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In many plant species, somatic cell differentiation is accompanied by endoreduplication, a process during which cells undergo one or more rounds of DNA replication cycles in the absence of mitosis, resulting in nuclei with multiples of 2C DNA amounts (4C, 8C, 16C, etc.). In some orchids, a disproportionate increase in nuclear DNA contents has been observed, where successive endoreduplication cycles result in DNA amounts 2C + P, 2C + 3P, 2C + 7P, etc., where P is the DNA content of the replicated part of the 2C nuclear genome. This unique phenomenon was termed "progressively partial endoreplication" (PPE). We investigated processes behind the PPE in Ludisia discolor using flow cytometry (FCM) and Illumina sequencing. In particular, we wanted to determine whether chromatin elimination or incomplete genome duplication was involved, and to identify types of DNA sequences that were affected. Cell cycle analysis of root tip cell nuclei pulse-labeled with EdU revealed two cell cycles, one ending above the population of nuclei with 2C + P content, and the other with a typical "horseshoe" pattern of S-phase nuclei ranging from 2C to 4C DNA contents. The process leading to nuclei with 2C + P amounts therefore involves incomplete genome replication. Subsequent Illumina sequencing of flow-sorted 2C and 2C + P nuclei showed that all types of repetitive DNA sequences were affected during PPE; a complete elimination of any specific type of repetitive DNA was not observed. We hypothesize that PPE is part of a highly controlled transition mechanism from proliferation phase to differentiation phase of plant tissue development., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Nuclear genome size is an inherited quantitative trait of eukaryotic organisms with both practical and biological consequences. A detailed analysis of major families is a promising approach to fully understand the biological meaning of the extensive variation in genome size in plants. Although Orchidaceae accounts for ∼10% of the angiosperm diversity, the knowledge of patterns and dynamics of their genome size is limited, in part due to difficulties in flow cytometric analyses. Cells in various somatic tissues of orchids undergo extensive endoreplication, either whole-genome or partial, and the G1-phase nuclei with 2C DNA amounts may be lacking, resulting in overestimated genome size values. Interpretation of DNA content histograms is particularly challenging in species with progressively partial endoreplication, in which the ratios between the positions of two neighboring DNA peaks are lower than two. In order to assess distributions of nuclear DNA amounts and identify tissue suitable for reliable estimation of nuclear DNA content, we analyzed six different tissue types in 48 orchid species belonging to all recognized subfamilies. Although traditionally used leaves may provide incorrect C-values, particularly in species with progressively partial endoreplication, young ovaries and pollinaria consistently yield 2C and 1C peaks of their G1-phase nuclei, respectively, and are, therefore, the most suitable parts for genome size studies in orchids. We also provide new DNA C-values for 22 orchid genera and 42 species. Adhering to the proposed methodology would allow for reliable genome size estimates in this largest plant family. Although our research was limited to orchids, the need to find a suitable tissue with dominant 2C peak of G1-phase nuclei applies to all endopolyploid species., (© 2015 International Society for Advancement of Cytometry.)

Flow cytometry (FCM) has been widely used in plant science to determine the amount of nuclear DNA, either in absolute units or in relative terms, as an indicator of ploidy. The requirement for fresh material in some applications, however, limits the value of FCM in field research, including plant biosystematics, ecology and population biology. Dried plant samples have proven to be a suitable alternative in some cases (large-scale ploidy screening) although tissue dehydration is often associated with a decrease in the quality of FCM analysis. The present study tested, using time-scale laboratory and in situ field experiments, the applicability of glycerol-treated nuclear suspension for DNA flow cytometry. We demonstrate that plant nuclei preserved in ice-cold buffer + glycerol solution remain intact for at least a few weeks and provide estimates of nuclear DNA content that are highly comparable and of similar quality to those obtained from fresh tissue. The protocol is compatible with both DAPI and propidium iodide staining, and allows not only the determination of ploidy level but also genome size in absolute units. Despite its higher laboriousness, glycerol-preserved nuclei apparently represent the most reliable way of sample preservation for genome size research. We assume that the protocol will provide a vital alternative to other preservation methods, especially when stringent criteria on the quality of FCM analysis are required.

Background and Aims: Hieracium sub-genus Pilosella (hawkweeds) is a taxonomically complicated group of vascular plants, the structure of which is substantially influenced by frequent interspecific hybridization and polyploidization. Two kinds of species, 'basic' and 'intermediate' (i.e. hybridogenous), are usually recognized. In this study, genome size variation was investigated in a representative set of Central European hawkweeds in order to assess the value of such a data set for species delineation and inference of evolutionary relationships., Methods: Holoploid and monoploid genome sizes (C- and Cx-values) were determined using propidium iodide flow cytometry for 376 homogeneously cultivated individuals of Hieracium sub-genus Pilosella, including 24 species (271 individuals), five recent natural hybrids (seven individuals) and experimental F(1) hybrids from four parental combinations (98 individuals). Chromosome counts were available for more than half of the plant accessions. Base composition (proportion of AT/GC bases) was cytometrically estimated in 73 individuals., Key Results: Seven different ploidy levels (2x-8x) were detected, with intraspecific ploidy polymorphism (up to four different cytotypes) occurring in 11 wild species. Mean 2C-values varied approx. 4.3-fold from 3.53 pg in diploid H. hoppeanum to 15.30 pg in octoploid H. brachiatum. 1Cx-values ranged from 1.72 pg in H. pilosella to 2.16 pg in H. echioides (1.26-fold). The DNA content of (high) polyploids was usually proportional to the DNA values of their diploid/low polyploid counterparts, indicating lack of processes altering genome size (i.e. genome down-sizing). Most species showed constant nuclear DNA amounts, exceptions being three hybridogenous taxa, in which introgressive hybridization was suggested as a presumable trigger for genome size variation. Monoploid genome sizes of hybridogenous species were always between the corresponding values of their putative parents. In addition, there was a good congruency between actual DNA estimates and theoretical values inferred from putative parental combinations and between DNA values of experimental F(1) hybrids and corresponding established hybridogenous taxa., Conclusions: Significant differences in genome size between hawkweed species from hybridogenous lineages involving the small-genome H. pilosella document the usefulness of nuclear DNA content as a supportive marker for reliable delineation of several of the most problematic taxa in Hieracium sub-genus Pilosella (including classification of borderline morphotypes). In addition, genome size data were shown to have a good predictive value for inferring evolutionary relationships and genome constitution (i.e. putative parental combinations) in hybridogenous species.

Flow cytometry (FCM) using DNA-selective fluorochromes is now the prevailing method for the measurement of nuclear DNA content in plants. Ease of sample preparation and high sample throughput make it generally better suited than other methods such as Feulgen densitometry to estimate genome size, level of generative polyploidy, nuclear replication state and endopolyploidy (polysomaty). Here we present four protocols for sample preparation (suspensions of intact cell nuclei) and describe the analysis of nuclear DNA amounts using FCM. We consider the chemicals and equipment necessary, the measurement process, data analysis, and describe the most frequent problems encountered with plant material such as the interference of secondary metabolites. The purpose and requirement of internal and external standardization are discussed. The importance of using a correct terminology for DNA amounts and genome size is underlined, and its basic principles are explained.

The power of flow cytometry in field plant research may be greatly enhanced by analysis of nonfresh tissues. Previous attempts to use chemical fixatives, however, received only little attention because of protocol complexity and limited time, after which successful assays were achieved. This unit describes simple and rapid procedures for relative nuclear DNA content estimation in dehydrated tissues of vascular plants by DAPI flow cytometry. Histograms with reasonable resolution can be obtained in several-year-old specimens. The approach here is particularly suitable for reliable determination of DNA ploidy level, although detection of small variation in nuclear DNA content is also possible in many cases. Retrospective ploidy inference in silica-dry or herbarium vouchers and simplified transport of plant material from remote areas are among the principal benefits for plant biosystematics, ecology, and population biology.

Background: Only fresh plant material is generally used for rapid DNA ploidy estimation by flow cytometry (FCM). This requirement, however, substantially limits convenient FCM application in plant biosystematics, population biology, and ecology. As desiccation is a routine way for sample preservation in field botany, potential utilization of dehydrated tissues of vascular plants in FCM research was examined., Methods: Standard DAPI protocol was employed to evaluate the performance of 60 air-dried species, spanning more than 100-fold range of nuclear DNA amounts. Multiploid Vaccinium subg. Oxycoccus was selected as model taxon for detailed investigation and cytotype comparison., Results: A majority of analyzed plants yielded distinct peaks with reasonable coefficients of variation after several months of storage at room temperature. Fluorescence intensity of nuclei isolated from desiccated tissues was highly comparable with that for fresh material, allowing reliable DNA ploidy estimation. Deep-freezer preservation substantially extended Vaccinium samples lifetime (at least to 3 years) and maintained high histogram resolution., Conclusions: The introduced approach eliminates the need for fresh material in many vascular plants and thus opens new prospects for plant FCM. Convenient cytotype investigation in field research and retrospective ploidy determination in already herbarized samples are among the principal advantages.

The ploidy level of plants of the Vaccinium sect. Oxycoccus (HILL) W.D.J. KOCH sampled in the Czech Republic, Germany, Austria and Poland was determined by chromosome counting and/or by flow cytometry. Forty-five characters were measured and scored in the morphometric analysis. Principal component analysis, cluster analysis, canonical discriminant analysis and classificatory discriminant analysis were used in the statistical analyses. Diploid (2n=24), tetraploid (2n=48) and hexaploid (2n=72) populations were confirmed and a new ploidy level pentaploid hybrid plants (2n=60) was revealed. Results of the multivariate morphometric analysis support the separation of the two native species. Diploid V. microcarpum (TURCZ. exRUPR.) SCHMALH. differs from the polyploids by smaller size of petals, shorter style and stamens (stamens have long filaments and short anthers), glabrous pedicels, mostly solitary flowers, earlier flowering and by occurrence predominantly in Polytrichum strictum tufts. The low taxonomic significance of some features often used in keys for their separation (shape of fruits, insertion of prophylla, pubescence of filaments) was confirmed. V. oxycoccos L. includes three ploidy levels. The hexaploids represent the most abundant ploidy level in the area studied. They show a slightly bigger size of petals, longer bracts, prophylla, style, sepal tips and wider seeds in comparison with the tetraploids. Pentaploid cranberries are hitherto known only from the Czech Republic. They differ particularly in the low proportion of fully-developed tetrads. [ABSTRACT FROM AUTHOR]

Abstract: Contact zones of different cytotypes provide an opportunity to address evolutionary mechanisms underlying the origin, establishment and maintenance of karyological diversity at intraspecific level. We explored the fine-scale distribution of ploidy levels of Oxalis obtusa in seven mixed-ploidy sites in the Western and Northern Cape Provinces, South Africa, and searched for potential isolating barriers promoting assortative mating. Five different ploidy levels (2x, 3x, 4x, 6x and 8x) were detected among 336 samples collected at 112 microsites. The studied sites were inhabited by two (2 sites), three (4 sites) or even all five (1 site) different cytotypes. Despite their sympatric growth, different ploidies show some spatio-ecological segregation. The greatest differences among microsites hosting different cytotypes were found in precipitation parameters. There is a clear altitudinal gradient in ploidy composition in the most ploidy-variable site, Pakhuis Pass. Our results show that a combination of niche partitioning and clumping of same-ploidy individuals due to vegetative reproduction seems to be efficient reproductive barriers, which limit inter-ploidy gene flow in the zones of ploidy contact and contribute to the long-term maintenance of cytotype mixtures in O. obtusa. [Copyright &y& Elsevier]