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为构建玉米多重PCR检测体系,提高分子标记检测效率,利用10份代表性玉米材料对238对InDel引物进行单重PCR评估,共得到192对扩增效率高、稳定性好的引物。根据软件评估结果、扩增质量、产物范围、染色体均匀分布原则从192对引物中优选出30对综合表现较好的引物形成扩增产物范围在80~200 bp和200~400 bp的两组核心引物组合,每套组合中有10对引物分布在不同染色体上。在核心引物组合的基础上综合考虑染色体分布、碱基片段范围、引物荧光颜色,逐一添加引物,最终形成两组玉米20重PCR体系,一组40重荧光标记毛细管电泳。
In order to construct a multiplex PCR system for maize and improve the efficiency of molecular marker detection, 238 pairs of InDel primers were evaluated by single PCR using 10 representative maize materials. A total of 192 pairs of primers with high efficiency and good stability were obtained. According to the results of software evaluation, amplification quality, product range and chromosomal distribution principle, 30 pairs of 192 pairs of primers were selected as the best performing primers to form two groups of nucleotides with amplification products ranging from 80 to 200 bp and 200 to 400 bp Primer combinations, with 10 pairs of primers distributed on different chromosomes per set. Based on the combination of core primers, chromosomal distribution, base fragment range and primer fluorescent color were taken into account, and two sets of PCR systems of corn were finally formed, and a set of 40-fold fluorescently labeled capillary electrophoresis was finally formed.