A high-throughput method for the detection of homoeologous gene deletions in hexaploid wheat

To facilitate the screening for specific homoeologous gene deletions in hexaploid wheat, we have developed a TaqMan qPCR-based method that allows high-throughput detection of deletions in homoeologous copies of any gene of interest, provided that sufficient polymorphism (as little as a single nucleotide difference) amongst homoeologues exists for specific probe design. We used this method to identify deletions of individual TaPFT1 homoeologues, a wheat orthologue of the disease susceptibility and flowering regulatory gene PFT1 in Arabidopsis. This method was applied to wheat nullisomic-tetrasomic lines as well as other chromosomal deletion lines to locate the TaPFT1 gene to the long arm of chromosome 5. By screening of individual DNA samples from 4500 M2 mutant wheat lines generated by heavy ion irradiation, we detected multiple mutants with deletions of each TaPFT1 homoeologue, and confirmed these deletions using a CAPS method. We have subsequently designed, optimized, and applied this method for the screening of homoeologous deletions of three additional wheat genes putatively involved in plant disease resistance.We have developed a method for automated, high-throughput screening to identify deletions of individual homoeologues of a wheat gene. This method is also potentially applicable to other polyploidy plants.Identification of plant gene function is important not only to better understand how plants grow and develop, but also for potential exploitation of this information for molecular and/or traditional crop breeding. However, extensive gene duplication and polyploidy events during evolution mean that plants carry multiple copies of the same gene as well as large numbers of closely-related genes [1]. Therefore, for gene function studies, it is important to determine whether different copies of the same or related genes are functionally redundant or whether polymorphisms have been selected through evolution and led to functional specialisation [2,3]. Reverse g