摘要
基于火龙果Ty1-copia类反转录转座子的长末端重复序列信息设计引物,采用5因素4水平L16(45)的完全随机正交试验,对Mg2+浓度、dNTPs浓度、Taq DNA聚合酶用量、引物浓度和DNA含量进行优化,建立火龙果IRAP分子标记反应体系。结果表明,25μL反应体系含基因组DNA 20ng、2.5mmol/L MgCl2、0.10mmol/L dNTPs、10×PCR Buffer 2.5μL、Taq DNA聚合酶0.75U、0.25μmol/L LTR引物,0.080g/mL的非变性PAGE胶适于IRAP多态性检测。
Based on the LTR sequences of dragon fruit (Hylocereus spp. ) Tyl-copia retrotrans- posons, primers for IRAP were designed. The complete random orthogonal design L16 (45 ) was used to optimize the IRAP-PCR system at four levels and five factors, namely, Mg2+ , dNTP, Taq DNA polymerase, primers and DNA template. The results showed that the optimal system for IRAP amplification was 25 μL mixture containing 20 ng of template DNA,2.5 mmol/L MgCl2,0. 10 mmol/L dNTPs,2.5μL of 10× PCR Buffer,0.75 U of Taq DNA polymerase, 0. 25μmol/LLTR primer. PAGE with 0. 080 g/mL non-denaturing polyacrylamide gel could yield reproducible, clear and reliable bands, which facilitate the identification of dragon fruit germplasms,the elucidation of their genetic diversity as well as assessment of genetic variation.
出处
《华中农业大学学报》
CAS
CSCD
北大核心
2014年第4期33-38,共6页
Journal of Huazhong Agricultural University
基金
国家自然科学基金项目(31260464
31060256)
贵州省重大专项(20126006-01)