甘蓝迟抽薹基因的RAPD标记及种质资源的筛选
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摘要
甘蓝是一种世界性蔬菜，由于其具有适应性广，耐寒性强和一定的耐热性等特点，因此在引入我国后发展很快，分布也很广。但近些年来，由于气候条件的反常、栽培方式的变更，甘蓝未熟抽薹的现象时有发生，为解决这一问题，最经济有效的手段就是培育出迟抽薹甘蓝品种。本试验是利用混合分离群体法（Bulked Segregant Analysis,BSA）将F2群体中研究的抽薹性状的个体分为两组，构建两DNA池，然后利用RAPD标记分析，在两个混合池之间寻找与甘蓝迟抽薹基因连锁的分子标记，并利用该标记来鉴定22份甘蓝材料的抗抽薹性。本试验具体得出如下结论：
1. 以亲本抗抽薹材料C和亲本易抽薹材料F在春化处理后的抽薹时间为标准，确定了F2群体在春化处理后，抽薹情况是，早抽薹植株：中间型：晚抽薹植株为1：2：1，因此得出了甘蓝的抽薹性状虽然为数量性状，但在迟抽薹基因中可能存在一个类似于质量性状的主效基因。
2. 通过亲本C、F对105条随机引物进行筛选，共有87个引物获得了扩增产物，出现的谱带总数为592条。从中选出具有多态性的引物共19条，其中在两DNA池中扩增出条带清晰，重复性、多态性好的引物只有一条即N1。N1引物在两DNA池之间扩增出一差异片段N1-750，经DNA池的各单株及F2两极端群体的单株检测，N1-750确实与甘蓝迟抽薹基因相连锁，连锁距离为7.9cM。
3. 用筛选出来的19个引物对22份材料进行RAPD分析，其中6个引物扩增出的条带清晰，重复性好，共得到44条多态性条带。经UPGMA聚类分析，以遗传距离0.627为阈值，将供试的22份材料聚为七类。
4. 用已标记出的连锁基因N1-750验证了材料a、b、c和d为抗抽薹材料，u和v属于易抽薹材料，同时鉴定出e、g、i、j、k和m属于抗抽薹材料。
Cabbage is a popular kind of vegetable in the word with the characters of cold-resistant and hot-resistant. So it is widespread in china and developed fast after being introduced. But the phenomenon that crudity cabbage bolting happened frequently because climate was abnormal and cultivating patterns changed. The most economic medium solving the problem is to cultivate a variety of cabbage bolting late. This experiment used the method of bulked segregant analysis, divided bolting characters of F2 groups into two sections and established DNA pool. In order to find the gene linked together with that bolting late in the mix DNA pool by RAPD Marker analysis, further appraise the characters of resisting bolting. The main results were as follows:
First, after F2 plants were treated with vernalization, the ratio of bolting was 1:2:1 among early bolting variety, medium variety and late variety by the standard of bolting time of resisting bolting and easy bolting parent material after treated with vernalization. So there may be main gene similar to quality characters despite being a quantity character.
Second, through Parent C and parent F, 105 random primer were selected, and 87 primer acquired amplified products, 592 bands were gotten in all, 19 bands were polymorphism., and the good polymorphism and repeating primer with clear bands was only one, which was N1 in the two DNA poles. N1 amplified one different fragment that was N1-750, which linking cabbage bolting gene was confirmed through examining the individual plant of the two DNA poles and F2 group at each ends, and link distance was 7.9cM.
Third, through RAPD method 22 materials were analyzed by selected 19 primers, only 6 primers were amplified clear and repeating bands, which got 44 polymorphism bands. through UPGMA analysis, 22 materials were categorized seven kinds according to the threshold that is the inheritance distance of 0.627.
Forth, by N1-750 which is the marked linking gene. a, b, c and d were confirmed resisting bolting materials; u and v is liable bolting materials; and e, g, i, j, k and m were appraised resisting bolting materials.
Postgraduate: Cao Weirong
Speciality: Genetics and Breeding
Supervisor: Prof.Wang Chao
1. 以亲本抗抽薹材料C和亲本易抽薹材料F在春化处理后的抽薹时间为标准，确定了F2群体在春化处理后，抽薹情况是，早抽薹植株：中间型：晚抽薹植株为1：2：1，因此得出了甘蓝的抽薹性状虽然为数量性状，但在迟抽薹基因中可能存在一个类似于质量性状的主效基因。
2. 通过亲本C、F对105条随机引物进行筛选，共有87个引物获得了扩增产物，出现的谱带总数为592条。从中选出具有多态性的引物共19条，其中在两DNA池中扩增出条带清晰，重复性、多态性好的引物只有一条即N1。N1引物在两DNA池之间扩增出一差异片段N1-750，经DNA池的各单株及F2两极端群体的单株检测，N1-750确实与甘蓝迟抽薹基因相连锁，连锁距离为7.9cM。
3. 用筛选出来的19个引物对22份材料进行RAPD分析，其中6个引物扩增出的条带清晰，重复性好，共得到44条多态性条带。经UPGMA聚类分析，以遗传距离0.627为阈值，将供试的22份材料聚为七类。
4. 用已标记出的连锁基因N1-750验证了材料a、b、c和d为抗抽薹材料，u和v属于易抽薹材料，同时鉴定出e、g、i、j、k和m属于抗抽薹材料。
Cabbage is a popular kind of vegetable in the word with the characters of cold-resistant and hot-resistant. So it is widespread in china and developed fast after being introduced. But the phenomenon that crudity cabbage bolting happened frequently because climate was abnormal and cultivating patterns changed. The most economic medium solving the problem is to cultivate a variety of cabbage bolting late. This experiment used the method of bulked segregant analysis, divided bolting characters of F2 groups into two sections and established DNA pool. In order to find the gene linked together with that bolting late in the mix DNA pool by RAPD Marker analysis, further appraise the characters of resisting bolting. The main results were as follows:
First, after F2 plants were treated with vernalization, the ratio of bolting was 1:2:1 among early bolting variety, medium variety and late variety by the standard of bolting time of resisting bolting and easy bolting parent material after treated with vernalization. So there may be main gene similar to quality characters despite being a quantity character.
Second, through Parent C and parent F, 105 random primer were selected, and 87 primer acquired amplified products, 592 bands were gotten in all, 19 bands were polymorphism., and the good polymorphism and repeating primer with clear bands was only one, which was N1 in the two DNA poles. N1 amplified one different fragment that was N1-750, which linking cabbage bolting gene was confirmed through examining the individual plant of the two DNA poles and F2 group at each ends, and link distance was 7.9cM.
Third, through RAPD method 22 materials were analyzed by selected 19 primers, only 6 primers were amplified clear and repeating bands, which got 44 polymorphism bands. through UPGMA analysis, 22 materials were categorized seven kinds according to the threshold that is the inheritance distance of 0.627.
Forth, by N1-750 which is the marked linking gene. a, b, c and d were confirmed resisting bolting materials; u and v is liable bolting materials; and e, g, i, j, k and m were appraised resisting bolting materials.
Postgraduate: Cao Weirong
Speciality: Genetics and Breeding
Supervisor: Prof.Wang Chao
引文
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盖树鹏，孟祥栋.利用RAPD技术进行植物性状标记及辅助选择.生物技术通报.1999，6：33–37
郭蓓，邱丽娟，邵桂花等.大豆耐盐基因的PCR标记.中国农业科学.2000，33（1）：10–16
姜自锋，林乃铨，徐梅.RAPD技术及其应用中的一些问题.福建农林大学学报.2002，31（3）：356–360
贾继增.分子标记种质资源鉴定和分子标记育种.中国农业科学.1996,29(4): 1–10
李竟雄,宋同明著.植物细胞遗传学.北京:科学出版社.1997,1–51
李丽敏. 春早熟甘蓝未熟抽薹的原因及防止对策.中国蔬菜.2001（3）：40–41
李庆典，程斐.大白菜抽薹特性的研究进展.莱阳农学院学报.1997,14（4）：265–268
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刘春林，官春云.植物RAPD标记的可靠性研究.生物技术通报.1999（2）：31–32
刘旭.遗传标记和遗传图谱构建.作物品种资源.1997,3: 29–32
刘勋甲，郑用琏，尹艳.遗传标记的发展及分子标记在农作物遗传育种中的应用Ⅱ.分子标记在农作物遗传育种中的应用及原理.湖北农业科学.1998(3): 27–32
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师建领.采种方法对春甘蓝未熟抽薹现象的影响.种子科技.1999（3）
宋洪元,雷建军,李成琼等.应用RAPD标记鉴定结球甘蓝品种.西南农业大学学报.2002,24（1）：38–41
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