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Genotyping Common Bean for the Potyvirus Resistance Alleles I and bc-1² with a Multiplex Real-Time Polymerase Chain Reaction Assay

A multiplex real-time polymerase chain reaction (PCR) assay was developed to simultaneously genotype plants for the I and bc-1² alleles, which condition resistance in beans to Bean common mosaic virus and Bean common mosaic necrosis virus. A segregating F₂ population was derived from the cross between pinto bean breeding line P94207-189A (bc-1 bc-1 I I) and Olathe (bc-12 bc-1² i i). Real-time PCR assays were developed that were specific for each allele, and a multiplex PCR reaction could unambiguously assign F₂ plants to one of nine genotypes. Remnant F₁ plants were used as a comparative reference sample. PCR results among this sample fit a normal distribution for both real-time PCR assays, and 99% probability distributions were determined for heterozygotes. F₂ plants were genotyped based on results relative to the probability distributions for heterozygotes. F₂ plants also were genotyped for the I and bc-1² alleles by performing F₃ family progeny tests for virus resistance. Agreement between the two methods was 100% (198/198) for the bc-1² allele, and 92.4% (183/198) for the I allele. Erroneous genotyping was due to recombination between the amplicon and the I allele. Realtime PCR assays provide a robust method for genotyping seedlings and, in some cases, may eliminate the need for progeny testing.