23个油橄榄品种的RAPD分析
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摘要
本研究采用RAPD技术对23个引种的油橄榄品种进行分类和鉴定研究。首先针对油橄榄基因组DNA的提取改良基因组DNA的提取方法，以获得较高质量的油橄榄基因组DNA用于RAPD分析。然后对油橄榄RAPD反应体系体系进行优化，以建立最佳RAPD扩增体系，筛选出适合油橄榄RAPD分析的引物对样品DNA进行PCR扩增。对样品的RAPD图谱进行分析，获得各样品的RAPD分子特征，最后统计和分析数据，同时对样品间的亲缘关系进行分析和讨论。研究结果如下：
(1)本研究采用改良CTAB法，分别从新鲜和硅胶保存的油橄榄嫩叶中提取DNA，与其他部位提取效果相比量大，纯度高、褐化程度低；得到的DNA产率达到194—352ng／mg，OD_(260)／OD_(280)为1.82—2.08。通过琼脂糖凝胶电泳分析，DNA完整性较好，达到RAPD试验的要求。
(2)建立了油橄榄RAPD最佳反应体系：PCR扩增的总体积为20μL，包括40ng的模板DNA，10×PCR buffer 2μL，1.5mmol／L MgCl_2，0.2mmol／L dNTP，0.25μmol／L引物，Taq酶1U。扩增程序为：94℃预变性2min，94℃变性45s，36℃退火1min，72℃2min，40个循环后在72℃延伸4min，结束后在4℃条件下保存。最后用1.5％的琼脂糖凝胶电泳检测其扩增产物。
(3)采用优化后的反应体系对80个引物进行筛选，经过三次重复，筛选出多态性和扩增重复性好、谱带清晰且较多的11个引物，用于油橄榄全部DNA样品的RAPD分析。
(4)应用筛选出11个引物进行扩增，共产生127条带，其中78条为多态性带，占61.4％，平均每个引物扩增的DNA带数为11.55条，多态性DNA的带数为7.09条。扩增出的DNA片段在300—2000 bp之间，其中尤以500—1200 bp之间最多。根据扩增结果建立了品种间亲缘关系的UPGMA聚类图，将23个品种可划分为2大类。
(5)对23个油橄榄品种的分子特征进行了分析，获得了4个品种的RAPD特异标记即阿维金娜在S_(1011)—600bp、小果卡林在S_(1019)—1400bp和S_(1025)—2400bp、佛奥在S_(1025)—400bp以及科拉蒂娜在S_(1011)—650bp分别有各自的特异带。
Random amplified polymorphic DNA (RAPD) was used to study the classificationand identification of 23 olive (Olea europaea L.) cultivars. Firstly, the method of DNAextraction was modified for olive DNA extraction to acquire high quality olive DNA thatwas used for RAPD analysis. Secondly, RAPD reaction systems were selected forestablishing optimal RAPD ampification system. The primers that were sieved were usedfor sample DNA PCR amplfication. Finally, RAPD fmgerprint maps and experiment datawere counted and analysed to acquire molecule characteristic of olive samples.Phylogenetic relationships of twenty three olive varieties were studied based onphylogenetic tree by use of UPGMA method.
The main results of this research are showed as follows
①The improved CTAB method was the best one for the extraction of olive genomicDNA. DNA could be extracted from fresh young leaves and young leaves that werepreserved by silica gel. DNA from young and tender leaves had better purity and the outputwas high: the output was 194-352ng/mg, OD_(260)/OD_(280) of DNA was1.82-2.08. Theintegrality of olive genomic DNA was preferable which was detected by agarose gelelectrophoresis. The extracted olive genomic DNA was satisfactory for RAPD analysis.
②Optimal ampification system of RAPD was established. DNA was amplified in 20μL using the reaction mixtures containing 1×PCR buffer, 1.5mmol/L MgCl_2, 0.2mmol/Lof dNTPs, 1U of Taq Polymerase, 0.25μmol/L of primer, 40ng of template DNA. The PCRreactions were performed in a thermal cycler (PTC-100) programmed for 1 cycle of 2 minat 94℃followed by 40 cycles of 45 sec at 94℃, 1 min at 36℃and 2 min at 72℃for denaturing, primer annealing and extension, respectively. The last cycle was followed byincubation for 4 min at 72℃. Amplification products were conserved at 4℃. AmplificationRAPD products were analysed by gel electrophoresis run in 1.5％agarose in 0.5×TBEbuffer.
③The 11 primers which produced the highest number of polymorphic bands andshowed consistent and reproducible results were sieved from eighty 10bp primers usingoptimal ampification system. All the reactions were performed three times.
④Eleven 10bp primers which were selected from eighty arbitrary primers wereapplied to the amplification. Total 127 bands were produced, in which 78 bands (61.4％)were polymorphic. The average numbers of DNA bands and polymorphic DNA bandsamplified by each primer were 11.55 and 7.09 respectively. Amplification DNA segmentsize was between 300bp and 2000bp, and the many segments centralized between 500bpand 1500bp. A dendrogram showing genetic relationships was constructed through anunweighted pair-group method (UPGMA) and the 23 cultivars were clustered into 2 maingroups.
⑤There were specific bands in four cultivars, ie Arbequina S_(1011)-600bp, KaliniotS_(1019)-1400bp and S_(1025)-2400bp, Frantoio S_(1025)-400bp, Coratina S_(1011)-650bp, and thesebands can be used in olive germplasm identification.
(1)本研究采用改良CTAB法，分别从新鲜和硅胶保存的油橄榄嫩叶中提取DNA，与其他部位提取效果相比量大，纯度高、褐化程度低；得到的DNA产率达到194—352ng／mg，OD_(260)／OD_(280)为1.82—2.08。通过琼脂糖凝胶电泳分析，DNA完整性较好，达到RAPD试验的要求。
(2)建立了油橄榄RAPD最佳反应体系：PCR扩增的总体积为20μL，包括40ng的模板DNA，10×PCR buffer 2μL，1.5mmol／L MgCl_2，0.2mmol／L dNTP，0.25μmol／L引物，Taq酶1U。扩增程序为：94℃预变性2min，94℃变性45s，36℃退火1min，72℃2min，40个循环后在72℃延伸4min，结束后在4℃条件下保存。最后用1.5％的琼脂糖凝胶电泳检测其扩增产物。
(3)采用优化后的反应体系对80个引物进行筛选，经过三次重复，筛选出多态性和扩增重复性好、谱带清晰且较多的11个引物，用于油橄榄全部DNA样品的RAPD分析。
(4)应用筛选出11个引物进行扩增，共产生127条带，其中78条为多态性带，占61.4％，平均每个引物扩增的DNA带数为11.55条，多态性DNA的带数为7.09条。扩增出的DNA片段在300—2000 bp之间，其中尤以500—1200 bp之间最多。根据扩增结果建立了品种间亲缘关系的UPGMA聚类图，将23个品种可划分为2大类。
(5)对23个油橄榄品种的分子特征进行了分析，获得了4个品种的RAPD特异标记即阿维金娜在S_(1011)—600bp、小果卡林在S_(1019)—1400bp和S_(1025)—2400bp、佛奥在S_(1025)—400bp以及科拉蒂娜在S_(1011)—650bp分别有各自的特异带。
Random amplified polymorphic DNA (RAPD) was used to study the classificationand identification of 23 olive (Olea europaea L.) cultivars. Firstly, the method of DNAextraction was modified for olive DNA extraction to acquire high quality olive DNA thatwas used for RAPD analysis. Secondly, RAPD reaction systems were selected forestablishing optimal RAPD ampification system. The primers that were sieved were usedfor sample DNA PCR amplfication. Finally, RAPD fmgerprint maps and experiment datawere counted and analysed to acquire molecule characteristic of olive samples.Phylogenetic relationships of twenty three olive varieties were studied based onphylogenetic tree by use of UPGMA method.
The main results of this research are showed as follows
①The improved CTAB method was the best one for the extraction of olive genomicDNA. DNA could be extracted from fresh young leaves and young leaves that werepreserved by silica gel. DNA from young and tender leaves had better purity and the outputwas high: the output was 194-352ng/mg, OD_(260)/OD_(280) of DNA was1.82-2.08. Theintegrality of olive genomic DNA was preferable which was detected by agarose gelelectrophoresis. The extracted olive genomic DNA was satisfactory for RAPD analysis.
②Optimal ampification system of RAPD was established. DNA was amplified in 20μL using the reaction mixtures containing 1×PCR buffer, 1.5mmol/L MgCl_2, 0.2mmol/Lof dNTPs, 1U of Taq Polymerase, 0.25μmol/L of primer, 40ng of template DNA. The PCRreactions were performed in a thermal cycler (PTC-100) programmed for 1 cycle of 2 minat 94℃followed by 40 cycles of 45 sec at 94℃, 1 min at 36℃and 2 min at 72℃for denaturing, primer annealing and extension, respectively. The last cycle was followed byincubation for 4 min at 72℃. Amplification products were conserved at 4℃. AmplificationRAPD products were analysed by gel electrophoresis run in 1.5％agarose in 0.5×TBEbuffer.
③The 11 primers which produced the highest number of polymorphic bands andshowed consistent and reproducible results were sieved from eighty 10bp primers usingoptimal ampification system. All the reactions were performed three times.
④Eleven 10bp primers which were selected from eighty arbitrary primers wereapplied to the amplification. Total 127 bands were produced, in which 78 bands (61.4％)were polymorphic. The average numbers of DNA bands and polymorphic DNA bandsamplified by each primer were 11.55 and 7.09 respectively. Amplification DNA segmentsize was between 300bp and 2000bp, and the many segments centralized between 500bpand 1500bp. A dendrogram showing genetic relationships was constructed through anunweighted pair-group method (UPGMA) and the 23 cultivars were clustered into 2 maingroups.
⑤There were specific bands in four cultivars, ie Arbequina S_(1011)-600bp, KaliniotS_(1019)-1400bp and S_(1025)-2400bp, Frantoio S_(1025)-400bp, Coratina S_(1011)-650bp, and thesebands can be used in olive germplasm identification.
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