Investigations on Nuclear DNA Content and DNA Synthesis in Plants and Fungi Using Flow Cytometry and Fluorescence Microscopy

A special issue of Genes (ISSN 2073-4425). This special issue belongs to the section "Molecular Genetics and Genomics".

Deadline for manuscript submissions: closed (15 May 2021) | Viewed by 34349

Special Issue Editors


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Guest Editor
Department of Agricultural Biotechnology, Laboratory of Molecular Biology and Cytometry, UTP University of Science and Technology, Kaliskiego Ave. 7, 85-796 Bydgoszcz, Poland
Interests: cell cycle; endoreduplication; genome size; ploidy; nuclear dynamics; seed enhancement and germination; flow cytometry; confocal microscopy

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Co-Guest Editor
Department of Agricultural Biotechnology, Laboratory of Molecular Biology and Cytometry, UTP University of Science and Technology, Kaliskiego Ave. 7, 85-796 Bydgoszcz, Poland
Interests: cell cycle; endoreduplication; polysomaty; genome size; flow cytometry

E-Mail Website
Co-Guest Editor
Department of Agricultural Biotechnology, Laboratory of Molecular Biology and Cytometry, UTP University of Science and Technology, Kaliskiego Ave. 7, 85-796 Bydgoszcz, Poland
Interests: DNA content; DNA molecular markers; genome size; polyploidy; flow cytometry

Special Issue Information

Dear Colleagues,

Knowledge on nuclear DNA content and DNA synthesis activity during the cell cycle and endoreduplication is important not only for fundamental research but also in plant breeding and seed production and technology. Flow cytometry has become the method of choice for genome size estimation, ploidy analysis, establishment of cell cycle activity, and endoreplication intensity in plants. This method, however, does not provide information on the localization of nuclei of different ploidies in intact tissues; to elucidate this, fluorescence (confocal) microscopy is the technique used. The aim of this Special Issue is to provide insights into current research involving flow cytometry (including nuclei and chromosome sorting) and/or fluorescence microscopy to evaluate plant systematics, evolution, genome stability in in vitro cultures, responses to environmental stresses, and DNA synthesis during plant growth and development. Additionally, contributions on nucleoplasmic ratio and the relationship between nuclear and organellar genomes are welcome. Papers on applications of fungi flow cytometry and confocal microscopy in medical mycology, plant pathology, fermentation industry and environmental sciences as well as in fundamental studies on fungal biology and physiology will also be accepted.

In this Special Issue, we would like to include original articles, short communications, and reviews of recent applications of nuclear flow cytometry and fluorescence microscopy in fundamental plant research, as well as in agricultural and horticultural practices.

Prof. Elwira Sliwinska
Guest Editor

Dr. Monika Rewers
Dr. Iwona Jedrzejczyk
co-Guest Editors

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Keywords

  • Cell cycle activity
  • Nuclear DNA content
  • DNA synthesis
  • Endoreduplication
  • Polysomaty
  • Genome size
  • Ploidy
  • Somaclonal variation
  • Nucleocytoplasmic ratio
  • Nuclear dynamics
  • Mycology
  • Fungal biology and physiology

Published Papers (10 papers)

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Editorial

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3 pages, 179 KiB  
Editorial
Investigations on Nuclear DNA Content and DNA Synthesis in Plants and Fungi Using Flow Cytometry and Fluorescence Microscopy
by Elwira Sliwinska
Genes 2022, 13(3), 417; https://doi.org/10.3390/genes13030417 - 25 Feb 2022
Viewed by 1350
Abstract
The twenty-first century has been an era of extensive genome exploration and modifications, using advanced methods such as genome sequencing and editing [...] Full article

Research

Jump to: Editorial

25 pages, 3732 KiB  
Article
Chromosome Number, Ploidy Level, and Nuclear DNA Content in 23 Species of Echeveria (Crassulaceae)
by Guadalupe Palomino, Javier Martínez-Ramón, Verónica Cepeda-Cornejo, Miriam Ladd-Otero, Patricia Romero and Jerónimo Reyes-Santiago
Genes 2021, 12(12), 1950; https://doi.org/10.3390/genes12121950 - 3 Dec 2021
Cited by 6 | Viewed by 4037
Abstract
Echeveria is a polyploid genus with a wide diversity of species and morphologies. The number of species registered for Echeveria is approximately 170; many of them are native to Mexico. This genus is of special interest in cytogenetic research because it has a [...] Read more.
Echeveria is a polyploid genus with a wide diversity of species and morphologies. The number of species registered for Echeveria is approximately 170; many of them are native to Mexico. This genus is of special interest in cytogenetic research because it has a variety of chromosome numbers and ploidy levels. Additionally, there are no studies concerning nuclear DNA content and the extent of endopolyploidy. This work aims to investigate the cytogenetic characteristics of 23 species of Echeveria collected in 9 states of Mexico, analyzing 2n chromosome numbers, ploidy level, nuclear DNA content, and endopolyploidy levels. Chromosome numbers were obtained from root tips. DNA content was obtained from the leaf parenchyma, which was processed according to the two-step protocol with Otto solutions and propidium iodide as fluorochrome, and then analyzed by flow cytometry. From the 23 species of Echeveria analyzed, 16 species lacked previous reports of 2n chromosome numbers. The 2n chromosome numbers found and analyzed in this research for Echeveria species ranged from 24 to 270. The range of 2C nuclear DNA amounts ranged from 1.26 pg in E. catorce to 7.70 pg in E. roseiflora, while the 1C values were 616 Mbp and 753 Mbp, respectively, for the same species. However, differences in the level of endopolyploidy nuclei were found, corresponding to 4 endocycles (8C, 16C, 32C and 64C) in E. olivacea, E. catorce, E. juarezensis and E. perezcalixii. In contrast, E. longiflora presented 3 endocycles (8C, 16C and 32C) and E. roseiflora presented 2 endocycles (8C and 16C). It has been suggested that polyploidization and diploidization processes, together with the presence of endopolyploidy, allowed Echeveria species to adapt and colonize new adverse environments. Full article
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17 pages, 4761 KiB  
Article
On the Origin of Tetraploid Vernal Grasses (Anthoxanthum) in Europe
by Zuzana Chumová, Terezie Mandáková and Pavel Trávníček
Genes 2021, 12(7), 966; https://doi.org/10.3390/genes12070966 - 24 Jun 2021
Cited by 5 | Viewed by 2582
Abstract
Polyploidy has played a crucial role in the evolution of many plant taxa, namely in higher latitudinal zones. Surprisingly, after several decades of an intensive research on polyploids, there are still common polyploid species whose evolutionary history is virtually unknown. Here, we addressed [...] Read more.
Polyploidy has played a crucial role in the evolution of many plant taxa, namely in higher latitudinal zones. Surprisingly, after several decades of an intensive research on polyploids, there are still common polyploid species whose evolutionary history is virtually unknown. Here, we addressed the origin of sweet vernal grass (Anthoxanthum odoratum) using flow cytometry, DNA sequencing, and in situ hybridization-based cytogenetic techniques. An allotetraploid and polytopic origin of the species has been verified. The chromosome study reveals an extensive variation between the European populations. In contrast, an autopolyploid origin of the rarer tetraploid vernal grass species, A. alpinum, has been corroborated. Diploid A. alpinum played an essential role in the polyploidization of both European tetraploids studied. Full article
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17 pages, 4760 KiB  
Article
Flow Cytometry-Based Determination of Ploidy from Dried Leaf Specimens in Genomically Complex Collections of the Tropical Forage Grass Urochloa s. l.
by Paulina Tomaszewska, Till K. Pellny, Luis M. Hernández, Rowan A. C. Mitchell, Valheria Castiblanco, José J. de Vega, Trude Schwarzacher and Pat (J.S.) Heslop-Harrison
Genes 2021, 12(7), 957; https://doi.org/10.3390/genes12070957 - 23 Jun 2021
Cited by 17 | Viewed by 3678
Abstract
Urochloa (including Brachiaria, Megathyrus and some Panicum) tropical grasses are native to Africa and are now, after selection and breeding, planted worldwide, particularly in South America, as important forages with huge potential for further sustainable improvement and conservation of grasslands. We [...] Read more.
Urochloa (including Brachiaria, Megathyrus and some Panicum) tropical grasses are native to Africa and are now, after selection and breeding, planted worldwide, particularly in South America, as important forages with huge potential for further sustainable improvement and conservation of grasslands. We aimed to develop an optimized approach to determine ploidy of germplasm collection of this tropical forage grass group using dried leaf material, including approaches to collect, dry and preserve plant samples for flow cytometry analysis. Our methods enable robust identification of ploidy levels (coefficient of variation of G0/G1 peaks, CV, typically <5%). Ploidy of some 348 forage grass accessions (ploidy range from 2x to 9x), from international genetic resource collections, showing variation in basic chromosome numbers and reproduction modes (apomixis and sexual), were determined using our defined standard protocol. Two major Urochloa agamic complexes are used in the current breeding programs at CIAT and EMBRAPA: the ’brizantha’ and ’humidicola’ agamic complexes are variable, with multiple ploidy levels. Some U. brizantha accessions have odd level of ploidy (5x), and the relative differences in fluorescence values of the peak positions between adjacent cytotypes is reduced, thus more precise examination of this species is required. Ploidy measurement of U. humidicola revealed aneuploidy. Full article
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14 pages, 1489 KiB  
Article
Characteristics of the Diploid, Triploid, and Tetraploid Versions of a Cannabigerol-Dominant F1 Hybrid Industrial Hemp Cultivar, Cannabis sativa ‘Stem Cell CBG’
by Seth Crawford, Brendan M. Rojas, Eric Crawford, Matthew Otten, Thecla A. Schoenenberger, Andrea R. Garfinkel and Hsuan Chen
Genes 2021, 12(6), 923; https://doi.org/10.3390/genes12060923 - 17 Jun 2021
Cited by 18 | Viewed by 5933
Abstract
Hemp (Cannabis sativa L.) has recently become an important crop due to the growing market demands for products containing cannabinoids. Unintended cross-pollination of C. sativa crops is one of the most important threats to cannabinoid production and has been shown to reduce [...] Read more.
Hemp (Cannabis sativa L.) has recently become an important crop due to the growing market demands for products containing cannabinoids. Unintended cross-pollination of C. sativa crops is one of the most important threats to cannabinoid production and has been shown to reduce cannabinoid yield. Ploidy manipulation has been used in other crops to improve agronomic traits and reduce fertility; however, little is known about the performance of C. sativa polyploids. In this study, colchicine was applied to two proprietary, inbred diploid C. sativa inbred lines, ‘TS1-3’ and ‘P163’, to produce the tetraploids ‘TS1-3 (4x)’ and ‘P163 (4x)’. The diploid, triploid, and tetraploid F1 hybrids from ‘TS1-3’ × ‘P163’, ‘TS1-3 (4x)’ × ‘P163’, and ‘TS1-3 (4x)’ × ‘P163 (4x)’ were produced to test their fertilities, crossing compatibilities, and yields. The results indicated a reduction in fertility in the triploids and the tetraploids, relative to their diploid counterparts. When triploids were used as females, seed yields were less than 2% compared to when diploids were used as females; thus, triploids were determined to be female infertile. The triploids resulting from the crosses made herein displayed increases in biomass and inflorescence weight compared to the diploids created from the same parents in a field setting. Statistical increases in cannabinoid concentrations were not observed. Lastly, asymmetric crossing compatibility was observed between the diploids and the tetraploids of the genotypes tested. The results demonstrate the potential benefits of triploid C. sativa cultivars in commercial agriculture. Full article
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15 pages, 1931 KiB  
Article
Genome Size, Chromosome Number and Morphological Data Reveal Unexpected Infraspecific Variability in Festuca (Poaceae)
by Gloria Martínez-Sagarra, Sílvia Castro, Lucie Mota, João Loureiro and Juan A. Devesa
Genes 2021, 12(6), 906; https://doi.org/10.3390/genes12060906 - 11 Jun 2021
Cited by 10 | Viewed by 3196
Abstract
Polyploidy has played an important evolutionary role in the genus Festuca (Poaceae), and several ploidy levels (ranging from 2n = 2x = 14 to 2n = 12x = 84) have been detected to date. This study aimed to estimate the genome [...] Read more.
Polyploidy has played an important evolutionary role in the genus Festuca (Poaceae), and several ploidy levels (ranging from 2n = 2x = 14 to 2n = 12x = 84) have been detected to date. This study aimed to estimate the genome size and ploidy level of two subspecies belonging to the F. yvesii polyploid complex by flow cytometry and chromosome counting. The phenotypic variation of the cytotypes was also explored, based on herbarium material. The genome size of F. yvesii subsp. lagascae has been estimated for the first time. Nuclear 2C DNA content of F. yvesii subsp. summilusitana ranged from 21.44 to 31.91 pg, while that of F. yvesii subsp. lagascae was from 13.60 to 22.31 pg. We report the highest ploidy level detected for Festuca (2n = 14x = 98) and previously unknown cytotypes. A positive correlation between holoploid genome size and chromosome number counts shown herein was confirmed. The morphometric approach showed a slight trend towards an increase in the size of some organs consistent with the variation in the ploidy level. Differences in characters were usually significant only among the most extreme cytotypes of each subspecies, but, even in this case, the high overlapping ranges prevent their distinction. Full article
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16 pages, 1571 KiB  
Article
Can Knowledge of Genetic Distances, Genome Sizes and Chromosome Numbers Support Breeding Programs in Hardy Geraniums?
by Mehrdad Akbarzadeh, Katrijn Van Laere, Leen Leus, Jan De Riek, Johan Van Huylenbroeck, Stefaan P.O. Werbrouck and Emmy Dhooghe
Genes 2021, 12(5), 730; https://doi.org/10.3390/genes12050730 - 13 May 2021
Cited by 13 | Viewed by 3451
Abstract
Breeding programs in ornamentals can be facilitated by integrating knowledge of phylogenetic relatedness of potential parents along with other genomic information. Using AFLP, genetic distances were determined for 59 Geranium genotypes, comprising 55 commercial cultivars of the three subgenera of a total collection [...] Read more.
Breeding programs in ornamentals can be facilitated by integrating knowledge of phylogenetic relatedness of potential parents along with other genomic information. Using AFLP, genetic distances were determined for 59 Geranium genotypes, comprising 55 commercial cultivars of the three subgenera of a total collection of 61 Geranium genotypes. A subgroup of 45 genotypes, including intragroup and intergroup hybrids, were selected and further characterized for genome sizes and chromosome numbers. The variation in genome size ranged from 1.51 ± 0.01 pg/2C to 12.94 ± 0.07 pg/2C. The chromosome numbers ranged from 26 to 108–110 with some hybrids showing an aberrant number of chromosomes based on their parents’ constitution. All chromosome numbers of Geranium are an even number, which presumes that unreduced gametes occur in some cross combinations. Overall, parental difference in genome size and chromosome number were not limiting for cross compatibility. Good crossing compatibility was correlated to a Jaccard similarity coefficient as parameter for parental relatedness of about 0.5. Additionally, parent combinations with high differences in the DNA/chromosome value could not result in a successful cross. We expect that our results will enable breeding programs to overcome crossing barriers and support further breeding initiatives. Full article
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21 pages, 7247 KiB  
Article
Endopolyploidy Variation in Wild Barley Seeds across Environmental Gradients in Israel
by Anna Nowicka, Pranav Pankaj Sahu, Martin Kovacik, Dorota Weigt, Barbara Tokarz, Tamar Krugman and Ales Pecinka
Genes 2021, 12(5), 711; https://doi.org/10.3390/genes12050711 - 10 May 2021
Cited by 4 | Viewed by 2491
Abstract
Wild barley is abundant, occupying large diversity of sites, ranging from the northern mesic Mediterranean meadows to the southern xeric deserts in Israel. This is also reflected in its wide phenotypic heterogeneity. We investigated the dynamics of DNA content changes in seed tissues [...] Read more.
Wild barley is abundant, occupying large diversity of sites, ranging from the northern mesic Mediterranean meadows to the southern xeric deserts in Israel. This is also reflected in its wide phenotypic heterogeneity. We investigated the dynamics of DNA content changes in seed tissues in ten wild barley accessions that originated from an environmental gradient in Israel. The flow cytometric measurements were done from the time shortly after pollination up to the dry seeds. We show variation in mitotic cell cycle and endoreduplication dynamics in both diploid seed tissues (represented by seed maternal tissues and embryo) and in the triploid endosperm. We found that wild barley accessions collected at harsher xeric environmental conditions produce higher proportion of endoreduplicated nuclei in endosperm tissues. Also, a comparison of wild and cultivated barley strains revealed a higher endopolyploidy level in the endosperm of wild barley, that is accompanied by temporal changes in the timing of the major developmental phases. In summary, we present a new direction of research focusing on connecting spatiotemporal patterns of endoreduplication in barley seeds and possibly buffering for stress conditions. Full article
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9 pages, 519 KiB  
Communication
Genome Size Diversity in Rare, Endangered, and Protected Orchids in Poland
by Monika Rewers, Iwona Jedrzejczyk, Agnieszka Rewicz and Anna Jakubska-Busse
Genes 2021, 12(4), 563; https://doi.org/10.3390/genes12040563 - 13 Apr 2021
Cited by 10 | Viewed by 3118
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 [...] Read more.
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. Full article
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16 pages, 2443 KiB  
Article
Spatial and Temporal Patterns of Endopolyploidy in Mosses
by Marianna Paľová, Dajana Ručová, Michal Goga and Vladislav Kolarčik
Genes 2021, 12(1), 27; https://doi.org/10.3390/genes12010027 - 27 Dec 2020
Cited by 5 | Viewed by 2942
Abstract
Somatic polyploidy or endopolyploidy is common in the plant kingdom; it ensures growth and allows adaptation to the environment. It is present in the majority of plant groups, including mosses. Endopolyploidy had only been previously studied in about 65 moss species, which represents [...] Read more.
Somatic polyploidy or endopolyploidy is common in the plant kingdom; it ensures growth and allows adaptation to the environment. It is present in the majority of plant groups, including mosses. Endopolyploidy had only been previously studied in about 65 moss species, which represents less than 1% of known mosses. We analyzed 11 selected moss species to determine the spatial and temporal distribution of endopolyploidy using flow cytometry to identify patterns in ploidy levels among gametophytes and sporophytes. All of the studied mosses possessed cells with various ploidy levels in gametophytes, and four of six species investigated in sporophytic stage had endopolyploid sporophytes. The proportion of endopolyploid cells varied among organs, parts of gametophytes and sporophytes, and ontogenetic stages. Higher ploidy levels were seen in basal parts of gametophytes and sporophytes than in apical parts. Slight changes in ploidy levels were observed during ontogenesis in cultivated mosses; the youngest (apical) parts of thalli tend to have lower levels of endopolyploidy. Differences between parts of cauloid and phylloids of Plagiomnium ellipticum and Polytrichum formosum were also documented; proximal parts had higher levels of endopolyploidy than distal parts. Endopolyploidy is spatially and temporally differentiated in the gametophytes of endopolyploid mosses and follows a pattern similar to that seen in angiosperms. Full article
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