采用RAPD标记研究西瓜种质资源的亲缘关系
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摘要
本试验以四川农业大学保存的西瓜种质资源为材料(共26个材料)。通过田间、室内的调查进行西瓜的生物学性状的统计分析；采用改良CTAB法对西瓜属基因组DNA进行提取；对西瓜的RAPD反应体系进行了优化；筛选适用于本试验的随机引物;计算材料间的遗传距离并对材料进行聚类分析.结果如下：
1、通过田间和室内对26个西瓜材料的生物学性状进行观察统计。结果表明这些材料在倒蔓期、开花期、坐果节位、结果数、果型,果重、果皮硬度、果皮颜色、果肉色、果皮厚、皮肉界限、种子大小多少颜色、TSS等方面均存在一定的差异。
2、本研究使用的改良cTAB法和SDS法分别对26个西瓜材料进行了基因组DNA的提取,经分析表明,改良后的cTAB法更适用于西瓜属基因组DNA的提取,提取的DNAOD260/OD280比值在1.83-2.11之间,这说明所提取的DNA的质量较高,能够用于RAPD的分析。
3、采用正交试验设计的方法,建立了西瓜RAPD分析的优化反应体系。所得最优体系为：每25μL反应体系中含引物0.4μmol/L、Mg2+ 1.5mmol/L、Taq DNA聚合酶2.5U、dNTPs250μmol/L、1×Buffer、模板DNA1Ong/μl；适宜扩增条件为预变性94℃,时间5min；变性94℃,时间30s；退火37℃,时间1min；延伸72℃,时间1min；循环次数35圈,72℃保温7min,4℃保存。
4、从60个随机引物中筛选出3个,用这3个引物对26份西瓜材料的DNA样品进行扩增,共扩增出了56条,平均每个引物18.67条,其中51条具有多态性,占总条带数的91.07%,平均为每个引物17条多态性带。
5、利用计算机软件DPS软件统计RAPD数据,Genetic Distance法计算材料间遗传距离。得出所研究的26个材料遗传距离介于0.1628-0.5694之间；用UPGMA进行聚类,当相似系数为0.31时,可以将26份材料分为8大类群：1个华北生态地理型,1个华东生态地理型,1个日本生态地理型,1个西北生态地理型,1个美国生态型,1个华南生态型,2个中间类型。
26 materials coming from Sichuan Agricultural University were used in this study. We investigated and statistically analyzed the biological characters,and extracted the genomic DNA by improved CTAB method. In the paper,the reaction system of RAPD were optimzed and the suitable primers were screened, and cluster analysis were performed according to their genetic distance.The resutls as following:
1.The biological characters of these 26 watermelons were investigated and statistically analyzed through field and labtorary experiments.The results showed there were some differences in the pour rattan stage, blooming stage, fruit setting node, fruit number, fruit type,fruit heavy, peel hardness, peel color, flesh color, peel thickness, the boundary of peel and flesh, seed size,seed number seed color,TSS etc among these materials.
2.26 watermelon materials genomic DNA were extracted by improved CTAB method and SDS method. The results showed the improved CTAB method would be better. OD260/OD280 of DNA was 1.83-2.11, and DNA quality was high that could be used for RAPD amplification.
3.The optimal RAPD-PCR system of watermelon was established with orthogonal experiment design. The optimized PCR amplification system was 1×Buffer,0.4μmol/L primer,1.5mmol/L Mg2+,2.5 U Taq enzyme,250μmol/L dNTPs,10 ng/μl template DNA,add ddH2O to terminal volume 25μl. The suitable PCR procedure was one cycle denaturing at 94℃5min,40 cycles which involves denaturing at 94℃for 30s,annealing at 37℃for 30s and extending at 72℃for 1 min,one cycle extending at 72℃for 7min.
4.The analysis of the 26 watermelon materials with 3 RAPD primers which were screened from 60 random primers generated 56 bands,with an average of 18.67 bands per primer. And 51 (91.07%) of them were polymorphic, with an average of 17 polymorphic bands per primer.
5. Using DPS software to deal with all date, and the genetic distance was analyzed by genetic distance method. Cluster analysis indicated the genetic distance of 26 materials was 0.1628-0.5694, and all the materials could be divided into eight groups with the similarity coefficient at 0.31.They were respectively one North China ecogeographic group, one East China group, one Japan group, one Northwest China group, one American group, one South China group and two medial groups.
1、通过田间和室内对26个西瓜材料的生物学性状进行观察统计。结果表明这些材料在倒蔓期、开花期、坐果节位、结果数、果型,果重、果皮硬度、果皮颜色、果肉色、果皮厚、皮肉界限、种子大小多少颜色、TSS等方面均存在一定的差异。
2、本研究使用的改良cTAB法和SDS法分别对26个西瓜材料进行了基因组DNA的提取,经分析表明,改良后的cTAB法更适用于西瓜属基因组DNA的提取,提取的DNAOD260/OD280比值在1.83-2.11之间,这说明所提取的DNA的质量较高,能够用于RAPD的分析。
3、采用正交试验设计的方法,建立了西瓜RAPD分析的优化反应体系。所得最优体系为：每25μL反应体系中含引物0.4μmol/L、Mg2+ 1.5mmol/L、Taq DNA聚合酶2.5U、dNTPs250μmol/L、1×Buffer、模板DNA1Ong/μl；适宜扩增条件为预变性94℃,时间5min；变性94℃,时间30s；退火37℃,时间1min；延伸72℃,时间1min；循环次数35圈,72℃保温7min,4℃保存。
4、从60个随机引物中筛选出3个,用这3个引物对26份西瓜材料的DNA样品进行扩增,共扩增出了56条,平均每个引物18.67条,其中51条具有多态性,占总条带数的91.07%,平均为每个引物17条多态性带。
5、利用计算机软件DPS软件统计RAPD数据,Genetic Distance法计算材料间遗传距离。得出所研究的26个材料遗传距离介于0.1628-0.5694之间；用UPGMA进行聚类,当相似系数为0.31时,可以将26份材料分为8大类群：1个华北生态地理型,1个华东生态地理型,1个日本生态地理型,1个西北生态地理型,1个美国生态型,1个华南生态型,2个中间类型。
26 materials coming from Sichuan Agricultural University were used in this study. We investigated and statistically analyzed the biological characters,and extracted the genomic DNA by improved CTAB method. In the paper,the reaction system of RAPD were optimzed and the suitable primers were screened, and cluster analysis were performed according to their genetic distance.The resutls as following:
1.The biological characters of these 26 watermelons were investigated and statistically analyzed through field and labtorary experiments.The results showed there were some differences in the pour rattan stage, blooming stage, fruit setting node, fruit number, fruit type,fruit heavy, peel hardness, peel color, flesh color, peel thickness, the boundary of peel and flesh, seed size,seed number seed color,TSS etc among these materials.
2.26 watermelon materials genomic DNA were extracted by improved CTAB method and SDS method. The results showed the improved CTAB method would be better. OD260/OD280 of DNA was 1.83-2.11, and DNA quality was high that could be used for RAPD amplification.
3.The optimal RAPD-PCR system of watermelon was established with orthogonal experiment design. The optimized PCR amplification system was 1×Buffer,0.4μmol/L primer,1.5mmol/L Mg2+,2.5 U Taq enzyme,250μmol/L dNTPs,10 ng/μl template DNA,add ddH2O to terminal volume 25μl. The suitable PCR procedure was one cycle denaturing at 94℃5min,40 cycles which involves denaturing at 94℃for 30s,annealing at 37℃for 30s and extending at 72℃for 1 min,one cycle extending at 72℃for 7min.
4.The analysis of the 26 watermelon materials with 3 RAPD primers which were screened from 60 random primers generated 56 bands,with an average of 18.67 bands per primer. And 51 (91.07%) of them were polymorphic, with an average of 17 polymorphic bands per primer.
5. Using DPS software to deal with all date, and the genetic distance was analyzed by genetic distance method. Cluster analysis indicated the genetic distance of 26 materials was 0.1628-0.5694, and all the materials could be divided into eight groups with the similarity coefficient at 0.31.They were respectively one North China ecogeographic group, one East China group, one Japan group, one Northwest China group, one American group, one South China group and two medial groups.
引文
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