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ISSN : 1225-5009(Print)
ISSN : 2287-772X(Online)
Flower Research Journal Vol.31 No.S pp.5-5
DOI : https://doi.org/10.11623/frj.2023.31.S.5

High-throughput Genotyping Assay on Single Pollen Nucleus of Barley (Hordeum vulgare) via Crystal Digital PCR System to Observe Impact of Epigenetic Drugs on Meiotic Recombination Rates

Yun-Jae Ahn1,2, Joerg Fuchs3, Andreas Houben3, and Stefan Heckmann3*

1Department of Horticultural Science, Kyungpook National University, Daegu 41566, Korea
2Institute of Agricultural Science and Technology, Kyungpook National University, Daegu 41566, Korea
3Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), OT Gatersleben, Corrensstraße 3, 06466 Stadt Seeland, Germany

Abstract

New allelic combinations derived from meiotic recombination during sexual reproduction are the foundations in plant breeding research. Several methods exist to measure meiotic recombination rates in eukaryotes in large scales of segregating populations employing molecular markers or counting chiasmata through microscope to score crossovers (COs) directly are widely used, however, both can be time-consuming and burdensome in some research facilities. Therefore, we established efficient measurement of meiotic recombination rates promptly in gametes before fertilization process, which provides shortened time for meiotic recombination detection in a large number of pollen nuclei samples from single flower. In addition, measuring meiotic recombination rates in the gametes allows researchers to include segregation distortion in measurements. Moreover, by employing highly-sensitive Crystal digital PCR system, measuring meiotic recombination rates in a single pollen nucleus of barley with low amount of DNA content (≈ 5 pg) became feasible without additional Whole Genome Amplification (WGA) step before the genotyping assay. The efficiency of genotyping was enhanced by pre-treating pollen nuclei with a restriction enzyme, thus improve accessibility of PCR components towards nucleus. In total, more than 42,000 individual pollen nuclei were genotyped and meiotic recombination rates of four chromosomal intervals were measured, and reliability of measurements was confirmed by offspring plants in segregating populations. Furthermore, the established system was confirmed that it is compatible with other crops.

초록

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  2. Journal Abbreviation : 'Flower Res. J.'
    Frequency : Quarterly
    Doi Prefix : 10.11623/frj.
    ISSN : 1225-5009 (Print) / 2287-772X (Online)
    Year of Launching : 1991
    Publisher : The Korean Society for Floricultural Science
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