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Flow cytometry: a quick method to determine ploidy levels in honeybush (Cyclopia spp.)

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Abstract

Cyclopia (honeybush) species are widely-used in the production of a South African herbal tea and are endemic to the fynbos region of the Western and Eastern Cape Provinces. Honeybush is still one of the orphan agriculture crops and recent breeding efforts by researchers are hampered by the lack of basic genetic information e.g. basic chromosome numbers and ploidy levels. This study determined nuclear DNA content and ploidy level of various genotypes of three Cyclopia species using flow cytometry and cytological counting of chromosomes. Nuclei analysis of young leaves of C. genistoides, C. longifolia and C. subternata were done using a flow cytometer, while root tip squashes were carried out in order to correlate flow cytometry results. Flow cytometry analysis indicated differences in the nuclear DNA content among and within species whilst the DNA ploidy level only differed among species. Cyclopia genistoides had a higher DNA ploidy level (≥ 10C) and DNA content (10.63 pg) than C. longifolia (6.09 pg) and C. subternata (5.99 pg), with no differences observed between the ploidy level of the latter two species (6C). The inferred ploidy level from nuclear DNA content by flow cytometry was consistent in all 30 genotypes of C. longifolia, and 24 of the 25 C. subternata and in only four of the 15 C. genistoides studied genotypes. These findings are important in breeding new cultivars with desired horticultural traits, thus improving the commercial characteristics for the sustainable production of honeybush.

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Acknowledgements

The authors thank the personnel of the ARC Infruitec-Nietvoorbij Crop Development Division for their assistance in the field sampling. Lastly, thanks to Gerrit Visser of the Plant Improvement Division (Biotechnology), ARC-Tropical and Subtropical Crops (ARC-TSC); Lize Engelbrecht, Rozanne Adams and Dumisile Lumkwana of the Central Analytical Facilities (CAF) at the University of Stellenbosch for Flow cytometry training and sample preparation and analysis.

Funding

This study was partially supported by funding from the Department of Science and Technology (DST), Monetary Treasury Economic Fund (MTEF), National Research Foundation (NRF), ARC Infruitec-Nietvoorbij and Tshwane University of Technology.

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Authors and Affiliations

  1. Crop Development Division, Agricultural Research Council Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch, 7599, South Africa

    Mlamuli M. Motsa & Cecilia Bester

  2. Department of Horticulture, Faculty of Science, Tshwane University of Technology, Private Bag X680, Pretoria, 0001, South Africa

    Mlamuli M. Motsa & Margaretha M. Slabbert

  3. Plant Improvement (Biotechnology), Agricultural Research Council Tropical and Subtropical Crops (ARC-TSC), Private Bag X11208, Nelspruit, 1200, South Africa

    Karin Hannweg

  4. Biometry, Agricultural Research Council, Private Bag X8783, Pretoria, 0001, South Africa

    Mardé Booyse

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  1. Mlamuli M. Motsa
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  2. Cecilia Bester
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Correspondence to Cecilia Bester.

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Motsa, M.M., Bester, C., Slabbert, M.M. et al. Flow cytometry: a quick method to determine ploidy levels in honeybush (Cyclopia spp.). Genet Resour Crop Evol 65, 1711–1724 (2018). https://doi.org/10.1007/s10722-018-0648-z

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