Submitted to: Molecular Breeding
Publication Type: Peer reviewed journal
Publication Acceptance Date: 5/8/2002
Publication Date: 9/1/2002
Citation: VANDEMARK, G.J., MIKLAS, P.N. A FLUORESCENT PCR ASSAY FOR THE CODOMINANT INTERPRETATION OF A DOMINANT SCAR MARKER LINKED TO THE VIRUS RESISTANCE GENE BC-1(2) IN COMMON BEAN. MOLECULAR BREEDING, 10:193-201. 2002. 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. A dominant molecular marker, SBD1300, has been identified in beans that is linked to the bc-12 gene, whi confers resistance in bean to bean common mosaic virus (BCMV).To distinguish between plants with one and two copies of the resistance gene it has always been necessary to self-pollinate all plants that have the dominant SBD1300 marker and evaluate progeny plants in the greenhouse for resistance to BCMV. This process requires approximately four months for completion. This is done to avoid the incorporation into the population of the undesirable gene that is responsible for disease susceptibility, which is present in plants that only have a single copy of the resistance gene. 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 the bc-12 gene. Accuracy in assigning genotype to plants with this assay ranges from 98-100%. Plants can be evaluated at the seedling stage, resulting in considerable savings in time and costs, and reduced greenhouse space requirements. The use of this assay will allow for the precise and rapid genotyping of bean plants for the bc-12 resistance gene.
Technical Abstract: Our objective was to develop a rapid and accurate procedure to genotype plants for the bc-12 allele, which conditions resistance in common bean to bean common mosaic and bean common mosaic necrosis viruses. A quantitative PCR assay based on the detection of fluorescent labeled amplicons was developed to distinguish between homozygous (bc-12//bc-12), heterozygous (bc-12//bc-1) and null (bc-1//bc-1) F2 genotypes. Remnant F1 plants were used as a comparative reference sample. PCR results among this sample fit a normal distribution, and 99% and 95% confidence intervals for heterozygotes were determined. F2 plants for which no amplification was detected were classified as null (bc-1//bc-1) genotypes. F2 plants that fell within the confidence intervals for heterozygotes were classified as heterozygotes (bc-12//bc-1), while plants that fell outside the right tail of the heterozygote confidence intervals were classified as homozygotes (bc- 12//bc-12). F2 plants were also genotyped for the bc-12 allele by self- pollinating the plants and performing F3 family progeny tests for virus resistance. Agreement between the two different methods for genotyping plants was 100% (59/59) when PCR genotyping was based on results for a 99% heterozygote confidence interval, and 98.3% (58/59) when based on a 95% heterozygote confidence interval. This assay will accelerate breeding for virus resistance in bean by facilitating discrimination among plants that are heterozygous or homozygous for the bc-12 allele.