Measurement Of Nuclear DNA Content Research Articles

Flow cytometric analysis of pollen and spores: An overview of applications and methodology.

Pollen grains are the male gametophytes in a seed-plant life cycle. Their small, particulate nature and crucial role in plant reproduction have made them an attractive object of study using flow cytometry (FCM), with a wide range of applications existing in the literature. While methodological considerations for many of these overlap with those for other tissue types (e.g., general considerations for the measurement of nuclear DNA content), the relative complexity of pollen compared to single cells presents some unique challenges. We consider these here in the context of both the identification and isolation of pollen and its subunits, and the types of research applications. While the discussion here mostly concerns pollen, the general principles described here can be extended to apply to spores in ferns, lycophytes, and bryophytes. In addition to recommendations provided in more general studies, some recurring and notable issues related specifically to pollen and spores are highlighted.

Peaceful revolution in genome size: polyploidy in the Nabidae (Heteroptera); autosomes and nuclear DNA content doubling

Abstract Genome size and the position of 18S ribosomal DNA (rDNA) were analysed in two Himacerus, eight Nabis and two Prostemma species from the family Nabidae using flow cytometry and fluorescence in situ hybrization techniques. The karyotypes of Nabis biformis and Nabis maoricus, each with 2n = 16 + XY, and Prostemma aeneicolle, with 2n = 26 + XY, were recorded for the first time. All the species displayed one or two 18S rDNA signals on the X chromosome and up to two signals on the Y chromosome. Several females exhibited two different types of X chromosome breakage, namely within or outside of the 18S rDNA region. Measurements of nuclear DNA content revealed significant differences between all three genera under study. Most notably, the nuclear DNA content of Himacerus species, with 2n = 32/36 + XY (2C = 9–10 pg), was double that of Nabis species, with 2n = 16 + XY (2C = 4–6 pg). Therefore, the previously rejected theory of an autosomal polyploidy event in the evolution of the genus Himacerus is strongly supported by the results of the present study and is now being resurrected.

Nuclear DNA content variation within four species of Asian catfish of family Pangasidae and their two interspecific hybrids by flow cytometry

Nuclear DNA content (NDC) of species or population is believed to have been formed naturally by many mechanisms such chromosomal mutation, insertion and deletion, transposable element, and duplication. Additionally, hybridizations and species’ phylogenetic relationship may also contribute to the NDC diversity. This study was aimed to investigate the profile of NDC in four species Asian catfishes of the genera Pangasius including Pangasionodon hypophthalmus, Pangasius djambal, Pangasius nasusutus, Pangasius nieuwenhuisii, interspecific hybrid of female P. hypophthalmus and male P. djambal (Hybrid HD), and female P. hypophthalmus and male P. nasutus (Hybrid HN). Red blood cells (RBC) were taken from the respective species/groups and NDC measurement was performed in an Attune acoustic flowcytometer (ABI) using DAPI staining and chicken, Gallus domesticus, RBC was used as size reference. The results showed that the mean NDC of P. hypophthalmus, P. djambal. P. nasusutus, P. nieuwenhuisii, were 0.960±0.0254 pg, 1.017±0.0510 pg, 1.000±0.0410 pg, 1.074±0.0231 pg, which are within the range of NDC in the other catfish families The NDC values of Hybrid HD and Hybrid HN were1.005±0.0358 and 0.956± 0.0089, respectively. Among the pure line species, the NDC of P. hypophthalmus was the lowest and was different (P<0.05) from those of P. djambal and P. nieuwenhuisii but was not different (P>0.05) from that of the P. nasutus. The NDC of both Hybrid HD and Hybrid HN were not different form their respective parental lines. However, the NDC profiles of both hybrids were different in that the NDC of the former was in between while the latter was below their respective parental lines. Phylogenetically, the NDC diversity within Pangasiid catfish in this study was independent of their phylogenetic relationship based on cytoplasmic and nuclear markers.
 Keywords: Flow cytometry, nuclear DNA content, P.hypophthalmus, P. djambal, P. nasutus, P. nieuwenhuisii, interspecific hybrid.

Unravelling the complexity of salt marsh ‘Fucus cottonii’ forms (Phaeophyceae, Fucales)

Genetic affiliation, nuclear DNA content, and gamete functioning were examined in small salt marsh Fucus from three localities in western Ireland. Individuals with small and dioecious receptacles were found at all localities, but production of germlings was only evident at Locality 1. Here, the Fucus vegetation formed a morphological cline from F. vesiculosus with bladders in the mid-intertidal to small Fucus individuals lacking bladders in the salt marsh of the upper intertidal. Measurements of nuclear DNA content ranged from 1–1.8 pg at this locality, with F. vesiculosus individuals in the lower range. At the two other localities, the small salt marsh Fucus consisted of distinct morphological entities. Microsatellite analyses revealed that individuals at Locality 2 were derived mainly from F. vesiculosus, whereas those from Locality 3 were hybrids between F. vesiculosus and F. spiralis with greatest affiliation to F. spiralis. While the small salt marsh Fucus forms from Locality 2 had high nuclear DNA content (c. 4 pg) and were probably octoploids, the small salt marsh Fucus from Locality 3 formed two groups: one with high (3.9–4.6 pg) and one with low (1.5–1.9 pg) nuclear DNA content. Nuclear DNA content measured in individuals from Locality 3 varied between 1.1–2.8 pg in F. vesiculosus and 2–3.5 pg in F. spiralis, and showed a more or less stepwise increase in both species, consistent with polyploidy. We hypothesize that the small salt marsh Fucus forms originate from genome size changes in the parental taxa.

Karyotype characterization and nuclear DNA content measurement in Bromeliaceae: state of the art and future perspectives.

In Bromeliaceae, cytogenetic and flow cytometry analyses have been performed to clarify systematic and evolutionary aspects. Karyotyping approaches have shown the relatively high chromosome number, similar morphology and small size of the chromosomes. These facts have prevented a correct chromosome counting and characterization. Authors have established a basic chromosome number of x = 25 for Bromeliaceae. Recently, one karyomorphological analysis revealed that x = 25 is no longer the basic chromosome number, whose genome may have a polyploid origin. Besides cytogenetic characterization, the 2C DNA content of bromeliads has been measured. Nuclear DNA content has varied from 2C = 0.60 to 2C = 3.34 picograms. Thus, in relation to most angiosperms, the 2C DNA content of Bromeliaceae species as well as their chromosome size can be considered relatively small. In spite of some advances, cytogenetic and flow cytometry data are extremely scarce in this group. In this context, this review reports the state of the art in karyotype characterization and nuclear DNA content measurement in Bromeliaceae, emphasizing the main problems and suggesting prospective solutions and ideas for future research.

Karyological relationships in Indian species of Drimia based on fluorescent chromosome banding and nuclear DNA amount.

The genus Drimia (syn. Urginea), commonly called squill, represents a species complex, infrageneric delimitation being ill-defined due to morphological variability, population variation within species and polyploidy. In the present study, fluorescent chromosome banding and measurements of nuclear DNA content by flow cytometry were performed in five Indian species of Drimia: Drimia indica, Drimia polyantha, Drimia razii, Drimia wightii and Drimia coromandeliana to elucidate taxonomic relationship and obtain possible insights into the evolutionary processes within this group. All taxa analyzed exhibited similar karyomorphology with subtle differences accounted by nucleolar chromosomes. Nuclear DNA content ranged from 20.41pg/2C in D. polyantha to 40.80pg/2C in D. coromandeliana and was positively correlated with chromosome number (r = 0.67, P = 0.02) and total diploid chromatin length (r = 0.59, P = 0.06). Fluorescent chromosome banding revealed the presence of CMA(+ve)/DAPI(-ve) signals associated with nucleolar chromosomes presumably coincident with NOR in all species and unique CMA(+ve) signals in diploid populations of D. indica. Satellite polymorphism between homologous NOR-bearing chromosomes was observed which supports hybrid origin of the taxon. UPGMA dendrogram and scatter diagrams based on karyological parameters indicated a close relationship of D. indica, D. razii and D. polyantha while D. wightii and D. coromandeliana appeared distant. D. wightii appeared more close to D. indica than to all other species based on genome size and karyomorphology. As a whole, D. indica showed high intra-specific variability with populations exhibiting intergrading characters with other species. In conclusion, it is likely that hybridization followed by reproductive isolation of polymorphic forms arising by adaptation to different ecological niches resulted in species diversification of Drimia in India, probably from a common ancestor similar to D. indica.

The first report of an alloheptaploid from the genusFragaria(Rosaceae)

AbstractHybrid recognition in the genus Fragaria (strawberry, Rosaceae) is difficult when based on morphological characteristics since there is great similarity among Fragaria species, even between different ploidy levels. This study aimed to identify and characterise an alloheptaploid hybrid (2n = 7x = 49) Fragaria for the first time in the genus from Europe (Germany, Bavaria) using different techniques. The hybrid was documented based on the evidence obtained from ploidy levels using flow cytometric measurement of nuclear DNA content, identification of the parental origin using 14 RAPD primers, fertility assessment from artificial crosses and fruit set level. RAPD analysis showed that the hybrid shared five species-specific markers with diploid F. vesca, and three with hexaploid F. moschata, indicating these species to be the most likely parents. The mean fluorescent intensity reflected by nuclear DNA in the putative hybrid (703) fell within the range accepted for heptaploidy in Fragaria, while the corr...

Karyological relationships among some South American species of Solanum (Solanaceae) based on fluorochrome banding and nuclear DNA amount

Fluorescent chromosome banding and measurements of nuclear DNA content by image cytometry of Feulgen-stained cells were performed in one sample each of eight diploid (2n = 24) species of Solanum: S. endoadenium, S. argentinum, S. pseudocapsicum, S. atropurpureum, S. elaeagnifolium, S. sisymbriifolium, S. chenopodioides, and S. palustre. The species studied could be distinguished by heterochromatin amount, banding patterns, and genome size. They exhibited only GC-rich heterochromatin and showed a comparatively low heterochromatin amount (expressed as percentage of haplotype karyotype length), ranging from 2.10 in S. argentinum to 8.37 in S. chenopodioides. Genome size displayed significant variation between species, with 1C-values ranging from 0.75 pg (735 Mbp) in S. palustre to 1.79 pg (1,754 Mbp) in S. sisymbriifolium. No significant correlation between genome size and heterochromatin amount was observed, but intrachromosomal asymmetry index (A1) was negative and significantly correlated with heterochromatin amount. DNA content was positively and significantly correlated with karyotype length. DNA C-value distribution in the genus as well as karyotype affinities and relationships between species are discussed in relation to different infrageneric classifications of Solanum.

Molecular Identification of Armillaria gallica from the Niobrara Valley Preserve in Nebraska

Armillaria isolates were collected from a unique forest ecosystem in the Niobrara Valley Preserve in Nebraska, USA, which comprises a glacial and early postglacial refugium in the central plains of North America. The isolates were collected from diverse forest trees representing a unique mixture of forest types. Combined methods of rDNA sequencing and flow cytometric measurements of nuclear DNA content determined that all Armillaria isolates collected from the site were A. gallica.

Integral method for measuring the quantity of cellular DNA content by digital microphotography

Quantitative measurements of nuclear DNA content based on Feulgen reaction and the analysis of CCD images has been proposed. The measurements were performed in the monochrome CCD option (650 × 514 pixels) with a wavelength of 551 nm. The linear dependence of photomatrix element signals on the falling light was shown with a multigrade light absorption filter. The optimal microscope and camera settings and an approach for elimination of the optic blur are proposed. It was found that the contribution of background fluorescence of Feulgen-stained nuclei into the measurements was negligible. Densitometric measurements of the DNA content in blood cells of four vertebrate species (Gallus domesticus, Danio rerio, Homo sapiens, Rana arvalis) were consistent with the literature data. The precision of our approach is comparable to other known cytometry methods (http://www.genomesize.com). The current improvement of CCD technical parameters and the widespread use of CCD cameras in biological applications give perspectives for the development of the suggested approach for measuring the quantity of cellular DNA.

New Record Holders for Maximum Genome Size in Eudicots and Monocots

This paper presents the largest genome sizes reported so far for angiosperms: for the monocotsTrillium hagae(Melanthiaceae) pg and for the eudicotsViscum album(Santalaceae) with pg. They were found during ongoing measurements of nuclear DNA content in more than 4000 accessions. Moreover, it is demonstrated that both of the values for the largest genome size so far for the monocots ofFritillaria assyriacaand for the eudicots ofViscum cruciatumneed to be corrected.

Cytogenetic characterization of Hydrangea involucrata Sieb. and H. aspera D. Don complex (Hydrangeaceae): genetic, evolutional, and taxonomic implications

The subsection Asperae of genus Hydrangea L. (Hydrangeaceae) has been investigated for three reasons: several ambiguous classifications concerning Hydrangea aspera have been published, unexpected differences in genome size among seven accessions have been reported Cerbah et al. (Theor Appl Genet 103:45–51, 2001), and two atypical chromosome numbers (2n = 30 for Hydrangea involucrata and 2n = 34 for H. aspera) have been found when all other species of the genus present 2n = 36. Therefore, these two species and four subspecies of Hydrangea in all 29 accessions were analyzed for their genome size, chromosome number, and karyotype features. This investigation includes flow cytometric measurements of nuclear DNA content and bases composition (GC%), fluorochrome banding for detection of GC- and AT-rich DNA regions, and fluorescent in situ hybridisation (FISH) for chromosome mapping of 5 S and 18 S-5.8 S-26 S rDNA genes. In the H. aspera complex, the genome size ranged from 2.98 (subsp. sargentiana) to 4.67 pg/2C (subsp. aspera), an exceptional intraspecific variation of 1.57-fold. The mean base composition was 40.5% GC. Our report establishes the first karyotype for the species H. involucrata, and for the subspecies of H. aspera which indeed present different formulae, offering an element of discrimination. FISH and fluorochrome banding revealed the important differentiation between these two species (H. involucrata and H. aspera) and among four subspecies of the H. aspera complex. Our results are in agreement with the Chinese classification that places the groups Kawakami and Villosa as two different species: Hydrangea villosa Rehder and Hydrangea kawakami Hayata. This knowledge can contribute to effective germplasm management and horticultural use.

Karyotype and genome size of Iberochondrostoma almacai (Teleostei, Cyprinidae) and comparison with the sister-species I. lusitanicum

This study aimed to define the karyotype of the recently described Iberian endemic Iberochondrostoma almacai, to revisit the previously documented chromosome polymorphisms of its sister species I.lusitanicum using C-, Ag-/CMA3 and RE-banding, and to compare the two species genome sizes. A 2n = 50 karyotype (with the exception of a triploid I.lusitanicum specimen) and a corresponding haploid chromosome formula of 7M:15SM:3A (FN = 94) were found. Multiple NORs were observed in both species (in two submetacentric chromosome pairs, one of them clearly homologous) and a higher intra and interpopulational variability was evidenced in I.lusitanicum. Flow cytometry measurements of nuclear DNA content showed some significant differences in genome size both between and within species: the genome of I. almacai was smaller than that of I.lusitanicum (mean values 2.61 and 2.93 pg, respectively), which presented a clear interpopulational variability (mean values ranging from 2.72 to 3.00 pg). These data allowed the distinction of both taxa and confirmed the existence of two well differentiated groups within I. lusitanicum: one that includes the populations from the right bank of the Tejo and Samarra drainages, and another that reunites the southern populations. The peculiar differences between the two species, presently listed as “Critically Endangered”, reinforced the importance of this study for future conservation plans.

Book review: Flow cytometry with plant cells: Analysis of genes, chromosomes and genomes

Book review: Flow cytometry with plant cells: Analysis of genes, chromosomes and genomes

Measurement of nuclear DNA content of the genus Trifolium L. as a measure of genebank accession identity

The collection, identification and maintenance of genebank accessions of the genus Trifolium is a major task because of the large number of genera and their occasional morphological similarity. We investigated whether the measurement of nuclear DNA content can serve as an additional criterion for identification of mislabeled accessions. Relative nuclear DNA content was determined by flow cytometry measurements for a total of 151 genebank accessions of 23 Trifolium species with notable agronomical value. Among 23 species analyzed, 15 were found to possess a uniform relative nuclear DNA content, with intraspecific variability of the majority of analyzed species lower than 5%. Within six Trifolium species, 1–2 atypical accessions with outstanding differences in relative DNA content, chromosome number and morphological features were found. In T. hybridum and partially in T. ambiguum these outstanding differences could be ascribed to variations in accession ploidy level. For the remaining atypical accessions, the determined nuclear DNA content, chromosome number and morphological features were not related to those characteristic of the species. Additionally taxonomic identity of atypical accessions was determined using ITS region sequencing and morphological observations. We propose flow cytometric measurements of nuclear DNA content as simple and reliable technique which can be used at seedling stage to verify identity and genetic stability of Trifolium genebank accessions.

Cytochemistry and C-values: The Less-well-known World of Nuclear DNA Amounts

In the plant sciences there are two widely applied technologies for measuring nuclear DNA content: Feulgen absorbance cytophotometry and flow cytometry (FCM). While FCM is, with good reasons, increasingly popular among plant scientists, absorbance-cytophotometric techniques lose ground. This results in a narrowing of the methodological repertoire, which is neither desirable nor beneficial. Both approaches have their advantages, but static cytophotometry seems to pose more instrumental difficulties and material-based problems than FCM, so that Feulgen-based data in the literature are often less reliable than one would expect. The purpose of this article is to present a selective overview of the field of nuclear DNA content measurement, and C-values in particular, with a focus on the technical difficulties imposed by the characteristics of the biological material and with some comments on the photometrical aspects of the work. For over 20 years it has been known that plant polyphenols cause problems in Feulgen DNA cytophotometry, since they act as major staining inhibitors leading to unreliable results. However, little information is available about the chemical classes of plant metabolites capable of DNA staining interference and the mechanisms of their inhibition. Plant slimes are another source of concern. In FCM research to uncover the effects of secondary metabolites on measurement results has begun only recently. In particular, the analysis of intraspecific genome size variation demands a stringent methodology which accounts for inhibitors. FCM tests for inhibitory effects of endogenous metabolites should become obligatory. The use of dry seeds for harvesting embryo and endosperm nuclei for FCM and Feulgen densitometry may often provide a means of circumventing staining inhibitors. The importance of internal standardization is highlighted. Our goal is a better understanding of phytochemical/cytochemical interactions in plant DNA photometry for the benefit of an ever-growing list of plant genome sizes.

Production and Characterization of Intergeneric Hybrids between Dichroa febrifuga and Hydrangea macrophylla

The potential of producing an intergeneric hybrid between Dichroa febrifuga Lour. and Hydrangea macrophylla (Thunb.) Ser. was investigated. Reciprocal hybridizations were made between a D. febrifuga selection (GUIZ 48) and diploid (‘Veitchii’) and triploid (‘Kardinal’ and ‘Taube’) cultivars of H. macrophylla. Embryo rescue was employed for about one-third of the crosses that produced fruit, and the rest were allowed to mature on the plant and seed-collected and germinated. Reciprocal hybrids, which were verified with simple sequence repeat markers, were produced from both embryo rescue and seed germination and with both diploid and triploid H. macrophylla cultivars. Hybrids were intermediate in appearance between parents, but variability in leaf, inflorescence, and flower size and flower color existed among the hybrids. A somatic chromosome number of 2n = 6x = 108 was tentatively proposed for D. febrifuga GUIZ 48. Chromosome counts and flow-cytometric measurements of nuclear DNA content indicated that some of the hybrids may be aneuploids, but neither analysis was definitive. Although hybrids with H. macrophylla as the pistillate parent did not form pollen-producing anthers, D. febrifuga × H. macrophylla hybrids had normal-appearing anthers that produced abundant pollen. F2 and BC1 progeny were obtained using D. febrifuga × ‘Veitchii’ hybrids. This work documents the first step in an effort to combine desirable horticultural features from D. febrifuga and H. macrophylla.

Flow cytometric measurements do not reveal different ploidy levels in Minthostachys (Lamiaceae)

Ten of the 17 species of the taxonomically difficult Andean mint genus Minthostachys (Lamiaceae) were submitted to flow cytometric measurements of nuclear DNA content to test the hypothesis of the occurrence of different ploidy levels within the genus. Nuclear DNA content was found to vary from 1.643 to 1.775 pg, i.e by only ca. 8% between individual accessions, thus providing no evidence for polyploidy in Minthostachys. While these results do not preclude the possibility that the genus contains polyploid species nor the occurrence of heteroploidy with nearly identical nuclear DNA contents, they suggest that polyploidy did not play a major role in its diversification.

Estimation of nuclear DNA content in plants using flow cytometry

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.

Interploidal gene flow and introgression in bryophytes: Sphagnum girgensohnii × S. russowii, a case of spontaneous neotriploidy

Hybrid zones between female sporophyte-bearing haploid Sphagnum girgensohnii and male allodiploid S. russowii were studied. The existence of hybrids was initially hypothesized based on incidence of conspicuously large sporophytes in S. girgensohnii in the presence of male S. russowii. Measurements of nuclear DNA content, morphology and isozymes provide the first evidence of spontaneous hybridization across ploidal levels in bryophytes. Viable spores from the hybrid capsules yielded triploid protonemata and juvenile gametophores in culture. Isozyme profiles of the triploid gametophores displayed unbalanced heterozygous patterns containing two alleles. As S. russowii is an allodiploid species with S. girgensohnii and S. rubellum as progenitors, isozyme banding patterns of the triploids are interpreted as consisting of a single allele copy from the S. rubellum genome in S. russowii, and two copies of another allele at the same locus inherited from S. girgensohnii. The triploid sporophytes can be considered as F2 hybrids formed by introgression. The allotriploid gametophores are formed through diplospory or syndiploidy in the hybrid capsules. Comparison of hybrid sporophyte morphology to S. girgensohnii and S. russowii confirmed that hybrid capsules are larger and with an abnormally swollen sporophyte foot. The spore sizes of the hybrid capsules were significantly larger than spores from normal, intraspecific S. girgensohnii capsules. The percentage of germinated spores was <5% in cultures from hybrid sporophytes, which is much lower than in intraspecific sporophytes. The new knowledge opens the way for re-evaluation of polyploid evolution in bryophytes with emphasis on gametic non-reduction, introgression and two-step models of polyploid formation.