The systematic value of nuclear DNA content for all species of Narcissus L. (Amaryllidaceae)

The taxonomy of all species of Narcissus (Amaryllidaceae), an important horticultural crop, has not been investigated recently. As a new approach, genome size was determined by flow cytometry with propidium iodide from 375 accessions. The somatic nuclear DNA contents (2C) were shown to range from 14 to 38 pg for the diploids. Narcissus assoanus and N. gaditanus are, based on their nuclear DNA content, removed from section Apodanthi and placed in a new section Juncifolii. The different ploidy levels and species involved were entangled for N . “fernandesii” s.l. and a new allotetraploid form is named here. Section Pseudonarcissus was much more heterogeneous in nuclear DNA content than expected. Sixty-five accessions of N. pseudonarcissus possessed, with 23.7 pg, similar amounts of DNA. However, several species from this section were clearly distinctive in nuclear DNA content. It runs from the diploid N. primigenius with 21.7 pg to the also diploid N. nevadensis with 38.2 pg. Also N. abscissus and N. moleroi are with about 26 pg clearly different from N. pseudonarcissus. For the first time, in 11 accessions, hexaploidy was found in N. pseudonarcissus ssp. bicolor. A new section Nevadensis with 30–39 pg of nuclear DNA was split off from the section Pseudonarcissus with now 21–27 pg. A nonoploid N. dubius with 96.3 pg has by far the highest amount of nuclear DNA and can be calculated to have the highest ploidy ever reported in Narcisssus. The total number of Narcissus species was determined as 36, nine more than in Flora Europaea and they were divided up in two subgenera and 11 sections. Flow cytometry is shown to produce easily obtainable and original systematic data that lead to new insights. Genome size or C-value turns out to be one of the most salient features to define the status of the species in the genus Narcissus.     

Nuclear DNA content of Pinus sylvestris (L.) as determined by laser flow cytometry

Nuclear DNA content was determined in nuclei isolated from needles, stems and roots of in vitro grown seedlings and from megagametophytes and embryo of mature seeds in three accessions of Pinus sylvestris L. One accession was from Inari, northern Finland at timber line, and two accessions were from the Alpine region in Italy. Nuclei were mechanically isolated by a chopping method, stained with propidium iodide, and DNA content was determined using an EPICS PROFILE laser flow cytometer. Nuclei isolated from leaves of barley (Hordeum vulgare L. cv. Sultan; 2C=11.12 pg) were used as an internal standard for measurement of pine nuclei. Mean 1C nuclear DNA content of P. sylvestris was 27.88 pg as determined from megagametophyte tissue. Mean 2C value was 52.25 pg as determined from stem and root tissue, and 55.58 pg as determined from embryo tissue. The ratio of 2C to 1C value was 1.87 and 1.99, respectively. Extracts of nuclei from needles contained propidium iodide-absorbing debris which may have interfered with measurements and resulted in lower 2C values than those obtained from stem and root.     

DNA Content Variation and Its Significance in the Evolution of the Genus Micrasterias (Desmidiales,Streptophyta)

It is now clear that whole genome duplications have occurred in all eukaryotic evolutionary lineages, and that the vast majority of flowering plants have experienced polyploidisation in their evolutionary history. However, study of genome size variation in microalgae lags behind that of higher plants and seaweeds. In this study, we have addressed the question whether microalgal phylogeny is associated with DNA content variation in order to evaluate the evolutionary significance of polyploidy in the model genus Micrasterias. We applied flow-cytometric techniques of DNA quantification to microalgae and mapped the estimated DNA content along the phylogenetic tree. Correlations between DNA content and cell morphometric parameters were also tested using geometric morphometrics. In total, DNA content was successfully determined for 34 strains of the genus Micrasterias. The estimated absolute 2C nuclear DNA amount ranged from 2.1 to 64.7 pg; intraspecific variation being 17.4–30.7 pg in M. truncata and 32.0–64.7 pg in M. rotata. There were significant differences between DNA contents of related species. We found strong correlation between the absolute nuclear DNA content and chromosome numbers and significant positive correlation between the DNA content and both cell size and number of terminal lobes. Moreover, the results showed the importance of cell/life cycle studies for interpretation of DNA content measurements in microalgae.     

Patterns of endopolyploidy and 2C nuclear DNA content (Feulgen) inScilla (Liliaceae)

The Feulgen-DNA content of 3558 nuclei from 21 different tissues and organs ofScilla decidua (Liliaceae) was measured by a scanning cytophotometer interfaced to a computer. The basic nuclear DNA content (2 C value) was 13.62 pg, and 71 per cent of the nuclei were polyploid. The highest DNA values were found in the antipodal cells of the ovule, and the elaiosomes of the seeds (512 C). In addition to polyploidy, the 2 C values exhibited tissue-specific variation which was statistically significant (0.05% level of probability), It is suggested that differential DNA replication and endopolyploidization may be basic factors in the complex mechanism of cell and tissue differentiation.Dedicated to Professor Dr.Lothar Geitler in honour of his 80th birthday.     

DURATION OF THE NUCLEAR CYC LE IN TRADESCANTIA PALUDOSA ROOT TIPS AS MEASURED WITH H3-THYMIDINE

Wimber , Donald E. (Brookhaven National Lab., Upton, N. Y.) Duration of the nuclear cycle in Tradescantia paludosa root tips as measured with H³-thymidine. Amer. Jour. Bot. 47(10): 828–834. Illus. 1960.—The duration of the nuclear cycle and its various subdivisions were measured in Tradescantia root tips by autoradiographic techniques. H³-thymidine was used as a nuclear label and was supplied to the roots for 0.5 hr. After labeling, the roots were allowed to grow in the absence of label for periods up to 38 hr. By determining the percentage of divisions labeled at the various times of fixation, a reconstruction of the nuclear cycle could be made. The average cycle was determined as 20 hr. in duration, DNA synthesis 10.8 hr., presynthetic interphase 4 hr., postsynthetic interphase 2.7 hr., prophase 1.6 hr., metaphase 0.3 hr. and anaphase-telophase 0.6 hr. Approximate standard deviations for the duration of some of the subdivisions were calculated.     

Nuclear DNA Content of 26 Orchid (Orchidaceae) Genera with Emphasis onDendrobium

2C DNA content values for 70 orchid species from 26 genera,including 37Dendrobiumspecies from eight taxonomic sections,were analysed using flow cytometry. The resulting nuclear DNAcontent values for species other thanDendrobiumranged from 1.91pg 2C^-1to 15.19 pg 2C^-1nuclei forCadetia tayloriandVanilla phaeantha,respectively.Dendrobiumnuclear DNA content values ranged from1.53 pg 2C^-1to 4.23 pg 2C^-1nuclei forD. cruentumandD. spectabile,respectively. DNA content measurements varied greatly withinDendrobiumsectionsLatouria and Spatulata. Nuclear DNA content values for the sixspecies analysed within Latouria ranged from 1.88 pg 2C^-1nucleiforD. macrophyllumto 4.23 pg 2C^-1nuclei forD. spectabile. NuclearDNA content values for the 16 species analysed within Spatulataranged from 1.69 pg 2C^-1nuclei forD. discolorto 4.05 pg 2C^-1nucleiforD. samoense. The least variation in DNA content was foundwithin the section Phalaenanthe, with nuclear DNA content valuesof 1.79 pg  2C^-1, 1.86 pg 2C^-1and 1.98 pg 2C^-1forD. bigibbum,D.affineandD. phalaenopsis, respectively.Copyright 1998 Annalsof Botany Company Orchidaceae,Dendrobium, flow cytometry, propidium iodide, nuclear DNA, genome size, 2C values.     

Genome size and environmental factors in the genus Pinus

Nuclear 1C DNA content in haploid megagametophyte tissue of 18 North American and one exotic Pinus species was determined using scanning microspectrophotometry. The nuclear DNA content in root meristematic cells of Zea mays L. ssp. mays, inbred line Va35 (4C = 10.31 pg) was used as a standard. DNA content measured by microspectrophotometry was verified using laser flow cytometry with two additional standards, Hordeum vulgare cv. Sultan (2C = 11.12 pg) and P. eldarica (2C = 47.30 pg). DNA values obtained by both methods were significantly correlated (r = 0.987). The 1C nuclear DNA content ranged from 21 pg to 31 pg. The ratio of DNA content in embryo tissue of P. eldarica to that in megagametophyte tissue was 1.72 by scanning microspectrophotometry and 1.74 by laser flow cytometry. To date, this is the most comprehensive data set available for North American Pinus species. Relationships between genome size of 18 North American Pinus species and climatic factors and indices of growth were investigated using regression and correlation analyses. Positive correlations were observed between nuclear DNA content and growth indices, minimum seed-bearing age, and seed dimensions. Strong negative correlations were observed between nuclear DNA content and two climatic factors, the lowest mean annual and monthly precipitation (excluding January) and the highest mean monthly spring air temperature. These correlations suggest that the large genome size and its variation in Pinus are adapted responses to the habitats of these species.     

Mosaic individuals found in genetically manipulated northern pike (Esox lucius) using flow cytometry

The purpose of this study was to investigate abnormal larvae produced in the experimental induction of gynogenetic northern pike ( Esox lucius L.), using a flow cytometry method. We reported for the first time the mosaic individuals found in genetically manipulated northern pike. Two types of abnormal larvae were obtained after eggs were fertilized with UV-irradiated sperm and then exposed to heat shock treatment. One type had malformations frequently associated with haploid syndrome and were proven to be haploids. The other type had a similar body size and appearance as diploids but with curved body and a swirling swimming pattern. Individuals of the second type were proven to be mosaics containing both 1n (59.5 ± 14.7% mean ± SD) and 2n (29.7 ± 13.3%) cell populations. The ratio of 1n:2n cell populations ranged from 80.6:10.6 to 38.5:44.7. There was no significant difference in the relative nuclear DNA content and cell size between 1n and 2n cell populations of the mosaic individuals as compared to haploid cells and diploid cells, respectively. The nuclear DNA content of the northern pike was estimated to be 2.07 ± 0.06 pg ( n  = 10) given that rainbow trout has a red blood cell nucleus DNA content of 4.66 pg.     

Nuclear DNA content and the control of chloroplast replication in wheat leaves

During development of the first leaf of breadwheat (Triticum aestivum L.) the number of chloroplasts per mesophyll cell increases between three- and four-fold. To establish if chloroplast replication is accompanied by endoreduplication, the nuclear DNA content of the cells was determined by chemical assay of isolated nuclei from mesophyll protoplasts and by microdensitometry of nuclei in mesophyll tissue. The DNA content of the nuclei was constant (27 to 32 pg) at each phase of chloroplast replication. Approximately 93% of the cells had a nuclear DNA content close to the 2C value of 32 pg. It is concluded that chloroplast replication is not dependent on nuclear endoreduplication in seedling leaves of wheat.     

Cytogeography of Cenchrus ciliaris (Poaceae) in Tunisia

Belonging to the genus Cenchrus with 16–22 species, Cenchrus ciliaris L. (syn. Pennisetum ciliare (L.) Link, buffelgrass) is a perennial, common in warmer regions of both hemispheres, growing as a C4 grass in a wide range of habitats. In the present study we determined chromosome number and nuclear DNA content (2C DNA) for 28 natural populations collected from northern to southern Tunisia. Three ploidy levels were found: one tetraploid population (2n?=?4x?=?36), three pentaploid (2n?=?5x?=?45), and 24 hexaploid populations (2n?=?6x?=?54). The hexaploid chromosome number has already been reported for Tunisian populations of C. ciliaris but tetraploid and pentaploid (2n?=?45) are new for this area. The tetraploid population was found in the semi-arid north; pentaploids were mostly on the northern side of the arid region, while the hexaploids were located mainly in the arid southern Tunisian and Saharan region. 2C DNA values, assessed using flow cytometry, correlated with chromosome counts. Nuclear DNA content ranged from 2C?=?3.03 to 4.61 pg, revealing three ploidy levels corresponding to 4x, 5x, 6x, and mean 2C DNA amounts were of 3.03, 3.7 and 4.48 pg, respectively. Each cytotype produced viable pollen. Flow cytometric seed screening neither proved nor disproved apomixis. The most frequent hexaploid populations seem best adapted to arid conditions in southern Tunisia. The monoploid value, 1Cx, was constant. The existence of pentaploid cytotype suggests hybridization ability between tetraploids and hexaploids. It appears that polyploidization is the major evolutionary mechanism in the speciation of C. ciliaris.     

Nuclear DNA characterization of two species ofVicia:Vicia bithynica L. andVicia narbonensis L.

The speciesVicia bithynica andVicia narbonensis, from the same subgeneric section ofVicia faba, show variations in nuclear DNA content Nuclear DNAs, extracted from root tips of the twoVicia species, were characterized by thermal denaturation, analytical ultracentrifugation and reassociation kinetics. The thermal denaturations of DNA, the number of DNA components reassociating with second order kinetics, the proportion of repeated DNA sequences, the frequency of the repeated DNA classes are reported and compared to previous data onVicia faba DNA. Feulgen absorptions at different thresholds of optical density⁺ of interphase nuclei in cytological preparations of the root meristems ofV. bithynica andV. narbonensis are determined and compared withV. faba analogous determinations. The results, confirming that plant genome is highly flexible, are discussed in relation to other data on the interspecific variations of the nuclear DNA content.     

Nuclear DNA content in the polyploid complex Turnera ulmifolia (Turnera L., Passifloraceae)

Turnera ulmifolia constitutes a well-studied polyploid complex with allo- and autopolyploid species ranging from 2 to 8x. Flow cytometry was used to determine nuclear DNA content, and to estimate 2C- and 1Cx-values with the aim of analysing the genome size in Turnera in terms of polyploid speciation. The 2C-value and 1Cx-value were evaluated in 12 species of the T. ulmifolia complex. Nuclear DNA content was estimated by flow cytometry of nuclei stained with propidium iodide. The 2C DNA content ranged from 1.38 to 1.83?pg in diploids, from 2.67 to 3.96?pg in tetraploids, from 2.73 to 4.31?pg in hexaploids, and from 3.53 to 5.90?pg in octoploids. The 1Cx-value ranged from 0.44 to 0.99?pg. The Turnera ulmifolia complex showed an increase in total DNA content in the ploidy level, but not in the expected proportion. The general tendency indicated a decrease in the 1Cx-value with increasing chromosome number, with T. grandidentata 4x being an outstanding exception. The 1Cx-values in the allooctoploids T. aurelii and T. cuneiformis differed by 1.6-fold from each other, probably as a result of different evolutionary histories following divergence from the last common ancestor.     

VARIATION OF NUCLEAR DNA CONTENT IN HELIANTHUS ANNUUS (ASTERACEAE)

Nuclear 2C DNA content was determined by laser flow cytometry for 13 diploid (2n = 34) lines (cultivated varieties and inbred lines) of Helianthus annuus. Mean DNA amount of second leaf nuclei varied from 6.01 to 7.95 pg (32%) among lines. Mean DNA content varied up to 19% within lines. Variability in mean DNA content exceeding 27% and 48% was detected among leaves from different nodes of plants of the open-pollinated variety, Californicus, and the inbred line, RHA 299, respectively. The root tip and shoot tip nuclei of H. annuus have been reported to consist of a mixture of aneuploid (17 to 33 chromosomes) and diploid (34 chromosomes) cells, a condition called aneusomaty. Chromosome counts of root tips and an analysis of the distribution of DNA content of large numbers of nuclei from leaves indicate that aneusomaty either does not occur, or is not common, among the lines investigated. The intraspecific, intraline, and intraplant variation in DNA content in H. annuus support the concept that a sizable portion of a plant genome is unstable and subject to rapid changes in DNA amount.     

Flow cytometric measurement of nuclear DNA content inCapsicum (Solanaceae)

Nuclei were isolated from leaf tissue of differentCapsicum species and the relative fluorescence intensity was measured by flow cytometry after propidium iodide staining.Pisum sativum nuclei with known nuclear genome size (9.07 pg) were used as internal standard to determine nuclear DNA content of the samples in absolute units. The 2C DNA contents ranged between 7.65 pg inC. annuum and 9.72 pg inC. pubescens, and the general mean of the genus was 8.42 pg. These values correspond, respectively, to 1C genome size of 3.691 (C. annuum), 4.690 (C. pubescens) and 4.063 (general mean) Mbp. In general, white-flowered species proved to have less DNA, with the exception ofC. praetermissum, which displayed a 2C DNA content of 9.23 pg. It was possible to divide the studied species into three main groups according to their DNA content, and demonstrate differences in DNA content within two of the three species complexes established on the basis of morphological traits.     

Substantial Genome Size Variation in Taraxacum stenocephalum (Asteraceae, Lactuceae)

There are only a few exceptions to the rule that polyploidy in Taraxacum is associated with agamospermy. One of them is the sexual, tetraploid species Taraxacum stenocephalum. Incidentally, remarkable variation in karyology was found in this species. The present study aims to confirm this variation by an extensive screen of nuclear DNA content. Individuals from two large populations in the Lesser and Greater Caucasus, Georgia were analyzed using flow cytometry to ascertain intraspecific nuclear DNA content variation. Across the whole data set comprising all 159 individuals, a 1.223-fold difference was detected based on propidium iodide (PI) analyses. To verify this finding, we compared flow-cytometric data obtained using DAPI (4′,6-diamidino-2-phenylindole) and PI staining using a representative subset of individuals. This comparison revealed a 1.194-fold difference in DNA content for DAPI and a 1.219-fold difference for PI. Mean nuclear genome size in absolute terms (2C value ± s.d.) was estimated at 4.38?±?0.21 pg, ranging from 4.01 pg to 4.89 pg, despite the invariable chromosome counts of 2n?=?32. A regression analysis comparing the datasets for DAPI and PI staining found a strong correlation between data obtained by the DAPI and PI dyes (R?=?0.976; P?=?0.0001). Simultaneous high-resolution flow-cytometric analyses also proved the accuracy of our findings. We discuss possible sources of these large differences in DNA content within Taraxacum stenocephalum. Further research is needed to identify the source of this remarkable variation.     

Intraplant Nuclear DNA Content Variation in Diploid Nuclei of Maize (Zea mays L.)

Diploid nuclei from stem, mesocotyl, nodal root and root tiptissue of two maize hybrids were examined with respect to theirDNA content. The nuclei were isolated and stained with DAPIand passed through a flow cytometer-cell sorter. The titrationcurve for each tissue was determined. Significant variationwas observed among nuclei of different tissue types. Stem androot tips had the highest diploid nuclear DNA amounts while2-week-old mesocotyl had the lowest diploid nuclear DNA amount.These results provide evidence that during plant developmentand differentiation, the amount of DNA within a diploid nucleuschanges through loss of specific DNA sequences. This study alsodemonstrates the sensitivity of flow cytometry in detectingsmall intraplant variation in nuclear DNA. Key words: Flow cytometry, fluorochrome DAPI, DNA content, tissue differentiation, plant development     

Nuclear DNA content in some species of Lessingianthus (Vernonieae, Asteraceae) by flow cytometry

The nuclear DNA content was determined for the first time in 25 species of the South American genus Lessingianthus H.Rob. (Vernonieae, Asteraceae) by flow cytometry. This analysis constitutes the first estimation of the genome size for the Vernonieae tribe. The 2C- and 1Cx-values were calculated in all the species. The 2C-value ranged from 2.04 to 14.34 pg. The 1Cx-value ranged from 0.995 to 1.43 pg. The general tendency indicated a decrease in the 1Cx-value with increasing ploidy level, with some exceptions, in some species the 1Cx-value increased with the ploidy increase. The measuring of DNA content allowed reporting a new cytotype for L. polyphyllus (Sch.Bip.) H.Rob.     

A comparison of internal standards for plant cytophotometry

Plant cell nuclei were compared with chicken erythrocyte nuclei for use as internal standards for microspectrophotometry. The amount of DNA per nucleus and the coefficient of variation for measurement of individual nuclei were determined for cells from dormant embryos of Pinus taeda and Pinus coulteri, from onion root tips and from chicken erythrocytes. The chicken erythrocytes had the least variability and thus were best suited for use as a standard. Onion root tips were least suitable, with a coefficient of variation 2 1/2 times that of erythrocytes. Although onion root tips have been used as an internal standard in other studies, their mitotic activity, in contrast with the nonreplication of DNA of mature erythrocytes, is reflected in a broad distribution of nuclei with values in the 2C-4C range. Coulter pine mature embryos were at the 3C level, whether dry or hydrated, while loblolly pine embryos were in the 2C state. This confirms previous reports. The coefficient of variability for the pine embryo cells was 1 1/2 times that of erythrocytes for nonhydrated seeds and twice the erythrocyte value for hydrated seeds. The larger 2C values for pine (26 pg for P. taeda and 17 pg for P. coulteri) are closer to values expected for many plant species than the 3 pg level of the chicken erythrocytes. Dormant P. taeda embryo cells (2C) are suggested as an alternative where the experimental material has large DNA values and/or chicken erythrocytes are difficult to procure. Large sample size is recommended for the plant materials if they are to be used as internal standards in Feulgen cytophotometry.     

Amounts of nuclear DNA in marine halophytes

The amount of nuclear DNA, expressed as the C-value, was estimated for 13 marine halophytic plant species from six families. Plant material was collected in the nature reserve of the Strunjan saltpan in the Northern Adriatic and comprised all halophytic species inside the investigated area. Reproductive region of the shoot or root tips of halophytes were dissected, nuclei were Feulgen stained and 2C-values were measured by DNA image cytometry as follows: Crithmum maritimum (4.38 pg DNA), Artemisia caerulescens (6.43 pg), Aster tripolium (21.43 pg), Inula crithmoides (3.63 pg), Atriplex portulacoides (1.83 pg), A. prostrata (1.51 pg), Salicornia europaea (2.75 pg), Salsola soda (2.62 pg), Sarcocornia fruticosa (5.91 pg), Suaeda maritima (2.11 pg), Limonium angustifolium (5.06 pg), Puccinellia palustris (8.15 pg) and Ruppia cirrhosa (4.65 pg). With the exception of the C-value estimate for A. caerulescens, which has been listed in the Plant DNA C-values Database, the C-values represent the first estimates for all the examined species. In addition, the C-value for R. cirrhosa is also the first report for the family Ruppiaceae. The investigated halophytes had a smaller genome size compared to other known C-values for species within a particular family and also when compared to the mean values of dicots and monocots. The study also showed that halophylic annuals have a smaller genome size (2.49 pg) than perennial ones (7.45 pg DNA).     

A Flow Cytometric Analysis of the Nuclear 2C DNA Content in 17 Phaseolus Species (53 Genotypes)

The genus Phaseolus is characterized by a highly stable karyotype of 2n = 22. Despite this constancy, the size of the chromosomes varies, and crossing of species is possible only in a few cases. We determined the 2C nuclear DNA content of a number of Phaseolus species, cultivars and genotypes by flow cytometry, in order to realize the interspecific and intraspecific variation of the 2C value. The data range from 1.03 pg to 2.18 pg without any clear correlation to systematic relationships. The mean DNA values of wild and cultivated forms, as well as those of Andean and Mesoamerican genotypes, do not differ significantly. The variation is interpreted in terms of some nucleotypic adaptations. The data may be useful for molecular biological analyses, as well as for biotechnological and classical breeding programmes.