A fluorescent PCR assay for the codominant interpretation of a dominant SCAR marker linked to the virus resistance allele bc-1 2 in common bean

Abstract

Our objective was to develop a rapid and accurate procedure to genotype common bean plants for the bc-12 allele, which conditions resistance to bean common mosaic and bean common mosaic necrosis viruses. A segregating F2 population was derived from the cross between pinto bean breeding lines P94207-43 (bc-12//bc-12) and P94207-189 (bc-1//bc-1). 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 performing F3 family progeny tests for virus resistance. Agreement between the two methods for genotyping plants was 100% (59/59) when PCR genotyping was based on 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. The experimental design may be generally applicable towards developing other assays for the codominant interpretation of dominant markers in diploid plants.