火龙果IRAP分子标记反应体系的建立与优化

Establishment and optimization of IRAP marker methodology in dragon fruit

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.