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United States Department of Agriculture

Agricultural Research Service

Title: A MULTIPLEX REAL TIME PCR ASSAY FOR SIMULTANEOUSLY GENOTYPING BEANS FOR BC-12 AND I, TWO GENES CONDITIONING RESISTANCE TO BEAN COMMON MOSAIC VIRUS

Authors
item Vandemark, George
item Miklas, Phillip

Submitted to: Plant and Animal Genome Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: October 1, 2003
Publication Date: January 10, 2004
Citation: Vandemark, G.J., Miklas, P.N. 2004. A multiplex real time pcr assay for simultaneously genotyping beans for bc-12 and i, two genes conditioning resistance to bean common mosaic virus. Plant and Animal Genome Conference Proceedings. p. 210.

Technical Abstract: Our objective was to simultaneously genotype plants for the bc-12 and I 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 (I I bc-1 bc 1) x Olathe (i i bc-12 bc-12). Real-time PCR assays were developed that were specific for each allele. The Taqman probes for bc-12 and I were labeled with the fluorochromes VIC and TAMRA, respectively, 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 for which no amplification was detected with both assays were classified as null (I I bc-1 bc-1) genotypes. F2 plants that fell within the probability distribution were classified as heterozygous for the respective allele, while plants that fell outside the right tail of the probability distribution were classified as homozygous. 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 97% (192/198) 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.

Last Modified: 9/1/2014
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