四引物扩增受阻突变体系PCR检测水稻香味基因badh2方法的优化

Optimization of the Method for Detection of Rice Fragrance Gene badh2 with Tetra-primer ARMS PCR

badh2是控制水稻香味的主效基因,其与等位非香基因Badh2功能差异主要在于第七外显子上的8 bp缺失及3个SNP,开发其易于分辨且稳定的功能标记将有助于香稻分子育种。本研究基于四引物扩增受阻突变体系PCR(Tetra Primer Amplification Refractory Mutation System PCR,Tetra-primer ARMS PCR)原理,在功能差异位点外侧两引物一致的情况下,分别设计与badh2和Badh2完全匹配的5'端添加错配碱基的靶引物。结果表明,经1%的琼脂糖凝胶电泳后,由完全匹配引物组成的PCR体系基本能分辨badh2/Badh2基因型,但目标条带较模糊;而引物中5'端引入错配碱基的PCR体系能产生更明晰、易于分辨的目标条带。本研究将有助于香稻分子育种及为其他类似分子标记的优化提供借鉴。

The badh2 is the major gene that controls the aroma of rice,and the functional difference between Badh2 and allelic non-aroma gene is mainly due to 8 bp deletion and 3 SNPs in the seventh exon.The development of easily distinguishable and stable functional markers will be helpful for molecular breeding of fragrant rice.In this study,based on the principle of the Tetra-primer amplification refractory mutation system PCR(Tetra-primer ARMS PCR),the target primers that completely matched badh2 or Badh2 and added mismatched bases at the 5'end were designed respectively under the condition that the two primers on the outside of the functional difference site were consistent.The results showed that after 1%agarose gel electrophoresis,the PCR system composed of fully matched primers could basically distinguish badh2/Badh2 genotypes,but the target bands were fuzzy.The PCR system with the introduction of mismatched bases at the 5'end of primers can produce more clear and easily distinguishable target bands.This study will be helpful for molecular breeding of fragrant rice and provide reference for optimization of other similar molecular markers.