Nucleus Genotyping Using Crystal Digital PCR™

Date:  15 July 2021 (Thursday)
Time: New York – 11:00 | London – 16:00 | Paris – 17:00 | Singapore – 23:00 | Tokyo – 00: 00 (16 July) | Sydney – 01:00 (16 July)
Duration: 60 minutes
Event structure: 45-minute presentation + 15-minute Q&A
Registration fee: Complimentary access
Webinar on-demand: Available to view until midnight 31st July 2021 (registration is required)

Novel allelic combinations generated through meiotic recombination assure genetic variation that is harnessed during plant breeding. Measuring meiotic recombination rates directly in gametes before fertilization enables the screening of large sample numbers from single individuals independent of molecular marker analysis in segregating populations or time-consuming crossover (CO) detection based on cytological observations. Up to now, single pollen nucleus genotyping in barley (Hordeum vulgare) requires a Whole Genome Amplification (WGA) step before PCR-based genotyping or single-cell sequencing due to the limited pollen nucleus DNA content (≈ 5 pg per haploid nucleus) thus restricting the number of samples analyzed.

Using Crystal Digital PCR™ on the naica® system from Stilla® Technologies, we developed a single pollen nucleus genotyping assay to measure meiotic recombination rates within four selected chromosomal intervals (two centromeric and two distal) in high-throughput without WGA. We improved the genotyping assay efficiency through a restriction enzyme pre-treatment of pollen nuclei and genotyped more than 42,000 individual pollen nuclei. Measured rates of meiotic recombination in hybrid pollen nuclei corresponded to that in segregating populations. Based on the 6-color naica® system the throughput was further increased by multiplexing our assays allowing the simultaneous analysis within two chromosomal intervals.

Nuclei from several crop plants with different nuclear sizes and amounts of DNA were shown to be compatible with the set-up, suggesting a broad application of Crystal Digital PCR™ -based single nuclei genotyping assays.