摘要
以10份大白菜杂交种及其亲本为研究对象,基于NCBI基因组数据库中EST序列信息,应用SSR标记及高分辨率溶解曲线技术筛选出用于大白菜杂交种纯度鉴定的SNP位点及复合SSR位点,并根据高通量分子检测技术种子纯度鉴定的需要,对单粒种子育苗条件、单株植株提取状态、快速DNA提取方法、复合PCR体系的建立等关键技术进行了摸索及优化。结果显示:经过48h破壳状态的单粒种子,采用改良CTAB法可于3h内得到较高质量的DNA;同时经7d达到苗期状态的单株叶片,采用chexe-100法亦可于40min内完成DNA提取;筛选得到的SNP位点可对6份大白菜杂交种进行纯度鉴定,复合SSR位点可对10份大白菜杂交种进行纯度鉴定。
@@@@In this research,10 Chinese cabbage〔Brassica campestris L. ssp. pekinensis(Lour) Olsson〕hybrids and their parents were taken as study objects.Based on the EST sequences from NCBI genome data base,a SNP site and multiplex EST-SSR sites,which could be used to identify the purity of Chinese cabbage hybrids,were screened by SSR marker and high resolution melting(HRM) technology.According to the requirement of seed purity identification by high throughput molecular technology,the key techniques such as seedling culture condition of single seed,extraction status of single grain plant, rapid DNA extraction method and establishment of multiplex PCR system were groped and optimized.Results showed that the single germinating seed after 48 h could gain DNA with higher quality by the improved CTAB method in 3 h. Meanwhile,plant leaves reached seeding stage after 7 d,could complete DNA extraction by chexe-100 method within 40 min.The SNP site obtained by screening could be used to indentify the purity of 6 Chinese cabbage hybrids,and the multiplex EST-SSR locus could be used to indentify the purity of 10 Chinese cabbage hybrids.
出处
《中国蔬菜》
北大核心
2013年第07X期31-38,共8页
China Vegetables
基金
天津市科技支撑计划项目(10ZCGYNC00400)
天津市应用基础及前沿技术研究计划项目(10JCYBJC09300)
