Location: Sugarcane Field StationTitle: Genotyping Sugarcane for the Brown Rust Resistance Locus Bru1 Using Unlabeled Probe Melting
|MIGNEAULT, ANDREW - UNIVERSITY OF FLORIDA|
Submitted to: Molecular Breeding
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/12/2015
Publication Date: N/A
Interpretive Summary: Brown rust is a major fungal disease of sugarcane in Florida, Louisiana, and other sugarcane growing regions. The gene known as Bru1 is an important source of genetic resistance to brown rust, and molecular markers are available to screen for the gene. However, the existing markers have two disadvantages. One of the markers (Bru1B) cannot distinguish between true and false negatives, while the other (Bru1A) requires multiple steps for detection, which increases the time and expense involved in using the marker. We developed an improved version of the Bru1A marker using a technique known as unlabeled probe melting. This technique measures the change in fluorescence of a DNA-binding dye as a mixture of target DNA and a short DNA probe is heated. Differences in DNA sequence in the region covered by the probe result in changes in the fluorescence measurements. Unlabeled probe melting was shown to be less expensive and faster than using the original Bru1A marker, and it eliminates the risk of false negative results when using the Bru1B marker. This new method allows sugarcane breeders to more efficiently screen new varieties for resistance to brown rust.
Technical Abstract: Brown rust, caused by the fungus Puccinia melanocephala, is a major disease of sugarcane (Saccharum spp.) 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 percent 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.