中国野生葡萄抗寒基因RAPD标记的研究
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摘要
2000年～2002年，通过田间葡萄杂交与播种育苗，获得玫瑰香×黑龙江实生的F_1代19株及北醇自交后代47株。运用RAPD技术，对中国野生葡萄12个种58个株系、欧洲葡萄20个品种、欧山杂交品种1个、欧美杂交品种2个、河岸葡萄2个株系及三个杂交组合(玫瑰香×黑龙江实生、北醇(?)、燕山(♀)×河岸-3)的122株F_1代共计205份材料进行了中国野生山葡萄抗寒基因分子标记研究。主要结果如下：
1．按照前人对中国野生葡萄抗寒性较系统的鉴定分析，首先以极抗寒亲本黑龙江实生(山葡萄)、不抗寒亲本玫瑰香(欧洲葡萄)及抗寒欧山杂种北醇为模板筛选引物。通过对110个随机引物的筛选，获得7个特异性引物。进一步用7个特异性引物对15份极抗寒野生葡萄种质及部分不抗寒野生种质扩增，结果显示，引物S241在供试的山葡萄9个株系及燕山(♀)中扩增出一条约670bP特异性DNA片段；另一个引物S238在山葡萄的6个株系中扩增出一条约800 bp特异性DNA片段。然后以三个杂交组合玫瑰香×黑龙江实生、北醇(?)及燕山(♀)×河岸-3的122株F_1代及其亲本为模板，引物S241分别在三个组合的部分后代中扩增出来自极抗寒亲本(山葡萄、燕山葡萄)的670 bp特异性DNA片段；引物S238分别在前两个组合的部分后代中扩增出来自极抗寒亲本(山葡萄)的800 bp特异性DNA片段，这表明S241_(670)和S238_(800)是与中国野生葡萄抗寒基因相连锁的RAPD标记。进一步以欧洲葡萄品种、中国野生葡萄株系、河岸葡萄株系、欧美杂种为试材，对所获RAPD标记的遗传特性进行了研究。结果证明这两个RAPD标记是来自中国野生葡萄山葡萄，且可以向后代遗传。抗寒基因RAPD标记也可以用作山葡萄做亲本的抗寒育种分子标记辅助选择的依据。RAPD标记S241_(670)。较S238_(800)有着更广泛的辅助选择作用。
2．中国野生葡萄抗寒基因RAPD标记的分离与遗传表现，表明抗寒性是多基因控制的数量性状遗传。
3．建立了适于葡萄抗寒基因RAPD分析的反应体系：在25μl反应混合液中，含模板DNA 25 ng，Mg~(2+)2.5 mM，dNTPs 0.25mM，Taq DNA聚合酶1.0 U，随机引物1μl。
19 hybrids derived from cross combination Muscat Hamburg * Heilongjiang seedling and 47 individuals of self-pollination cross of Beichun were obtained during 2000~ 2002. At the same time, RAPD markers linked to the hardiness genes of V. amuremis in Chinese wild Vitis were studied by using 205 plants, including 58 clones of 12 Chinese wild Vitis species, 20 cultivars of V. vinifera, a cultivar Beichun (V. vinifera x V. amnrensis), 2 cultivars from V. vinifera V. labrnsca, 2 clones of V. riparia and 122 FI individuals of the three crosses. The results were as follows:
1.According to the analysis of hardiness for Chinese wild Vitis identified systematically by other researchers, the three type DNA templates of grapes, including of the parent Heilongjiang seedling of V. amnrensis (the highest tolerance to hardiness) and another parent Muscat Hamburg of V. vinifera (the lower tolerance to hardiness), and a cultivar Beichun derived from cross V. vinifera x V. amnrensis (the higher tolerance to hardiness), were amplified and obtained polymorphic DNA band by seven primers of 110 random primers. Additionally, 15 DNA samples of the highest hardiness and some samples of lower hardiness among Chinese wild Vitis were amplified using these seven primers, and the specific fragment 670 bp appeared in 10 clones of V. amnrensis and Yanshan( ) of V. yeshanensis by primer S241, another specific fragment 800 bp appeared in 6 clones of V. amnrensis by primer S238. 122 FI individuals and their parents in the three crosses, including Muscat Hamburg x Heilongjiang seedling, Beichun x Beichun, a
nd Yanshan() x Hean-3, were amplified by the primers S241 and S238, and the specific DNA fragment S241e7o presented in some offspring of the three crosses and the specific DNA fragment S238soo presented in some offspring of the former two crosses, respectively. Therefore, The RAPD markers, S241670 and S238800 which derived from hardy parent, are linked to the hardiness gene in V. amnrensis of the Chinese wild Vitis. Thus, S241670 and S238800 could be used as the evidence for molecular marked-assisted selection in grape breeding of hardiness.
2.Based on the characteristic of the segregation and inheritance of the two RAPD markers linked to the hardiness genes in Chinese wild Vitis, it is suggested that hardiness
inheritance of grapes was a quantitatively inherited character controlled by polygenes.
3. An optimal RAPD reaction system was obtained which adapted to the RAPD analysis of hardiness gene in grapes. The optimized system include 25ng template DNA, 2.5mM Mg2+ concentration, 0.25mM dNTPs concentration, 1U Taq DNA polymerase and ml random primer in 25ul mixed solution.
1．按照前人对中国野生葡萄抗寒性较系统的鉴定分析，首先以极抗寒亲本黑龙江实生(山葡萄)、不抗寒亲本玫瑰香(欧洲葡萄)及抗寒欧山杂种北醇为模板筛选引物。通过对110个随机引物的筛选，获得7个特异性引物。进一步用7个特异性引物对15份极抗寒野生葡萄种质及部分不抗寒野生种质扩增，结果显示，引物S241在供试的山葡萄9个株系及燕山(♀)中扩增出一条约670bP特异性DNA片段；另一个引物S238在山葡萄的6个株系中扩增出一条约800 bp特异性DNA片段。然后以三个杂交组合玫瑰香×黑龙江实生、北醇(?)及燕山(♀)×河岸-3的122株F_1代及其亲本为模板，引物S241分别在三个组合的部分后代中扩增出来自极抗寒亲本(山葡萄、燕山葡萄)的670 bp特异性DNA片段；引物S238分别在前两个组合的部分后代中扩增出来自极抗寒亲本(山葡萄)的800 bp特异性DNA片段，这表明S241_(670)和S238_(800)是与中国野生葡萄抗寒基因相连锁的RAPD标记。进一步以欧洲葡萄品种、中国野生葡萄株系、河岸葡萄株系、欧美杂种为试材，对所获RAPD标记的遗传特性进行了研究。结果证明这两个RAPD标记是来自中国野生葡萄山葡萄，且可以向后代遗传。抗寒基因RAPD标记也可以用作山葡萄做亲本的抗寒育种分子标记辅助选择的依据。RAPD标记S241_(670)。较S238_(800)有着更广泛的辅助选择作用。
2．中国野生葡萄抗寒基因RAPD标记的分离与遗传表现，表明抗寒性是多基因控制的数量性状遗传。
3．建立了适于葡萄抗寒基因RAPD分析的反应体系：在25μl反应混合液中，含模板DNA 25 ng，Mg~(2+)2.5 mM，dNTPs 0.25mM，Taq DNA聚合酶1.0 U，随机引物1μl。
19 hybrids derived from cross combination Muscat Hamburg * Heilongjiang seedling and 47 individuals of self-pollination cross of Beichun were obtained during 2000~ 2002. At the same time, RAPD markers linked to the hardiness genes of V. amuremis in Chinese wild Vitis were studied by using 205 plants, including 58 clones of 12 Chinese wild Vitis species, 20 cultivars of V. vinifera, a cultivar Beichun (V. vinifera x V. amnrensis), 2 cultivars from V. vinifera V. labrnsca, 2 clones of V. riparia and 122 FI individuals of the three crosses. The results were as follows:
1.According to the analysis of hardiness for Chinese wild Vitis identified systematically by other researchers, the three type DNA templates of grapes, including of the parent Heilongjiang seedling of V. amnrensis (the highest tolerance to hardiness) and another parent Muscat Hamburg of V. vinifera (the lower tolerance to hardiness), and a cultivar Beichun derived from cross V. vinifera x V. amnrensis (the higher tolerance to hardiness), were amplified and obtained polymorphic DNA band by seven primers of 110 random primers. Additionally, 15 DNA samples of the highest hardiness and some samples of lower hardiness among Chinese wild Vitis were amplified using these seven primers, and the specific fragment 670 bp appeared in 10 clones of V. amnrensis and Yanshan( ) of V. yeshanensis by primer S241, another specific fragment 800 bp appeared in 6 clones of V. amnrensis by primer S238. 122 FI individuals and their parents in the three crosses, including Muscat Hamburg x Heilongjiang seedling, Beichun x Beichun, a
nd Yanshan() x Hean-3, were amplified by the primers S241 and S238, and the specific DNA fragment S241e7o presented in some offspring of the three crosses and the specific DNA fragment S238soo presented in some offspring of the former two crosses, respectively. Therefore, The RAPD markers, S241670 and S238800 which derived from hardy parent, are linked to the hardiness gene in V. amnrensis of the Chinese wild Vitis. Thus, S241670 and S238800 could be used as the evidence for molecular marked-assisted selection in grape breeding of hardiness.
2.Based on the characteristic of the segregation and inheritance of the two RAPD markers linked to the hardiness genes in Chinese wild Vitis, it is suggested that hardiness
inheritance of grapes was a quantitatively inherited character controlled by polygenes.
3. An optimal RAPD reaction system was obtained which adapted to the RAPD analysis of hardiness gene in grapes. The optimized system include 25ng template DNA, 2.5mM Mg2+ concentration, 0.25mM dNTPs concentration, 1U Taq DNA polymerase and ml random primer in 25ul mixed solution.
引文
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