ARS | Publication request: CODOMINANT INTERPRETATION OF DISEASE RESISTANCE LOCI USING MULTIPLEXED REAL TIME PCR.

Submitted to: American Society of Agronomy Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: July 17, 2004
Publication Date: November 13, 2004
Citation: Vandemark, G.J., Miklas, P.N. 2004. Codominant interpretation of disease resistance loci using multiplexed real time PCR. American Society of Agronomy 2004 Abstracts, Madison, WI. p 319.

Technical Abstract: Our objective was to simultaneously genotype plants for the I and bc-12 alleles, which condition resistance in beans to Bean common mosaic virus. A segregating F2 population was derived from the cross between pinto bean breeding line P94207-189A (bc-1 bc-1 I I) x 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 their results relative to the probability distributions for heterozygotes. F2 plants were also 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. Real-time PCR assays provide a robust method for genotyping seedlings and in some cases may eliminate the need for progeny testing.