Typing Mlo alleles for powdery mildew resistance in barley by single nucleotide polymorphism analysis using MALDI-ToF mass spectrometry

Abstract

Single nucleotide polymorphisms (SNPs) have been identified in a range of plant genomes. Development of rapid, low-cost methods to enable their validation and implementation as molecular markers is now required for practical applications. We report the development of single and multi-nucleotide primer extension assays to genotype co-dominant SNPs from small quantities of barley leaf tissue. In the single nucleotide primer extension assay, a genotyping primer with its 3′ end directly flanking a SNP was annealed to a target sequence and extended by a single dideoxynucleotide triphosphate complementary to the polymorphic base. In the multi-nucleotide primer extension assay, designed to facilitate allele calling, the genotyping primer with its 3′ end flanking the SNP was extended by either 1 or 2 nucleotides, depending on the allele encountered. Extension products were analysed using MALDI-ToF mass spectrometry and, making use of the molecular weight difference between DNA bases, the incorporated nucleotides were identified by the increase in mass of the extended primers. Based on a SNP identified in the barley Mlo gene, primer extension assays were designed and used for co-dominant marker-assisted selection of barley seedlings segregating for mlo-mediated resistance to powdery mildew. This allowed accurate selection of progeny lines carrying alleles for resistance to powdery mildew, including heterozygotes. Doubled haploid barley progenies were screened for Mlo alleles and a complete correlation between mlo/mlo genotype and resistant phenotype was found The method has been used by barley breeders for routine selection of barley genotypes resistant to powdery mildew.