학술논문
基于干种子DNA的洋葱杂交种纯度分子标记快速鉴定 / Rapid Identification of Onion Hybrid Purity by Molecular Markers Based on Dried Seed DNA
Document Type
Academic Journal
Author
孙亚玲; 李艳伟; 王振宝; 吴雄; 刘冰江; 杨妍妍; Sun Yaling; Li Yanwei; Wang Zhenbao; Wu Xiong; Liu Bingjiang; Yang Yanyan
Source
山东农业科学 / Shandong Agricultural Sciences. 55(11):169-175
Subject
Language
Chinese
ISSN
1001-4942
Abstract
为了寻找一种从洋葱种子中快速提取高质量DNA的方法,并能满足大量准确鉴定洋葱杂交种纯度的要求,本研究首先对全式金PlantZol试剂盒、天根快捷型植物基因组DNA提取系统、TPS法和CTAB法提取的洋葱种子DNA质量进行比较,筛选出最佳提取方法,并对不同发芽天数的种子及提取有效DNA的种子数量进行优化筛选,然后利用SCAR分子标记对洋葱杂交种的纯度进行鉴定,进一步验证洋葱种子DNA的提取质量.结果表明,天根快捷型植物基因组DNA提取系统未能成功得到洋葱干种子基因组总DNA;CTAB法提取的基因组总DNA带型整齐一致、单一明亮,条带无拖尾和降解现象,也没有明显的RNA和蛋白污染;洋葱干种子以 1粒和发芽3d提取的DNA更加完整,纯度更高.以CTAB法提取的1粒洋葱干种子基因组总DNA为模板,对杂交种Ms位点的基因型进行SCAR标记鉴定,PCR扩增条带清晰明亮,根据条带的迁移位置可以清楚区分洋葱不同细胞核基因型之间的差异.综上,本研究确定了CTAB法为洋葱干种子基因组总DNA提取的最佳方法,1 粒洋葱干种子获得的有效DNA完全可以满足杂交种分子标记鉴定的要求,实现洋葱杂交种纯度的早期鉴定.
In order to find a method for extracting high-quality DNA quickly from onion seeds and satisfy the requirements for accurate identification of the purity of a large number of onion hybrids,the DNA quality of onion seeds extracted by the TransGen PlantZol kit,TIANGEN rapid plant genomic DNA extraction system,TPS method and CTAB method were compared firstly in the study,and then the optimal extraction method was selected.The seeds with different germination days and the number of seeds for extracting effective DNA were also optimized and screened.Then,SCAR molecular markers were used to identify the purity of onion hybrid seeds to further verify the extraction quality of onion seed DNA.The results showed that the TIANGEN rapid plant genomic DNA extraction system failed to obtain the total genomic DNA of dried onion seed.The bands of DNA extracted by CTAB method were neat and consistent,single and bright,no tailing and degradation,and no obvious RNA and protein pollution.The DNA extracted from one dried onion seed and the germinated seeds after 3-day germination were more complete with higher purity.Using the total genomic DNA of one dried onion seed extracted by CTAB method as the template,SCAR markers were used to identify the Ms loci genotypes of onion hybrids.The PCR amplified bands were clear and bright,and the differences between different nuclear genotypes of onion could be clearly distinguished according to the migration positions of the bands.In summa-ry,this study determined that CTAB method was the best extraction method for the total genomic DNA from dried onion seeds,and the effective DNA obtained from one dried onion seed could satisfy the requirements of hybrid molecular marker identification,and realize the early identification of hybrid purity.
In order to find a method for extracting high-quality DNA quickly from onion seeds and satisfy the requirements for accurate identification of the purity of a large number of onion hybrids,the DNA quality of onion seeds extracted by the TransGen PlantZol kit,TIANGEN rapid plant genomic DNA extraction system,TPS method and CTAB method were compared firstly in the study,and then the optimal extraction method was selected.The seeds with different germination days and the number of seeds for extracting effective DNA were also optimized and screened.Then,SCAR molecular markers were used to identify the purity of onion hybrid seeds to further verify the extraction quality of onion seed DNA.The results showed that the TIANGEN rapid plant genomic DNA extraction system failed to obtain the total genomic DNA of dried onion seed.The bands of DNA extracted by CTAB method were neat and consistent,single and bright,no tailing and degradation,and no obvious RNA and protein pollution.The DNA extracted from one dried onion seed and the germinated seeds after 3-day germination were more complete with higher purity.Using the total genomic DNA of one dried onion seed extracted by CTAB method as the template,SCAR markers were used to identify the Ms loci genotypes of onion hybrids.The PCR amplified bands were clear and bright,and the differences between different nuclear genotypes of onion could be clearly distinguished according to the migration positions of the bands.In summa-ry,this study determined that CTAB method was the best extraction method for the total genomic DNA from dried onion seeds,and the effective DNA obtained from one dried onion seed could satisfy the requirements of hybrid molecular marker identification,and realize the early identification of hybrid purity.