Site-Specific Measurement of Meiotic Crossing-Over Rate with Droplet Digital PCR.

Authors

Olga Zimina, Mateusz Zelkowski, Andrew Ziesel, Wojciech P Pawlowski

Published in

Cold Spring Harbor protocols. Sep 10, 2026. Epub Sep 10, 2026.

Abstract

Understanding the frequency and distribution of meiotic crossovers (COs) is critical for both fundamental studies on meiosis and for practical applications in plant breeding, where controlling recombination can accelerate crop improvement. Determining CO rates at specific genomic loci has traditionally relied on labor-intensive methods that require the production and genotyping of large progenies. Here, we present a high-throughput protocol for site-specific quantification of meiotic COs in maize using droplet digital PCR (ddPCR). The method is based on genotyping individual pollen nuclei from hybrid plants to detect recombinant and nonrecombinant alleles at defined chromosomal intervals. By distributing several thousands of pollen nuclei into nanoliter-sized droplets and performing PCR with allele-specific fluorescent probes, this method allows precise quantification of CO frequency with high sensitivity. The protocol provides detailed guidance for nuclei isolation, probe master mix preparation, droplet generation, and data interpretation. This method can be easily adapted for use in other plants.

PMID:
42722405
Bibliographic data and abstract were imported from PubMed on 11 Sep 2026.

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