Estimation Of Nuclear DNA Content Research Articles

Nuclear DNA content and comparative FISH mapping of the 5s and 45s rDNA in wild and cultivated populations of Physalis peruviana L.

Physalis peruviana L. often known as goldenberry, has increased its commercial growth in the international market in recent years due to its nutritional value and antioxidant potential. This situation has enabled countries such as Peru to increase their production in order to meet the global demand. However, investigations about the genetic diversity of cultivated and wild populations of goldenberry are still in their early stages. FISH mapping of 5s and 45s rDNA loci and flow cytometry estimation of nuclear DNA content were used to assess genetic differences between wild and cultivated goldenberry populations from Ayacucho and Cajamarca. The majority of metaphases had six 5s rDNA sites for all populations and two and four 45s rDNA sites for the cultivated and wild populations, respectively. We were able to characterize nine different types of chromosomes based on their morphology, fluorescence, rDNA location, and conservation across populations by analyzing the chromosomes that contained rDNA. Furthermore, cultivated populations had more nuclear DNA (13.262±0.087 pg) than wild populations (12.955±0.086 pg). The results show genetic differences between wild and cultivated populations of goldenberry at molecular cytogenetic level as well as in genome size. These findings establish a precedent for future cytogenetic and genomic studies in goldenberry populations, enabling future breeding programs.

Mastering DNA Content Estimation by Flow Cytometry as an Efficient Tool for Plant Breeding and Biodiversity Research.

Flow cytometry gives a unique opportunity to analyze thousands of individual cells for multiple parameters in a course of minutes. The most commonly used flow cytometry application in plant biology is estimation of nuclear DNA content. This becomes an indispensable tool in different areas of plant research, including breeding, taxonomy, plant development, evolutionary biology, populational studies and others. DNA content analysis can provide an insight into natural ploidy changes that reflect evolutionary processes, such as interspecific hybridization and polyploidization. It is also widely used for processing samples with biotechnologically induced ploidy changes, for instance, plants produced by doubled haploid technology. Absolute genome size data produced by cytometric analysis serve as useful taxon-specific markers since genome size vary between different taxa. It often allows the distinguishing of species within a genus or even different subspecies. Introducing flow cytometry method in the lab is extremely appealing, but new users face a significant challenge of learning instrument management, quality sample preparation and data processing. Not only is flow cytometry a complex method, but plant samples have unique features that make plants a demanding research subject. Without proper training, researchers risk damaging the expensive instrument or publishing poor quality data, artifacts or unreproducible results. We bring together information from our experience, key papers and online resources to provide step by step protocols and give a starting point for exploring the abundant cytometry literature.

Nuclear DNA Content Estimation of Seaweed by Fluorimetry Analysis.

Fluorimetry analysis of nuclear DNA content allows identification of genome size and ploidy levels of different life phases, tissues, and populations in seaweed species. It is an easy method that saves time and resources compared to more complex techniques. Here we describe the methodology for measuring nuclear DNA content in seaweed species by DAPI fluorochrome staining and its comparison with the standard Gallus gallus erythrocytes nuclear content, one of the preferred internal standards. With this methodology, up to a thousand nuclei can be measured in a single staining session, allowing for a quick analysis of the studied species.

Estimation of Nuclear DNA Content in Some Aegilops Species: Best Analyzed Using Flow Cytometry.

The genera Triticum and Aegilops have been considered as the main gene pool of wheat due to their features, such as tolerance of all types of abiotic and biotic stresses. This study was conducted to evaluate the cytogenetic analyses in 115 native and wild populations from eleven Aegilops species using their nuclear DNA quantification. Mean 2C nuclear DNA contents of different ploidy levels in the wild wheat of Turkey and Iran were measured using the flow cytometry technique. The obtained results showed that the mean nuclear DNA content in diploid species varied from 10.09 pg/2C (Ae. umbellulata) to 10.95 pg/2C (Ae. speltoides var. ligustica) in Turkey. In Iranian diploids, the mean nuclear DNA content varied from 10.20 pg/2C (Ae. taushii) to 11.56 pg/2C (Ae. speltoides var. ligustica). This index in the tetraploid species of Turkey varied from 18.09 pg/2C (Ae. cylindrica) to 21.65 pg/2C (Ae. triaristata), and in Iranian species, it was from 18.61 pg/2C (Ae. cylindrica) to 21.75 pg/2C (Ae. columnaris). On the other hand, in the hexaploid species of Turkey, this index varied from 31.59 pg/2C (Ae. crassa) to 31.81 pg/2C (Ae. cylindrica); in the Iranian species, it varied from 32.58 pg/2C (Ae. cylindrica) to 33.97 pg/2C (Ae. crassa). There was a significant difference in the DNA content of Turkey and Iran diploid as well as tetraploid species; however, in hexaploid species, the difference was not significant. It was concluded that the variation in intraspecific genome size was very low in diploid and tetraploid populations; this means that the low variation is not dependent on geographic and climatic parameters. On the other hand, the interspecific variation is significant at the diploid and tetraploid populations. It is generally very difficult to distinguish Aegilops species from each other in natural conditions; meanwhile, in this study, all species could be, easily, quickly and unambiguously, distinguished and separated using the FCM technique.

Reference standards for flow cytometric estimation of absolute nuclear DNA content in plants.

The estimation of nuclear DNA content has been by far the most popular application of flow cytometry in plants. Because flow cytometry measures relative fluorescence intensities of nuclei stained by a DNA fluorochrome, ploidy determination, and estimation of the nuclear DNA content in absolute units both require comparison to a reference standard of known DNA content. This implies that the quality of the results obtained depends on the standard selection and use. Internal standardization, when the nuclei of an unknown sample and the reference standard are isolated, stained, and measured simultaneously, is mandatory for precise measurements. As DNA peaks representing G1/G0 nuclei of the sample and standard appear on the same histogram of fluorescence intensity, the quotient of their position on the fluorescence intensity axis provides the quotient of DNA amounts. For the estimation of DNA amounts in absolute units, a number of well‐established standards are now available to cover the range of known plant genome sizes. Since there are different standards in use, the standard and the genome size assigned to it has always to be reported. When none of the established standards fits, the introduction of a new standard species is needed. For this purpose, the regression line approach or simultaneous analysis of the candidate standard with several established standards should be prioritized. Moreover, the newly selected standard organism has to fulfill a number of requirements: it should be easy to identify and maintain, taxonomically unambiguous, globally available, with known genome size stability, lacking problematic metabolites, suitable for isolation of sufficient amounts of nuclei, and enabling measurements with low coefficients of variation of DNA peaks, hence suitable for the preparation of high quality samples.

Plant material selection, collection, preservation, and storage for nuclear DNA content estimation.

In theory, any plant tissue providing intact nuclei in sufficient quantity is suitable for nuclear DNA content estimation using flow cytometry (FCM). While this certainly opens a wide variety of possible applications of FCM, especially when compared to classical karyological techniques restricted to tissues with active cell division, tissue selection and quality may directly affect the precision (and sometimes even reliability) of FCM measurements. It is usually convenient to first consider the goals of the study to either aim for the highest possible accuracy of estimates (e.g., for inferring genome size, detecting homoploid intraspecific genome size variation, aneuploidy, among others), or to decide that histograms of reasonable resolution provide sufficient information (e.g., ploidy level screening within a single model species). Here, a set of best practices guidelines for selecting the optimal plant tissue for FCM analysis, sampling of material, and material preservation and storage are provided. In addition, factors potentially compromising the quality of FCM estimates of nuclear DNA content and data interpretation are discussed.

Nuclei isolation protocols for flow cytometry allowing nuclear DNA content estimation in problematic microalgal groups

Microalgae are fundamentally important organisms for global ecosystem functioning with high potential in biotechnology and its applications. The knowledge of their nuclear DNA content has become a prerequisite for many areas of microalgal research. Due to common presence of various pigments, secondary metabolites and complex cell walls, the nuclear DNA content estimation using flow cytometry (FCM) is, however, often laborious or even impossible with the currently used protocols. In this study the performance of six nuclei isolation protocols was compared on various problematic microalgae using FCM. The nuclei isolation methods involved osmotic bursting of cells, razor blade chopping of fresh biomass and two newly introduced protocols, razor blade chopping of desiccated biomass and bead beating. These techniques also involved the use of two different nuclei isolation solutions, Otto I + II solutions, and LB01 buffer. Performance of the particular protocols differed greatly, depending on the used nuclei isolation solution and microalgal group. The most successful method was a newly adopted chopping of desiccated biomass in LB01 buffer. This method seems more appropriate for nuclei isolation in filamentous microalgae; on the other hand, bead beating appears to be more suitable for nuclei isolation in solitarily living algae. Using the optimal protocol for a given species, their nuclear DNA content was estimated, resulting in first DNA content estimates for four investigated taxa (Chlamydomonas noctigama, Gonyostomum semen, Microglena sp. and Stigeoclonium sp.). The estimated DNA content spanned from 0.15 to 32.52 pg.

Isolation of plant nuclei for estimation of nuclear DNA content: Overview and best practices.

A critical aspect for obtaining accurate, reliable, and high-resolution estimates of nuclear DNA content is the release of nuclei from the cytoplasm in sufficient amounts, while maintaining their integrity throughout the analysis, protecting their DNA from degradation by endonucleases, and enabling stoichiometric DNA staining. In embryophytes, the most common method consists of chopping the plant material with a sharp razor blade to release nuclei into an isolation buffer, filtering the homogenate, and staining the nuclei in buffered suspension with a fluorochrome of choice. Despite the recent description of alternative approaches to isolate nuclei, the chopping procedure remains the most widely adopted method, due to its simplicity, rapidity, and effectiveness. In this review article, we discuss the specifics of nuclei isolation buffers and the distorting effects that secondary metabolites may have in nuclear suspensions and how to test them. We also present alternatives to the chopping procedure, options for filtering and fluorochromes, and discuss the applications of these varied approaches. A summary of the best practices regarding the isolation of plant nuclei for the estimation of nuclear DNA content is also provided.

Nuclear DNA content estimations and nuclear development patterns in Antarctic macroalgae

Nuclear DNA content of marine macroalgae is known for only about a 3% of the globally known taxa, and so far, the only available data concerning marine Antarctic macroalgae refer to the Phaeophyceae Ascoseira mirabilis and Desmarestia antarctica. As these data can be useful for species delimitation when combined with other taxonomical information, we aimed to expand the available number of measurements in the understudied Antarctic seaweed flora. To address this, we measured the nuclear DNA content of 12 Antarctic marine algae (seven Rhodophyceae and five Phaeophyceae). Nuclear DNA analyses were carried out by spectrofluorimetry and image analysis from samples conserved in Carnoy, using DAPI as DNA marker. For ten of these taxa, our values represent the first estimations to date. The nuclear DNA content estimates obtained for the Antarctic red algae examined vary between 2C = 0.38 pg in Gigartina skottsbergii and 2C = 1.63 pg in Neuroglossum delesseriae. In brown algae, the values range from 2C = 0.18 pg in both Desmarestia antarctica and Desmarestia menziesii to 2C = 0.96 pg in Phaeurus antarcticus. Furthermore, this study allowed us to identify nuclear developmental patterns for the first time in two Antarctic seaweeds (Ballia callitricha and Neuroglossum delesseriae).

A novel cysteine-rich peptide regulates cell expansion in the tobacco pistil and influences its final size

Plant morphogenesis is dependent on cell proliferation and cell expansion, which are responsible for establishing final organ size and shape during development. Several genes have been described as encoding components of the plant cell development machinery, among which are the plant peptides. Here we describe a novel cysteine-rich plant peptide (68 amino acids), encoded by a small open reading frame gene (sORF). It is specifically expressed in the reproductive organs of Nicotiana tabacum and is developmentally regulated. N- and C-terminal translational fusions with GFP in protoplasts have demonstrated that the peptide is not secreted. Knockdown transgenic plants produced by RNAi exhibited enlarged pistils due to cell expansion and the gene was named Small Peptide Inhibitor of Cell Expansion (SPICE). Estimation of nuclear DNA content using flow cytometry has shown that cell expansion in pistils was not correlated with endoreduplication. Decreased SPICE expression also affected anther growth and pollen formation, resulting in male sterility in at least one transgenic plant. Our results revealed that SPICE is a novel reproductive organ specific gene that controls cell expansion, probably as a component of a signal transduction pathway.

Estimation of nuclear DNA content and its variation among Indian Tea accessions by flow cytometry.

Nuclear DNA content and genome size variation among 36 Indian tea accessions were analyzed by flow cytometry. Initial standardization of protocols for isolation of nuclei, DNA staining and selection of an internal standard for tea accessions which have significantly high amount of phenolic secondary metabolites in their cytosol was carried out. Results obtained revealed that 2C DNA content of Indian tea is 7.46pg which correspondsto 1C genome size of 3673Mb. Inter accession variation in 2C DNA content was also observed among 35 diploid taxa ranging from 7.23 to 7.73pg which was significant at 1% probability level. The 2C DNA content of triploid (UPASI 3) was observed to be 11.47pg which is concurrent with the expected value. Results obtained showed that Assam and Cambod type tea accession have higher 2C DNA content of 7.73pg whereas Assam Cambod hybrids and Assam China hybrids have reduction in DNA content with 2C amounts, 7.23 and 7.32pg DNA respectively. The present study suggests that the species involved in origin of Indian tea must have differed in their genome sizes owing to significant inter accession variation in nuclear DNA content.

Nuclear genome size and chromosome number in the Mexican genusPittocaulon(Asteraceae)

Estimation of nuclear DNA content and chromosome number of four species of Pittocaulon (Asteraceae, Senecioneae), a small genus of Mexican succulent shrubs, was performed. The chromosome number 2n ...

The rising world of flow cytometric analysis of pollen grains.

Key terms flow cytometry; genome size; plant sciences; pollen grains; pulse analysis FLOW cytometry is nowadays recognized as a vital tool in plant sciences, with more and more applications in both basic and applied research (1). Due to the unique characteristics of plant material, i.e., complex three-dimensional tissues with the presence of rigid cell walls, specific methods had to be developed to produce suspensions of single particles of interest (2). These particles of interest include, nuclei, mitochondria, chloroplasts, and chromosomes, with most of the applications being focused around the estimation of nuclear DNA content (either in absolute or relative amounts), leading to increasing impacts in the fields of plant breeding and population biology. One of the few single particles produced by plants is the pollen grain. Still, most works focused on studying pollen grains have involved the extraction of nuclei or sperm cells, rather than using the intact pollen. This is mostly due to the autofluorescence and/or specific staining of the pollen exine, and to the particle size limitations of some flow cytometers (3). To add to this, isolation of high-quality nuclei from pollen grains is often difficult, at least in comparison with somatic tissues. Together, these difficulties have led to a very minute number of publications involving flow cytometric analysis of pollen grains in the last 25 years.

Challenges of flow-cytometric estimation of nuclear genome size in orchids, a plant group with both whole-genome and progressively partial endoreplication.

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.

Image cytometric analysis of nuclear and chromosomal DNA contents in two cytotypes of Aloe vera (L.) Burm. f.

Estimation of nuclear DNA content and distribution of DNA content in the chromosomal complement in diploid (2x =14) and triploid (3x =21) cytotypes of Aloe vera was done by using Feulgen image cytometric method. Feulgen densitometry analysis revealed that the DNA contents were directly proportional to chromosomal lengths in both diploid and triploid chromosomal complements. Analysis revealed that the mean DNA content of a chromosome in the diploid 2.42 pg was higher to the mean value of 2.15 pg of the triploid. Consequently, the mean DNA amount per basic genome (calculated by dividing the 2C value by ploidy) decreased with increasing ploidy. Estimation of nuclear DNA contents revealed that the triploid cytotype was 11 % less than the nuclear DNA content of diploid indicating genome downsizing or loss of DNA following polyploidy.

Development of Flow Cytometric Protocol for Nuclear DNA Content Estimation and Determination of Chromosome Number in Pongamia pinnata L., a Valuable Biodiesel Plant

The potentiality of Pongamia pinnata L. as a sustainable source of feedstock for the biodiesel industry is dependent on an extensive knowledge of the genome structure of the plant. Flow cytometry, with propidium iodide (PI) as the DNA stain, was used to estimate the nuclear DNA content of P. pinnata, with respect to Zea mays 'CE-777' as standard. The internal and pseudo-internal standardization was followed on account of the inhibitory effect of secondary compounds on PI intercalation. The antioxidants (PVP-40 and β-mercaptoethanol) were added to the nuclear isolation buffer for the reduction of inhibitory effect of P. pinnata cytosol. Nuclear DNA content estimation was done for P. pinnata leaves from different altitudes (37-117 m height from sea level) of Assam. Flow cytometry analysis indicated that the nuclear DNA content of P. pinnata is 2.66 pg with predicted 1C value of 1,300 Mb using Z. mays as standard. Coefficient of variation in flow cytometric analysis was within the limit of 5 % indicating that the results were reliable. Somatic chromosome numbers were counted from root-tip cells and was found to be 2n = 22 corresponding to the diploid level (x = 11). A decreasing trend in the nuclear DNA content was observed for the species of different altitudes.

Whole genome sequencing improves estimation of nuclear DNA content of Chinese hamster ovary cells

Whole genome sequencing improves estimation of nuclear DNA content of Chinese hamster ovary cells

Glycerol-treated nuclear suspensions—an efficient preservation method for flow cytometric analysis of plant samples

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.

Estimates of nuclear DNA content in red algal lineages.

The red algae are an evolutionarily ancient group of predominantly marine organisms with an estimated 6000 species. Consensus higher-level molecular phylogenies support a basal split between the unicellular Cyanidiophytina and morphologically diverse Rhodophytina, the later subphylum containing most red algal species. The Rhodophytina is divided into six classes, of which five represent early diverging lineages of generally uninucleate species, whose evolutionary relationships are poorly resolved. The remaining species compose the large (27 currently recognized orders), morphologically diverse and typically multinucleate Florideophyceae. Nuclear DNA content estimates have been published for <1 % of the described red algae. The present investigation summarizes the state of our knowledge and expands our coverage of DNA content information from 196 isolates of red algae. The DNA-localizing fluorochrome DAPI (4',6-diamidino-2-phenylindole) and RBC (chicken erythrocytes) standards were used to estimate 2C values with static microspectrophotometry. Nuclear DNA contents are reported for 196 isolates of red algae, almost doubling the number of estimates available for these organisms. Present results also confirm the reported DNA content range of 0.1-2.8 pg, with species of Ceramiales, Nemaliales and Palmariales containing apparently polyploid genomes with 2C = 2.8, 2.3 and 2.8 pg, respectively. Early diverging red algal lineages are characterized by relatively small 2C DNA contents while a wide range of 2C values is found within the derived Florideophyceae. An overall correlation between phylogenetic placement and 2C DNA content is not apparent; however, genome size data are available for only a small portion of red algae. Current data do support polyploidy and aneuploidy as pervasive features of red algal genome evolution.

Genome size in Anthurium evaluated in the context of karyotypes and phenotypes

Anthurium is an important horticultural crop from the family Araceae, order Alismatales, a lineage considered to have diverged from other monocots prior to the cereals. Genome size and its distribution in Anthurium were investigated to gain a basic understanding of genome organization in this large genus and to forge a firm foundation for advancement of molecular approaches for the study of Anthurium. Currently, genome size estimates have been reported for only two Anthurium samples. Bulk nuclear DNA content estimates were obtained by flow cell cytometry using leaf tissue collected from Anthurium species of different subgeneric groups and from commercial cultivars. The most current and well-supported topology of subgeneric, sectional relationships was applied to present genome size estimates in the context of reported chromosome counts, karyotypes, putative phylogenetic relationships, observed phenotypes and pedigree. Genome size estimates based on bulk nuclear DNA content for 77 accessions representing 34 species and 9 cultivars were obtained, including initial estimates for 33 Anthurium species, and both the smallest (Anthurium obtusum; Tetraspermium) and largest (Anthurium roseospadix; Calomystrium) Anthurium genome sizes reported to date. Genome size did not distinguish any subgeneric section, but ranged 5-fold (4.42-20.83 pg/2 C) despite consistent 2N= 30 chromosome counts. Intraspecies genome size variation >20 % is reported for Anthurium ravenii, A. watermaliense and A. gracile. Genome size estimates for Anthurium species spanning 13 recognized subgeneric sections indicate that genome size does not generally correlate with chromosome count or phylogenetic relationships. Mechanisms of genome expansion and contraction, including amplification and reduction of repetitive elements, polyploidy, chromosome reorganization/loss, may be involved in genome evolution in Anthurium as in other species. The new information on Anthurium genome sizes provides a platform for molecular studies supporting further research on genome evolution as well as cultivar development.