Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/10/2005
Publication Date: 5/1/2005
Citation: Vandemark, G.J., Miklas, P.N. 2005. Genotyping common bean for the potyvirus resistance alleles i and bc-12 with a multiplex real-time polymerase chain reaction assay. Phytopathology. 95:499-505.
Interpretive Summary: Molecular markers that are closely associated with important agronomic traits such as disease and pest resistance are widely used by plant breeders to accelerate progress of breeding programs. Unfortunately, 'dominant' molecular markers, in the case of diploid (2 copies of each chromosome) crop species, can only be used to determine if a plant has at least one copy of the gene of interest. Breeders need to be able to identify plants that have two copies of a gene of interest in order to assure that a variety produced from these plants uniformly expresses the trait. Two different genes, bc-12 and I, work together in beans to produce resistance to diseases caused by viruses. Prior to the work described here, only dominant molecular markers have been available to identify beans that have the bc-12 and I genes. To identify plants that have two copies of both resistance genes, breeders have had to self-pollinate plants that have the two dominant molecular markers and evaluate progeny plants in the greenhouse for resistance to BCMV. This process requires approximately four months for completion. Only one plant out of nine typically has two copies of both the bc-12 and I. This paper describes the development of a 'real-time fluorescent PCR' assay for determining at the seedling stage whether plants have zero, one, or two copies of both the I and bc-12 gene. The assay was 100% accurate for bc-12 and 93% accurate for the I gene. Plants can be evaluated at the seedling stage, resulting in up to thousands of dollars in savings per breeding cycle in labor and facilities costs. This assay provides a means of more rapidly and inexpensively producing improved bean varieties.
Technical Abstract: A multiplex real-time 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) 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 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. Real-time PCR assays provide a robust method for genotyping seedlings and in some cases may eliminate the need for progeny testing.