Mapping Meiotic Recombination DNA Double-Strand Breaks (DSBs) Hotspots -Methodological Advances and Challenges

Programmed DNA double-strand breaks (DSBs) initiate meiotic recombination at discrete genomic hotspots, and their precise mapping is crucial for understanding the molecular regulation of genome stability and evolution. A variety of genome-wide methods have been developed, each leveraging different biochemical principles - from direct capture of Spo11-oligonucleotide covalent intermediates to sequencing protein-bound single-stranded DNA (ssDNA), in situ DNA end labeling, and chromatin immunoprecipitation (ChIP). Direct approaches such as Spo11-oligo mappin, CC-seq and in situ DNA end labeling (END-seq) achieve nucleotide-resolution detection of the initial cleavage site, but typically require large amounts of starting material. ChIP ssDNA-based methods (e.g., ChIP-SSDS) enrich for recombinase associated proteins- bound (DMC1, RAD51, RPA) intermediates, reflecting an indirect proxy of DSB sites, and are limited to early resection stages. Together, while these complementary methods comprise a toolbox for dissecting meiotic hotspot landscapes, a more robust and facile approach for routine detection of meiotic DSB hotspots across diverse species remains to be developed.