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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

May 2005 , Volume 95 , Number  5
Pages  499 - 505

George J. Vandemark and Phillip N. Miklas

U.S. Department of Agriculture, Vegetable and Forage Crop Production Unit, Prosser, WA 99350

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Accepted for publication 10 January 2005.

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.

Additional keywords: protected I gene , quantitative PCR .

The American Phytopathological Society, 2005