Investigating Intraspecific Diversity of Metabolic Gene Clusters with MLPA and Synthetic Probes

Multiplex Ligation-Dependent Probe Amplification (MLPA) is a robust method for efficient genotyping known structural variations, especially duplications and deletions, in a population scale. It allows for the simultaneous assessment of multiple regions in the genome in a single assay. This feature makes MLPA especially useful in the analysis of metabolic gene clusters (MGCs), where three or more neighboring genes need to be genotyped to decipher the overall MGC structural pattern. By enabling fast and cost-effective screening of a large number of individuals or lines, MLPA may measure MGC intraspecific variation or reveal new MGC member(s), e.g., resulting from gene duplication. This in turn may provide insight into the potential diversity of biosynthetic pathways encoded by a given MGC. Currently, however, custom synthetic probes are required to target non-human genes with MLPA. Here we describe the process of designing such synthetic probes detecting MGC genes and conducting MLPA assays. MLPA with synthetic probes may be applied for MGC analysis in various species including plants, animals, or fungi.