PCR Markers Derived from Comparative Genomics for Detection and Identification of the Rice Pathogen Ustilaginoidea...

Ustilaginoidea virens is a fungal pathogen that causes the false smut disease in rice and reduces the yield and quantity of the grains. A nested polymerase chain reaction (PCR)-based assay was developed to detect U. virens using genes of U. virens as specific targets. Ninety-six candidate genes of U. virens were found through first-round homology screening against a local database comprising 46 genomes of fungi, bacteria, and plants, with a second-round comparison with the GenBank NR database to further identify genes unique to U. virens. Among 96 remaining candidate genes, 20 of them (GenBank accessions KY617806 to KY617825) were randomly selected for further testing and, eventually, six sets of nested PCR primers were developed after further sensitivity, specificity, and detection tests. All six sets could detect DNA of U. virens at as little as 1 to 10 fg/μl from field or lab samples. These primers may be used to detect infection by U. virens at early stages, for use in research toward mitigating disease spread, as well as for studying the ecology of U. virens. This study also serves to illustrate that a comparative genomics method may allow for selection and development of highly specific primers once draft or complete genomes are available.