EcoTILLING in Cochliobolus sativus Isolates Reveals Polymorphisms in the XYL1 and XYL2

Abstract

The fungus Cochliobolus sativus is a cereal pathogen that in addition to its role in plant disease has potential industrial applications. It has been shown to be an efficient producer of xylanase. The enzyme breaks down hemicellulose and therefore identification of isolates with high xylanase activity is one target for enhancing biofuel production. Genetic variants of XYL1 and XYL2 genes were found to be associated with altered xylanase activity. However, although these two major genes have been described, relatively little is known about the patterns of xylanase gene diversity in this pathogen. In this work, we have developed a low-cost EcoTILLING method for singlenucleotide polymorphism (SNP) and small indel discovery in XYL1 and XYL2 genes from fungal isolates that is suitable for most laboratories. The approach utilizes self-extracted single-strand-specific nucleases and standard agarose gel electrophoresis for polymorphism discovery. Gene-specific primer pairs were used to optimize enzymatic mismatch cleavage and polymorphism discovery on a panel of 56 C. sativus isolates. Based on the nucleotide polymorphisms detected, these isolates were organized into four haplotype groups. These haplotype groups were found to correlate with differences in xylanase production. Seven novel nucleotide variation was confirmed by sequencing. The discovered polymorphisms serve as genome specific markers for screening of additional C. sativus isolates. The ability of rapid and low-cost genotypic methods to categorize xylanase production may be of value in both basic research and industrial settings.