油豆角（Phaseolus vulgaris.L.）主要种质资源亲缘关系的RAPD分析
摘要
本研究通过田间观察实验和分子生物学实验，以27份菜豆品种作为试材，从生物学特征，RAPD分子标记两个方面进行了系统研究和探讨。结果如下：
1) 田间实验表明，油豆角是食荚菜豆中的优良变异品种群，是以其商品性为目标多年选择、变异的结果。黑龙江省油豆角种质资源丰富，但品种命名存在不系统，无规则的现象。在生物学特征方面，寻找能够在各个生长时期将油豆角从普通菜豆中区别出来的标记性状。实验结果表明，油豆角的花色是一个要深入研究的标记性状。
2) 在分子生物学实验部分，利用改良的SDS法提取DNA效果较好，利用RNA酶将可能杂质RNA的干扰去掉，仔细操作，获得在常温下可以长期保存的DNA。优化的RAPD扩增体系为：反应总体积25μl，其中含10倍扩增缓冲液2.5μl，Mg~(2+)2.5mmol／l，dNTP300μmol／l，引物为0.1mmol／l，基因组DNA为60ng，Taq酶1.5U，其他成分为无菌双蒸水。优化的程序为：94℃预变性10min，94℃变性1min，37℃退火1min，72℃延长1min，40个循环，72℃延长7min。用筛选出的14个引物对27品种进行了RAPD分析，共获得114条谱带，其中107条为多态带，多态百分率为89.9％。以遗传相似度0.690为阈值，经UPGMA聚类分析，可以将油豆角和普通菜豆分为两大类。以0.650为阕值，可以将油豆角和普通菜豆分为七大类。这与地理起源说基本吻合。
3) 分子生物学实验阶段发现引物SBSF-5可以将27份实验材料中的25个清晰准确地鉴别出来，具有一定的实用价值和商业前景。
The experiments were on the basis of the observation in the fields. 15 cultivars were selected out from 27 special cultivars . judging by biological characters and performance, which includes 12 for vine. and 3 for dwarf. The total amount of 27 cuhivars was systematically studied and discussed for two aspects: biological characters and RAPD molecular markers. The results showed:
1) The experiments on fresh bean in the fields showed that special local beans of Heilongjiang Province are good varieties of common bean, and they are the results of seed-selection of decades of years.. The natural resources of them in Heilongjiang province were abundant, but no systematically nomenclature existed. On the respect of biological characters, some color of blosom can distinguish them from common beans during the different periods of growth.
2) In the section of molecular biological experiments, the modified SDS method which was used to extract DNA got good results. Using RNAse could eliminate the disturbance of RNA. and got the qualified DNA with a long term of validity. The optimized RAPD systems were: total reaction volume 251. including 10 times amplification buffer solution 2.51. Mg2+ 2.5mmol/1. dNTP 300 mol/l, 0.1mmol/1, genome DNA 60ng. Taq enzymel.5U. Optimized program: 94 C 10 min; 94 C 1 min ,37 C 1 min, 72 C1mm . forty cycles; 72 C 7min one cycle; 4 C keeping. 27 cultivars were detected by RAPD through 14 selected arbitrary primers. A total of 117 loci were amplified among which 104 loci were polymorphic with a mean of bands per primer. The ratio of polymorphism is 89.9%. The genetic similar degree is 6.90 as threshold. Cluster (UPGMA) and principal coordinate analyses identified beans of Heilongjiang province as a distinct group with the rest. The result is in harmony with biological feature.Genetic similar degree is 6.5 as threshold,cluster (UPGMA) and coordinate analysis identified beans as 7groups which is similar to geographical areas.
3) The primer SBSF-5 can distinguished 25 cultivars by RAPD method.
Candidate:SunXiaodan Mayor. Vegetable breeding
Supervisor: Prof.QinZhiwei
1) 田间实验表明，油豆角是食荚菜豆中的优良变异品种群，是以其商品性为目标多年选择、变异的结果。黑龙江省油豆角种质资源丰富，但品种命名存在不系统，无规则的现象。在生物学特征方面，寻找能够在各个生长时期将油豆角从普通菜豆中区别出来的标记性状。实验结果表明，油豆角的花色是一个要深入研究的标记性状。
2) 在分子生物学实验部分，利用改良的SDS法提取DNA效果较好，利用RNA酶将可能杂质RNA的干扰去掉，仔细操作，获得在常温下可以长期保存的DNA。优化的RAPD扩增体系为：反应总体积25μl，其中含10倍扩增缓冲液2.5μl，Mg~(2+)2.5mmol／l，dNTP300μmol／l，引物为0.1mmol／l，基因组DNA为60ng，Taq酶1.5U，其他成分为无菌双蒸水。优化的程序为：94℃预变性10min，94℃变性1min，37℃退火1min，72℃延长1min，40个循环，72℃延长7min。用筛选出的14个引物对27品种进行了RAPD分析，共获得114条谱带，其中107条为多态带，多态百分率为89.9％。以遗传相似度0.690为阈值，经UPGMA聚类分析，可以将油豆角和普通菜豆分为两大类。以0.650为阕值，可以将油豆角和普通菜豆分为七大类。这与地理起源说基本吻合。
3) 分子生物学实验阶段发现引物SBSF-5可以将27份实验材料中的25个清晰准确地鉴别出来，具有一定的实用价值和商业前景。
The experiments were on the basis of the observation in the fields. 15 cultivars were selected out from 27 special cultivars . judging by biological characters and performance, which includes 12 for vine. and 3 for dwarf. The total amount of 27 cuhivars was systematically studied and discussed for two aspects: biological characters and RAPD molecular markers. The results showed:
1) The experiments on fresh bean in the fields showed that special local beans of Heilongjiang Province are good varieties of common bean, and they are the results of seed-selection of decades of years.. The natural resources of them in Heilongjiang province were abundant, but no systematically nomenclature existed. On the respect of biological characters, some color of blosom can distinguish them from common beans during the different periods of growth.
2) In the section of molecular biological experiments, the modified SDS method which was used to extract DNA got good results. Using RNAse could eliminate the disturbance of RNA. and got the qualified DNA with a long term of validity. The optimized RAPD systems were: total reaction volume 251. including 10 times amplification buffer solution 2.51. Mg2+ 2.5mmol/1. dNTP 300 mol/l, 0.1mmol/1, genome DNA 60ng. Taq enzymel.5U. Optimized program: 94 C 10 min; 94 C 1 min ,37 C 1 min, 72 C1mm . forty cycles; 72 C 7min one cycle; 4 C keeping. 27 cultivars were detected by RAPD through 14 selected arbitrary primers. A total of 117 loci were amplified among which 104 loci were polymorphic with a mean of bands per primer. The ratio of polymorphism is 89.9%. The genetic similar degree is 6.90 as threshold. Cluster (UPGMA) and principal coordinate analyses identified beans of Heilongjiang province as a distinct group with the rest. The result is in harmony with biological feature.Genetic similar degree is 6.5 as threshold,cluster (UPGMA) and coordinate analysis identified beans as 7groups which is similar to geographical areas.
3) The primer SBSF-5 can distinguished 25 cultivars by RAPD method.
Candidate:SunXiaodan Mayor. Vegetable breeding
Supervisor: Prof.QinZhiwei
引文
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