烟草白粉病抗性基因型的多重PCR检测体系及其在回交育种中的应用

为提高烟草白粉病隐性抗病基因（感病基因）NtMLO1（M1）和NtMLO2（M2）在分子标记辅助选择育种中基因型的选择效率，根据已报道的M1和M2突变基因序列，设计4对单基因特异性引物，组成2个双重PCR反应体系，分别用于同时扩增M1和M2突变型或M1和M2野生型等位基因。结果表明，建立的2个双重PCR体系扩增的特异性片段与单重PCR体系扩增的片段完全吻合，突变基因纯合体只在突变基因多重PCR体系中产生191和306 bp的特异性片段，野生基因纯合体只在野生基因多重PCR体系中产生437和652 bp的特异性片段，杂合体在2个反应体系中均有特异性片段。利用该体系可经过1次或2次PCR扩增准确检测出回交转育过程中回交或自交后代M1和M2的基因型，加快抗病品种育种进程。 

A multiplex PCR system for genotyping of powdery mildew resistance genes in tobacco and its application in backcross breeding

In order to improve the efficiency of genotype selection of recessive resistance genes (susceptibility genes) NtMLO1 (M1) and NtMLO2 (M2) to tobacco powdery mildew in molecular marker-assisted selection breeding, on the basis of the reported nucleotide sequences of M1 and M2 mutant genes, four pairs of primers were designed to develop two multiplex polymerase chain reaction systems, which were separately used to amplify wild-type or mutant alleles of M1 and M2 simultaneously. The results showed that the specific fragments amplified by the two established duplex PCR systems were identical with those amplified by the monoplex PCR systems. Mutant homozygote produced specific fragments of 191 and 306 bp only in mutant allele-specific multiplex PCR systems, and wild-type homozygote produced specific fragments of 437 and 652 bp only in wild-type allele-specific multiplex PCR systems, however heterozygote produced specific fragments in both multiplex PCR systems. These systems can be used to accurately detect the genotypes of M1 and M2 in backcross or selfed progenies during backcrossing via one or two multiplex PCR amplification, so as to speed up the breeding process of new disease-resistant cultivars.