Application of Bi-directional PCR Amplification of Specific Alleles in Single Nucleotide Polymorphism Genotyping of Tobacco

In order to improve the efficiency of molecular marker assisted breeding for PVY-resistant tobacco varieties,we dedicated to establish a simple and rapid bi-directional PCR amplification of specific alleles (Bi-PASA) method for detecting the single nucleotide polymorphism (SNP) G149C of the recessive PVY resistance gene eIF4E1 of the resistant germplasm Bankuncunshaiyan,and to verify the specificity,accuracy and practicality of the established detection method.It turned out that the established Bi-PASA detection method can effectively distinguish the three different genotypes for the G149C SNP of the eIF4E1 gene in a single PCR reaction: wildtype (GG),heterozygous mutant (GC) and homozygous mutant (CC).The genotypes of eIF4E1 in the F₂ population derived from K326/Bankuncunshaiyan were successfully identified by the Bi-PASA method.In addition,the genotypes of the F₂ population identified by the established Bi-PASA method were all the same with that identified by the conventional allele-specific PCR (AS-PCR) and direct sequencing,which suggested that the established Bi-PASA method is characterized by strong specificity,high accuracy and simple operation.Therefore,it can be better applied in the eIF4E1 gene marker assisted breeding.