Plant & Animal Genome IX Conference

Workshop: Barley
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TYPING BARLEY MLO ALLELES BY SINGLE NUCLEOTIDE POLYMORPHISMS ANALYSIS USING MALDI-TOF MASS SPECTROMETRY

Western Australian State Agricultural Biotechnology Centre, Murdoch University, Murdoch, WA, 6150, Australia

Recessive alleles of the barley Mlo gene confer broad spectrum and durable resistance to the fungal pathogen powdery mildew (Erysiphe graminis f.sp. hordeum). The Mlo alleles of susceptible varieties grown in Australia and the European recessive resistant variety Alexis were sequenced and aligned to identify DNA polymorhisms linked to the phenotype. Single Nucleotide Polymorphims (SNPs) were identified. To allow genetic analysis for marker-assisted breeding, a Single Nucleotide Primer Extension (SNuPE) assay was developed. After amplification, a primer with its 3Æ end directly flanking a selected mutation is extended by a single dideoxynucleoside-triphosphate (ddNTP). Each ddNTP differs in its mass with the resultant mass of the extended primer varying accordingly. Extended primers are analyzed using Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-ToF MS). The accuracy of mass determination allows identification of susceptible and resistant alleles as well as heterozygous individuals. Doubled haploid barley progenies were screened for their Mlo alleles and a complete correlation between genotype and phenotype was found.