Characterization of a Recombination Hotspot in Wheat Using Flow-Sorted Pollen Nuclei and Digital PCR

Allohexaploid bread wheat (Triticum aestivum, 2n=6x=42) behaves as diploid during meiosis, when the proper chromosome pairing is controlled by Ph1 locus. Crossing-over (CO) events mediated through homologous recombination are the source of new gene combinations and potentially interesting traits. However, COs are not evenly distributed along chromosomes and short regions (~1-4 kb) exhibiting significantly higher recombination rates (recombination hotspots) were observed. Previously, introgressive line of T. aestivum 8.1, resistant to powdery mildew was developed. The line contains a chromosome segment transferred from T. militinae into the subtelomeric region of long arm of chromosome 4A (4AL). In effort to clone resistance gene QPm.tut-4A from this line, a mapping population from a cross between T. aestivum cv. Chinese Spring and the line 8.1 was developed. Screening 7,500 individuals of the population identified a region with seven recombination events within 1.29 kb in the QPm.tut-4A locus. To characterize COs frequency in the locus, we have genotyped flow-sorted generative pollen nuclei using droplet digital PCR. This approach allows screening thousands of gametes, which can be easily obtained from only a few heterozygous plants and permits detection and quantification of recombinant molecules occurring even at low frequency. The method is generally applicable and provides fast and efficient method for high-resolution mapping recombination events along plant chromosomes to shed more light on the complex mechanisms underlying meiotic recombination. This work has been supported by the National Program of Sustainability I (No. LO1204), the Czech Science Foundation (Award 16-07155Y) and by Estonian Ministry of Agriculture.