genes G C A T T A C G G C A T Communication Genome Size Diversity in Rare, Endangered, and Protected Orchids in Poland Monika Rewers 1, * , Iwona Jedrzejczyk 1 , Agnieszka Rewicz 2 and Anna Jakubska-Busse 3   Citation: Rewers, M.; Jedrzejczyk, I.; Rewicz, A.; Jakubska-Busse, A. Genome Size Diversity in Rare, Endangered, and Protected Orchids in Poland. Genes 2021, 12, 563. https://doi.org/10.3390/ genes12040563 Academic Editor: Mario Ventura Received: 24 March 2021 Accepted: 11 April 2021 Published: 13 April 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Laboratory of Molecular Biology and Cytometry, Department of Agricultural Biotechnology, UTP University of Science and Technology, Kaliskiego Ave 7, 85-796 Bydgoszcz, Poland; jedrzej@utp.edu.pl 2 Department of Biogeography, Paleoecology and Nature Conservation, Faculty of Biology and Environmental Protection, University of Lodz, 1/3 Banacha Str., 90-237 Lodz, Poland; agnieszka.rewicz@biol.uni.lodz.pl 3 Departmentof Botany, Faculty of Biological Sciences, University of Wroclaw, Kanonia 6/8, 50-328 Wroclaw, Poland; anna.jakubska-busse@uwr.edu.pl * Correspondence: mrewers@utp.edu.pl Abstract: Orchidaceae is one of the largest and the most widespread plant families with many species threatened with extinction. However, only about 1.5% of orchids’ genome sizes have been known so far. The aim of this study was to estimate the genome size of 15 species and one infraspecific taxon of endangered and protected orchids growing wild in Poland to assess their variability and develop additional criterion useful in orchid species identification and characterization. Flow cytometric genome size estimation revealed that investigated orchid species possessed intermediate, large, and very large genomes. The smallest 2C DNA content possessed Liparis loeselii (14.15 pg), while the largest Cypripedium calceolus (82.10 pg). It was confirmed that the genome size is characteristic to the subfamily. Additionally, for four species Epipactis albensis, Ophrys insectifera, Orchis mascula, Orchis militaris and one infraspecific taxon, Epipactis purpurata f. chlorophylla the 2C DNA content has been estimated for the first time. Genome size estimation by flow cytometry proved to be a useful auxiliary method for quick orchid species identification and characterization. Keywords: flow cytometry; nuclear DNA content; Orchidaceae; propidium iodide; threatened species 1. Introduction The orchid family (Orchidaceae) is one of the largest and the most diverse group of flowering plants with both epiphytic and terrestrial perennial members [1–3]. It contains 700 genera and about 30,000 species successfully colonized almost every habitat on earth [4]. Even though, the tropical and subtropical regions are the most orchid-rich areas worldwide. In Europe, there are approximately 230 species [3], while about 56 ones in Poland [5,6]. The uniqueness of orchids is due to the exquisite flowers with great diversity in floral form, size, color, fragrance, and texture, as well as a long floral lifespan [7]. Some species are used in pharmacy, traditional medicine, and in the food industry [8,9]. The attractiveness of those plants for humans led to their excessive exploitation and together with their specific biology and environmental disruption cause that the orchids are the most threatened taxonomic group of plants [10]. Currently, nearly 800 species are listed as threatened on the International Union for Conservation of Nature (IUCN) [11] Red List and their number is constantly increasing. Therefore all known orchid species are protected by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). The Orchidaceae family is also one of the most diverse angiosperm families regarding genome size. The difference between the smallest known orchid genome (0.66 pg/2C in Trichocentrum maduroi) and the largest (110.8 pg/2C in Pogonia ophioglossoides) is almost 168-fold [12]. Nonetheless, it is noteworthy that genome size of only about 1.5% of orchids has been known so far [13]. Analyzing the available data, the variation in genome size seems to be specific to the orchid subfamily [12]. The Epidendroideae subfamily char- acterizes the highest variation in genome size between species (over 60-fold), although Genes 2021, 12, 563. https://doi.org/10.3390/genes12040563 https://www.mdpi.com/journal/genes