雄性不育性状表现优良。另外，含有ps-2基因的品系较易获得100%不育后代，其F1代育性正常，杂种可以用于生产。为了加快该不育的利用，本研究利用RFLP和CAPS的方法对其进行了定位研究，且用于辅助选育。本研究首先选用了四个探针及17种酶进行RFLP杂交，获得了CT269B探针与三个限制性酶包括TaqI，DraI和HindIII等，它们的组合可以在父母本及F1代间产生多态性。利用番茄连锁图谱设计的CAPS引物，对其父母本进行了筛选，从中获得了三对引物包括Tg506 (5’>cgcctgtgacctgtcctaa<3’/5’>ctgaaatggccaaagttgt<3’),Tg652(5’>atggccaaacaattgaacc<3’/5’>tgcatgccatgacttctca<3’),Tg516(5’>caaaaggctaggccaggaat <3’/ 5’>ggcacttcgcaaaactgaat <3’)，扩增后，它们可以在父母本间产生多态性。其中对Tg652利用F2代分离群体进行验证，结果表明，该标记与ps-2雄性不育连锁，连锁距离是13.75cM。另外，在实验过程中还成功转化了四个质粒到大肠杆菌中，且对CAPS标记的稳定性进行了探索，找到了最佳反应条件，为下一步研究奠定了基础。
The male sterility play a key role for tomato production. Some CMC lines have been introduced from Bulgaria and one inbred line named 383 has been selected for the further analysis and mapping in this study. The results from open field proved that this line gave a very stable sterility in Beijing climate. The advantage of ps-2 line was easy to obtain the progenies with 100% sterility. F1 hybrid restores the fertility and easily used for tomato production. In order to assist tomato breeding, PCR markers derived from tomato RFLP mapping have been used to map this gene comparing the genetic linkage map, which has been mapped in tomato chromosome 4. Four probes combined with 17 restriction enzyme has been used to identify the polymorphism. Probe CT269B gave the polymorphism between female and male parents after digested respectively by TaqI，DraI and HindIII. The newly designed PCR primers derived from tomato probes including Tg506(5’>cgcctgtgacctgtcctaa<3’/5’>ctgaaatggccaaagttgt<3’),Tg652(5’>atggccaaacaattgaacc<3’/5’>tgcatgccatgacttctca<3’),Tg516(5’>caaaaggctaggccaggaat <3’/ 5’>ggcacttcgcaaaactgaat <3’) produced the polymorphism between female and male parents. Among them, Tg65 was proved the tightly linkage with ps-2 gene by using F2 segregated population. The linkage distance is about 13.75 cM. In addition, four plasmids have been transferred into E. Coli. The stability of CAPs markers have been tested. All the above work will be benefit for the further research.
The male sterility play a key role for tomato production. Some CMC lines have been introduced from Bulgaria and one inbred line named 383 has been selected for the further analysis and mapping in this study. The results from open field proved that this line gave a very stable sterility in Beijing climate. The advantage of ps-2 line was easy to obtain the progenies with 100% sterility. F1 hybrid restores the fertility and easily used for tomato production. In order to assist tomato breeding, PCR markers derived from tomato RFLP mapping have been used to map this gene comparing the genetic linkage map, which has been mapped in tomato chromosome 4. Four probes combined with 17 restriction enzyme has been used to identify the polymorphism. Probe CT269B gave the polymorphism between female and male parents after digested respectively by TaqI，DraI and HindIII. The newly designed PCR primers derived from tomato probes including Tg506(5’>cgcctgtgacctgtcctaa<3’/5’>ctgaaatggccaaagttgt<3’),Tg652(5’>atggccaaacaattgaacc<3’/5’>tgcatgccatgacttctca<3’),Tg516(5’>caaaaggctaggccaggaat <3’/ 5’>ggcacttcgcaaaactgaat <3’) produced the polymorphism between female and male parents. Among them, Tg65 was proved the tightly linkage with ps-2 gene by using F2 segregated population. The linkage distance is about 13.75 cM. In addition, four plasmids have been transferred into E. Coli. The stability of CAPs markers have been tested. All the above work will be benefit for the further research.
引文
http://www.healthoo.com ,2003,12
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Staniaszek M. Marczewski W. Habdas H. and Potaczek H. Identification of RAPD markers linked to the ps gene and their usefulness for purity determination of breeding lines and F1 tomato hybrids. Biotechnology and Physiology, 1999 : 303-306
Atanassova B. Functional male sterility(ps-2) in tomato(Lycopesicon esculentum Mill.) and its application in breeding and hybrid seed production. Euphytica ,1999,107:13-21
Atanassova B. Linkage studies of the “positional sterility-2”mutant in tomato. J. Genet. & Breed. 1991, 45:293-296
Atanassova B.and Georgiev H. Investigation of tomato male sterile lines in relation to hybrid seed production. Acta Horticulturae 1986,190:553-557
李君明, Bistra Atanassova, 徐和金, 周永健,杨宝军.番茄ps-2雄性不育品系的初步观察. 园艺学报, 2001,(4):381(382
李君明, 宋燕，徐和金,周永健.番茄ps-2雄性不育系01222的育性观察.中国农学通报，2003,2:16-18
Stoilova T. Cholakova N. and Danailov Z. Distinguishing among male sterile and fertile lines of tomatoes(Lycopersicon esculentum Mill) by means of electrophoretic protein patterns. Biotechnology and Physiology.1999,306-309
Kaul M L H. Male sterility in higher plants. Springger Verlag. Press.Belin. 1988.
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Bishop C J. A stamenless male sterile tomato.Am.J.Bot.. 1954, 41:540-542
Polowick P L,Sawhney V K. Ultrastucture of the tapetal cell wall in the stamenless-2 mutant of tomato (Lycopersicon esculentum):correlation between structure and male-sterility. Proloplasma.1995,189:249-255
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of Heredity.1983,74:51-54
Peet MM, Batholomew M. Effect of night temperature on pollen characteristics，growth and fruitset in tomato (Lycopersicon esculentum Mill.). Journal of the American Society for Horticulture Science. 1996,121:514-519
Singh S, Sawhney V. Abscisic acid in a male sterile tomato mutant and its regulation by low temperature. J. Exp. Bot. 1998, 49: 199-203
Pressman E, Peet MM, Pharr DM. The effect of heat stress on tomato pollen characteristics is associated with changes in carbohydrate concentration in the developing anthers. Annals of Botany. 2002, 90( 5): 631-636
Goetz M, Godt DE, Guivarc h A, et al.. Induction of male sterility in plants by metabolic engineering of the carbohydrate supply. Proceedings of the National Academy of Sciences of the United States of America. 2001, 98(11): 6522-6527
Fellner M , Sawhney V K. Seed germination in a tomato male-sterile mutant is resistant to osmotic， salt and low-temperature stresses. Theor Appl.2001,102:215-221
Singh S, Sawhney V K, Pearce D W. Temperature effects on endogenous indole-3-acetic acid leve;s in leaves and stamens of the normal and male sterile ‘stamenless-2” mutant of tomato (Lycopersicon esculentum Mill.).Plant Cell and Environment. 1992,15:373-377
Koen J. Van den Heuvel, Rosalinde H. Van Lipzig, Gerard W. Barendse, et al.. Wullems Regulation of expression of two novel flower-specific genes from tomato (Solanum lycopersicum) by gibberellin. J. Exp. Bot. 2002, 53: 51-59
何长征, 萧浪涛, 刘志敏, 等. 植物激素与雄性不育关系的研究进展. 中国农学通报. 2002,18(3):65-69
Santokh Singh, Sawhney V K. Abscisic acid in a male sterile tomato mutant and its regulation by low temperature. Journal of Experimental Botany.1998,49:199-203
Martin F,Ruichuan Zhang.,Richard P, et al.. Reduced de-etiolation of hypocotyls growth in a tomato mutant is assiociated with hypersensitivity to， and high endogenous levels of， abscisic acid. Journal of Experimental Botany.2001,52:725-738
Kaur G, Ahuja I, Banga S S. Association of endogenous level of hormones with male sterility expression in alloplasmic lines of Brassica Juncea(L.)Coss. Annals of Agri-Bio Research. 2002,7(2):175-179
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Rastogi R, Sawhney V K. Polyamines and flower development in the male sterile stamenless-2 mutant of tomato (Lycopersicon esculentum).II， Effects of polyamines and their biosynthesis inhibitors on the development of normal and mutant flowers cultured in vitro. Plant Physiol.1990, 93:446-452
Susan W Gorman, Barrasiak D, Fairley C, et al.. A 610 Kb yac clone harbors 7cM of tomato (lycopersicon esculentum) DNA that includes the male sterile 14 gene and a hotspot for recombination. Mol.Gen.Genet..1996,251:52-59
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Atanassova B. Functional male sterility(ps-2) in tomato(Lycopesicon esculentum Mill.) and its application in breeding and hybrid seed production. Euphytica ,1999,107:13-21
Atanassova B. Linkage studies of the “positional sterility-2”mutant in tomato. J. Genet. & Breed. 1991, 45:293-296
Atanassova B.and Georgiev H. Investigation of tomato male sterile lines in relation to hybrid seed production. Acta Horticulturae 1986,190:553-557
李君明, Bistra Atanassova, 徐和金, 周永健,杨宝军.番茄ps-2雄性不育品系的初步观察. 园艺学报, 2001,(4):381(382
李君明, 宋燕，徐和金,周永健.番茄ps-2雄性不育系01222的育性观察.中国农学通报，2003,2:16-18
Stoilova T. Cholakova N. and Danailov Z. Distinguishing among male sterile and fertile lines of tomatoes(Lycopersicon esculentum Mill) by means of electrophoretic protein patterns. Biotechnology and Physiology.1999,306-309
Kaul M L H. Male sterility in higher plants. Springger Verlag. Press.Belin. 1988.
Gabelman W H. Male sterility in vegetable breeding. Brookhaven symp. Biol.1956,9:113-122
Chetelat R T. Revised list of monogenic stocks.TGC Report .2002,52:41-68
Rick C M. Genetis and development of nine male-sterilre tomato mutants. Hilgardia.1948,18:599-633
Bishop C J. A stamenless male sterile tomato.Am.J.Bot.. 1954, 41:540-542
Polowick P L,Sawhney V K. Ultrastucture of the tapetal cell wall in the stamenless-2 mutant of tomato (Lycopersicon esculentum):correlation between structure and male-sterility. Proloplasma.1995,189:249-255
余诞年,吴定华,陈竹君.番茄遗传学.湖南：湖南科学技术出版社,1999.5
Rick C M,Boynton J E. A temperature-sensitive male-sterile mutant of the tomato. Am.J.Bot..1967,54:601-611
Sawhney V K. Temperature control of male sterility in a tomato mutant. The Journal
of Heredity.1983,74:51-54
Peet MM, Batholomew M. Effect of night temperature on pollen characteristics，growth and fruitset in tomato (Lycopersicon esculentum Mill.). Journal of the American Society for Horticulture Science. 1996,121:514-519
Singh S, Sawhney V. Abscisic acid in a male sterile tomato mutant and its regulation by low temperature. J. Exp. Bot. 1998, 49: 199-203
Pressman E, Peet MM, Pharr DM. The effect of heat stress on tomato pollen characteristics is associated with changes in carbohydrate concentration in the developing anthers. Annals of Botany. 2002, 90( 5): 631-636
Goetz M, Godt DE, Guivarc h A, et al.. Induction of male sterility in plants by metabolic engineering of the carbohydrate supply. Proceedings of the National Academy of Sciences of the United States of America. 2001, 98(11): 6522-6527
Fellner M , Sawhney V K. Seed germination in a tomato male-sterile mutant is resistant to osmotic， salt and low-temperature stresses. Theor Appl.2001,102:215-221
Singh S, Sawhney V K, Pearce D W. Temperature effects on endogenous indole-3-acetic acid leve;s in leaves and stamens of the normal and male sterile ‘stamenless-2” mutant of tomato (Lycopersicon esculentum Mill.).Plant Cell and Environment. 1992,15:373-377
Koen J. Van den Heuvel, Rosalinde H. Van Lipzig, Gerard W. Barendse, et al.. Wullems Regulation of expression of two novel flower-specific genes from tomato (Solanum lycopersicum) by gibberellin. J. Exp. Bot. 2002, 53: 51-59
何长征, 萧浪涛, 刘志敏, 等. 植物激素与雄性不育关系的研究进展. 中国农学通报. 2002,18(3):65-69
Santokh Singh, Sawhney V K. Abscisic acid in a male sterile tomato mutant and its regulation by low temperature. Journal of Experimental Botany.1998,49:199-203
Martin F,Ruichuan Zhang.,Richard P, et al.. Reduced de-etiolation of hypocotyls growth in a tomato mutant is assiociated with hypersensitivity to， and high endogenous levels of， abscisic acid. Journal of Experimental Botany.2001,52:725-738
Kaur G, Ahuja I, Banga S S. Association of endogenous level of hormones with male sterility expression in alloplasmic lines of Brassica Juncea(L.)Coss. Annals of Agri-Bio Research. 2002,7(2):175-179
Galston A W, Sawhney R K. Polyamines in plant physiology. Plant Physiol.1990,94:406-410
Rastogi R, Sawhney V K. Polyamines and flower development in the male sterile stamenless-2 mutant of tomato (Lycopersicon esculentum).I， Level of polyamines and their biosynthesis in normal and mutant flowers. Plant Phsiol. 1990,93:439-445
Rastogi R, Sawhney V K. Polyamines and flower development in the male sterile stamenless-2 mutant of tomato (Lycopersicon esculentum).II， Effects of polyamines and their biosynthesis inhibitors on the development of normal and mutant flowers cultured in vitro. Plant Physiol.1990, 93:446-452
Susan W Gorman, Barrasiak D, Fairley C, et al.. A 610 Kb yac clone harbors 7cM of tomato (lycopersicon esculentum) DNA that includes the male sterile 14 gene and a hotspot for recombination. Mol.Gen.Genet..1996,251:52-59
Staniaszek M, Marczewski W, Habdas H, et al.. Identification of RAPD markers linked to the ps-gene and their usefulness for purity determination of breeding lines and F1 tomato hybrids. Biotechnology and Physiology .1998,303-305
Mariani C, Beuckeleer De, Truettner M J, et al.. Induction of male sterility in plants by a chimaeric ribonucleasegene.Nature.1990,347:737～741
赵广荣,宋远佞,邵景成, 等. 雄性不育基因工程及番茄杂种优势利用.甘肃农业科技.2000, 2：32-34
Burgess D G, Ralston E J, Hanson W G, et al.. A novel two –component system for cell lethality and its use in engineering nuclear male-sterility in plants. The Plant Journal. 31(1):113-125
Georgiev Hr. Heterosis in tomato breeding. In: prof Kalloo(ED),Genetic Improvement of Tomato,1991,99:83-98
Hong SB, Tucker ML. Molecular characterization of a tomato polygalacturonase gene abundantly expressed in the upper third of pistils from opened and unopened flowers. Plant Cell Reports. 2000, 19(7): 680-683
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