Genotyping Sugarcane for the Brown Rust Resistance Locus Bru1 Using Unlabeled Probe Melting

Abstract

Brown rust, caused by the fungus Puccinia melanocephala, is a major disease of sugarcane (Saccharum spp. hybrid) in Florida, Louisiana, and other sugarcane growing regions. The Bru1 locus has been used as a durable and effective source of resistance, and markers are available to select for the trait. The markers currently being used by the USDA Sugarcane Field Station in Canal Point, FL for Bru1 genotyping have two disadvantages. One marker (here Bru1B) is dominant, which means that a Bru1-negative individual cannot be distinguished from a failed PCR reaction. The second marker (here Bru1A) is codominant, but genotyping requires enzyme restriction and gel electrophoresis, adding time and cost to the analysis. A closed-tube, codominant assay for Bru1 would significantly decrease the time and cost currently required for Bru1 genotyping. By sequencing the Bru1A PCR product from Bru1-positive and Bru1-negative individuals, we indentified two SNPs (here Bru1A1 and Bru1A2) that alter the relevant restriction enzyme recognition sequence. An unlabeled probe assay was designed to target each of the SNPs. Unlabeled probe assays are DNA melting assays that rely on the dissociation of an oligonucleotide probe from its target DNA strand. The dissociation is detected by the decrease in fluorescence of double-stranded DNA binding dyes and is sensitive enough to detect single base pair changes. A comparison between the traditional genotyping method and unlabeled probe melting using 344 genotypes showed a 94.7 % success rate, with 100 % concordance between Bru1A and unlabeled probe genotyping. We also confirmed prior unpublished results that discovered a rare recombination between Bru1A and Bru1B. We have demonstrated that unlabeled probe melting can be used to detect the Bru1 locus, eliminates the risk of false negatives, and is faster and less expensive than the current method.