DNA amounts of roses (<Emphasis Type="Italic">Rosa</Emphasis> L.) and their use in attributing ploidy levels

The aims of the investigation were to characterise variability among the DNA amounts of roses and assess the predictability of ploidy levels from DNA amounts. Chromosome numbers in the genus Rosa range from 2n = 2x = 14 to 2n = 8 x = 56 and aneuploidy is rare. Published 2C DNA amounts range from 0.78 pg in R. xanthina Lindl. and R. sericea Lindl. (2n = 2x = 14) to 2.91 pg in R. canina L. (2n = 5x = 35). In this investigation, DNA amounts were estimated by flow cytometry of leaf nuclei stained with propidium iodide, using Petroselinum crispum (2C DNA amount = 4.46 pg) as the internal calibration standard. Ploidy levels based on DNA amounts (DNA ploidy) were assigned by comparing their DNA amounts with published DNA amounts and identifying peaks and intervening discontinuities in frequency distributions of DNA amounts. 2C DNA amounts ranged from 0.83 pg in R. ecae (2x = 2x = 14) to 3.99 pg in R. acicularis (2n = 8 x = 56). Differences in the 1Cx-values (2C DNA amount/ploidy values) were found among the taxonomic sections of Rosa. Ploidy levels could be confidently assigned to most species and cultivars, but the ploidy of some specimens in the section Caninae was uncertain for reasons attributed to genomic diversity and aneuploidy. Cytochimerism was detected in three cultivars of R. x alba. DNA ploidy was determined in 384 specimens representing 74 species and 5 horticultural classes.     

Variation in chromosomal DNA associated with the evolution of Arachis species.

The 2C nuclear DNA amounts were determined for 99 accessions, representing 23 Arachis species from 8 of 9 taxonomic sections, and two synthetic amphidiploids. Mean 2C DNA amounts varied by 15.20%, ranging from 10.26 to 11.82 pg, between accessions of Arachis hypogaea (2n = 4x = 40). Nuclear DNA content variation (5.33-5.91 pg) was also detected among Arachis duranensis (2n = 2x = 20) accessions. The intraspecific variation in the two species may have resulted from indirect selection for favourable genome sizes in particular environmental conditions. The accessions belonging to A. hypogaea ssp. hypogaea (mean value 11.27 pg) with longer life cycle had significantly larger mean DNA content than the accessions of A. hypogaea ssp. fastigiata (mean value 10.97 pg). For 20 diploid (2n = 2x = 20) species of the genus, 2C nuclear DNA amounts ranged from approximately 3 to 7 pg. The diploid perennial species of section Arachis have about 12% more DNA than the annual species. Comparisons of DNA amounts show that evolutionary rating is not a reliable guide to DNA amounts in generic sections of the genus; lower DNA values with evolutionary advancement were found in sections Heteranthae and Triseminatae, but the same was not true for sections Arachis and Caulorrhizae. Similarly, there is evidence of significant differences in DNA content between 4 ancient sections (Procumbentes, Erectoides, Rhizomatosae, and Extranervosae) of the genus. The occurrence of genome size plasticity in both A. duranensis and A. hypogaea provides evidence that A. duranensis could be one of the diploid progenitors of A. hypogaea. The DNA content in the two synthetic amphidiploids corresponded to the sum value estimated for parental species. Key words : Arachis species, genome size, Arachis hypogaea, Arachis duranensis, intraspecific variation.     

Intra- and interspecific variation in DNA content in Cistus (Cistaceae)

Flow cytometry, using propidium iodide and 4',6-diamidano-2-phenylindole staining, was used to estimate the nuclear DNA content (2C) and the proportion of A-T base pairs in 16 species of the Mediterranean genus Cistus. Genome sizes were shown to be constant within species, since no significant intraspecific variation in 2C DNA content was detected. At the genus level, up to about 1.5-fold differences in absolute DNA amounts were observed, ranging from 3.92 pg in C. crispus to 5.88 pg in C. monspeliensis. The (AT) : (GC) ratio was close to 1, and was similar for all species examined, ranging from 47.87% A-T content in C clusii, to 50.67% in C. populifolius. Pink-flowered species (subgenus Cistus) had lower DNA amounts than white-flowered species (subgenera Leucocistus and Halimioides). However, the distribution of DNA amounts in Cistus appeared to be continuous and did not permit a clear separation of infra-generic ranks in the genus.     

Chromosome numbers and DNA?C values in the genus Lippia (Verbenaceae)

The genus Lippia comprises herbs, shrubs, and small trees, including many species with medicinal properties. The species are distributed throughout South and Central America and Tropical Africa, but the majority of them occur in Brazil, Paraguay, and Argentina. The DNA?C value of 28 Brazilian species has been estimated by flow cytometry. Estimated DNA?C values ranged from 0.825?pg (L. corymbosa) to 2.150?pg (L.?brasiliensis). In addition, new chromosome numbers of 12 species have also been described, and meiotic cells with 12, 13, and 14 chromosome pairs were observed. A straightforward correlation between chromosome number and DNA?C value was not observed, probably due to two outlier species of Lippia that have been transferred from the genus Lantana. In general, the data confirm previous reports regarding the variation within the taxonomic sections and also suggest a new revision in section Zapania. Aspects of karyotypic evolution of the genus are also discussed.     

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.     

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.     

Genome evolution in the genus Sorghum (Poaceae)

BACKGROUND AND AIMS: The roles of variation in DNA content in plant evolution and adaptation remain a major biological enigma. Chromosome number and 2C DNA content were determined for 21 of the 25 species of the genus Sorghum and analysed from a phylogenetic perspective. METHODS: DNA content was determined by flow cytometry. A Sorghum phylogeny was constructed based on combined nuclear ITS and chloroplast ndhF DNA sequences. KEY RESULTS: Chromosome counts (2n = 10, 20, 30, 40) were, with few exceptions, concordant with published numbers. New chromosome numbers were obtained for S. amplum (2n = 30) and S. leiocladum (2n = 10). 2C DNA content varies 8.1-fold (1.27-10.30 pg) among the 21 Sorghum species. 2C DNA content varies 3.6-fold from 1.27 pg to 4.60 pg among the 2n = 10 species and 5.8-fold (1.52-8.79 pg) among the 2n = 20 species. The x = 5 genome size varies over an 8.8-fold range from 0.26 pg to 2.30 pg. The mean 2C DNA content of perennial species (6.20 pg) is significantly greater than the mean (2.92 pg) of the annuals. Among the 21 species studied, the mean x = 5 genome size of annuals (1.15 pg) and of perennials (1.29 pg) is not significantly different. Statistical analysis of Australian species showed: (a) mean 2C DNA content of annual (2.89 pg) and perennial (7.73 pg) species is significantly different; (b) mean x = 5 genome size of perennials (1.66 pg) is significantly greater than that of the annuals (1.09 pg); (c) the mean maximum latitude at which perennial species grow (-25.4 degrees) is significantly greater than the mean maximum latitude (-17.6) at which annual species grow. CONCLUSIONS: The DNA sequence phylogeny splits Sorghum into two lineages, one comprising the 2n = 10 species with large genomes and their polyploid relatives, and the other with the 2n = 20, 40 species with relatively small genomes. An apparent phylogenetic reduction in genome size has occurred in the 2n = 10 lineage. Genome size evolution in the genus Sorghum apparently did not involve a 'one way ticket to genomic obesity' as has been proposed for the grasses.     

Determination of ploidy level and nuclear DNA content in blueberry by flow cytometry

The technique of DNA flow cytometry was used to study variation in DNA content among different ploidy levels, as well as among diploid species, of Vaccinium section Cyanococcus. In a sample of plants of varying ploidy level, the relative fluorescence intensity (RFI) of nuclei stained with propidium iodide was a function of the number of chromosome sets (x), as represented by the linear equation RFI=3.7x-2.3 (r²=95%). The data indicated that DNA flow cytometry could be useful for the determination of ploidy level at the seedling stage in blueberry. They also suggest that conventional polyploid evolution has occurred in this section of the genus Vaccinium with an increase in nuclear DNA content concurrent with the increase in chromosome number. The nuclear DNA content of diploid species of Vaccinium section Cyanococcus was estimated from the relationship of the observed RFI to an internal known DNA standard (trout red blood cells). A nested analysis of variance indicated significant variation among species, as well as among populations within species, in nuclear DNA content, although this variation was small compared to the variation among ploidy levels. The variation in nuclear DNA content corresponded to the phylogenetic relationships among species determined from previous studies.     

Nuclear DNA Amounts in Mosses (Musci)

A comparative investigation into nuclear DNA amounts using flowcytometry and video-based Feulgen densitometry was carried outin 289 accessions of 138 different moss taxa (Bryatae), originatingfrom Austria, Switzerland, Spain, Mexico and the USA. Samplingincluded species from all major moss clades (except Sphagnum).Flow cytometry data agreed highly with the Feulgen data, whichonce again demonstrates the high reliability of both methodsfor DNA amount determination. For the first time, extensivedata on absolute C-values of mosses are available. Haploid DNAcontents (1C) ranged from 0.174 to 2.16 pg, which representsonly about a 12-fold variation. This low C-value variation isremarkable when compared to angiosperms which vary approx. 1000-fold.C-values are usually relatively constant within genera and evenfamilies; however, genera with varying C-values also exist.From the low frequency observed, secondary polyploidy playsonly a minor role in mosses. Possible reasons for the low C-valuevariation are discussed. Copyright 2000 Annals of Botany Company Mosses, Bryatae, genome size, nuclear DNA amounts, C-value variation, Feulgen, flow cytometry, densitometry, image analysis     

Flow cytometric analysis of genome size variation in some Passiflora species

Nuclear genome size variation was studied in eight taxa of Passiflora. Nuclear DNA content was estimated by flow cytometry of nuclei stained by propidium iodide. 2C DNA content ranged from 3.16-5.36 pg for diploids and 1.83 pg for tetraploid. Differences in nuclear genome size were observed among Passiflora species (pg): P. suberosa 1.83, P. edulis f. edulis 3.16, P. edulis f. flavicarpa (Brazil) 3.19, P. edulis f. flavicarpa (Mexico) 3.21, P. mucronata 3.40, Passiflora edmundoi 3.43, P. laurifolia 3.88, P. giberti 3.92, P. quadrangularis 5.36, the largest value being up to 192% greater than the smallest. The means of 2C DNA content were compared by the Tukey test, and the differences in genome size permitted the recognition of five taxa groups. The result was the same for the means 2C genome size (Mbp) values. The genetic parameters were studied with their respective estimators, phenotypic variance (sigma2F), genotypic variability (PhiG), and the genotypic determination index (H2). The genotypic determination index presented high magnitude estimates (greater than 99%) emphasizing the reliability of the results and demonstrating the efficiency of determining the DNA content in the species using only one leaf per plant. Passiflora species show great phenotypic variability and have different geographic distribution that might implicate in genetic diversity.     

Genome size variation in Central European species of Cirsium (Compositae) and their natural hybrids

BACKGROUND AND AIMS: Nuclear DNA amounts of 12 diploid and one tetraploid taxa and 12 natural interspecific hybrids of Cirsium from 102 populations in the Czech Republic, Austria, Slovakia and Hungary were estimated. METHODS: DAPI and PI flow cytometry were used. KEY RESULTS: 2C-values of diploid (2n = 34) species varied from 2.14 pg in C. heterophyllum to 3.60 pg in C. eriophorum (1.68-fold difference); the 2C value for the tetraploid C. vulgare was estimated at 5.54 pg. The DNA contents of hybrids were located between the values of their putative parents, although usually closer to the species with the smaller genome. Biennial species of Cirsium possessed larger nuclear DNA amounts than their perennial relatives. Genome size was negatively correlated with Ellenberg's indicator values for continentality and moisture and with eastern limits of distribution. A negative relationship was also detected between the genome size and the tendency to form natural interspecific hybrids. On the contrary, C-values positively corresponded with the spinyness (degree of spinosity). AT frequency ranged from 48.38 % in C. eriophorum to 51.75 % in C. arvense. Significant intraspecific DNA content variation in DAPI sessions was detected in C. acaule (probably due to the presence of B-chromosomes), and in tetraploid C. vulgare. Only the diploid level was confirmed for the Pannonian C. brachycephalum, generally considered to be tetraploid. In addition, triploidy was discovered for the first time in C. rivulare. CONCLUSIONS: Considerable differences in nuclear DNA content exist among Central European species of Cirsium on the diploid level. Perennial soft spiny Cirsium species of wet habitats and continental distributions generally have smaller genomes. The hybrids of diploid species remain diploid, and their DNA content is smaller than the mean of the parents. Species with smaller genomes produce interspecific hybrids more frequently.     

Nuclear DNA content of Vitis vinifera cultivars and ploidy level analyses of somatic embryo-derived plants obtained from anther culture

Flow cytometry was employed to determine the ploidy level of Vitis vinifera L. somatic embryo-derived plants obtained from anther culture. Only one among the 41 analysed plants (2.4%) presented somaclonal variation (tetraploidy); the other plants were diploid. No significant differences (P≤0.05) were detected between diploid and parental field plants. No haploid or aneuploid plants were observed. The nuclear DNA content of nine V. vinifera cultivars was also estimated using flow cytometry. A non-significant variation was found among the cultivars, with DNA content ranging from 1.17 pg/2C (cv. ‘Tinta Barroca’ and ‘Viosinho’) to 1.26 pg/2C (cv. ‘Cabernet Sauvignon’). These results and previous studies on other Vitis species suggest that Vitis genome is stable with regard to nuclear DNA content.     

Genome size variation in Arachis hypogaea and A. monticola re-evaluated.

Genome size variation within species is a frequently reported, but still a controversial problem. In the present study, we re-evaluated recently published Feulgen densitometric data on genome size and its infraspecific variation in Arachis hypogaea, and also conducted measurements in one accession of its wild relative A. monticola. The methods applied were propidium iodide flow cytometry and Feulgen densitometry using Pisum sativum as an internal standard. The 2C DNA contents previously published cannot be confirmed, but values obtained in this study are about half as large. Additionally, we could not reproduce the previously reported 1.15-fold variation within A. hypogaea; our data indicate genome size stability between respective accessions of this species. Based on 8.84 pg (2C) for Pisum sativum the DNA amounts (2C) were: 5.914 pg in A. hypogaea, and 5.979 pg in A. monticola.     

Nuclear DNA content variation and species relationships in the genus Lupinus (Fabaceae)

The 2C nuclear DNA content has been estimated by flow cytometry in 18 species and botanical forms of the genus Lupinus (family Fabaceae), using propidium iodide as a fluorescent dye. They represented distinct infrageneric taxonomic groups and differed in somatic chromosome numbers. Estimated 2C DNA values ranged from 0.97 pg in L. princei to 2.44 pg in L. luteus, which gives a more than 2.5-fold variation. Statistical analysis of the data obtained resulted in a grouping that supports the generally accepted taxonomic classification of the Old World lupins. The rough-seeded L. princei turned out to be an interesting exception, getting closer to smooth-seeded species. Results of DNA content analyses are discussed with regards to the phylogenetic relationships among the Old World lupins and some aspects of the evolution of the genus.     

2C DNA variation and relationships among New World species of the genus Lupinus (Fabaceae)

The 2C DNA values in 38 species and accessions of the genus Lupinus (Fabaceae) from the New World have been analysed using flow cytometry. They are representatives of North and South American species (the Atlantic and the Andean regions). Estimated 2C DNA values ranged from 1.08 pg in L. pusillus to 2.68 pg in L. albicaulis (both from North America), that is a variation of more than 2.5-fold. The variation for North American lupins was much higher than that for South American ones. Statistical analysis of the data resulted in a grouping that showed for North American lupins some correlation with the length of life cycle. Discussion concerns some aspects of the evolution of the genus.     

Cytophotometric estimation of in situ DNA content in several species of Araceae.

A wide variation in the in situ 4C DNA content, ranging from 15.02 pg to 54.09 pg was found in thirteen genera of the family Araceae. The obligate perennial species showed greater 4C DNA values compared with the facultative perennials and annuals. A remarkable heterogeneity in 4C nuclear DNA amounts was noted among obligate perennials. Intraspecific constancy in the amount of 4C DNA was recorded. The nuclear DNA content correlated positively with chromosome size, duration of the mitotic cycle, and annual to perennial growth forms. Despite an extensive variation in DNA content among the aroids investigated, each species was distinctly characterized by its specific nuclear DNA value, indicating its usefulness in taxonomic characterization and comparison of different aroids.     

Nuclear DNA Content Diversity in Chinese Soybean Introductions

Intraspecific nuclear DNA content has been documented in variousangiosperm species. The purpose of this study was to determinethe nuclear DNA content variation in soybean (Glycine max(L.)Merr.). Several studies have suggested that DNA content variationexists in soybean. Ninety soybean lines representing diversegeographic locations in China were analysed by flow cytometry.Nuclei were isolated and stained with either the fluorochromeDAPI or PI. After analysis, it was determined that PI stainednuclei more accurately assessed the total DNA content in soybean.A 12% variation in nuclear DNA content was observed among the90 lines. The amount of nuclear DNA in the lines was withinthe DNA range of United States cultivars previously examined.Nuclear DNA content variation in soybean is much less than thevariation reported in maize. These results could be due to thelack of polymorphism in large chromosomal elements containinglarge blocks of repetitive DNA. In addition, unlike maize, theamounts of DNA variation did not decrease as a result of moreintensive breeding in United States cultivars. Intraspecificnuclear DNA content variability is very different between thesetwo major agronomic species. Glycine max(L.) Merr.; germplasm; genome size     

Genome sizes of all 19 Araucaria species are correlated with their geographical distribution

Nuclear genome size was determined to investigate the relationships between all 19 species of Araucaria de Jussieu. Species from the two other genera of Araucariaceae, Wollemia and Agathis, were also studied. The genome size of 17 out of the 19 species of Araucaria are reported here for the first time. All Araucariaceae have the same chromosome number 2n?=?26. However, the nuclear DNA contents (2C value) for Araucaria range from 31.3 to 45.4?pg. There is a good correlation between genome size and division in sections, and geographical distribution. The two species from South America have 44.7 and 45.4?pg, the two species from Australia have 35.7 and 44.4?pg and the two species from New Guinea 34.7 and 40.4?pg. All 13 species of New Caledonia and the one from Norfolk Island have a similar, if not identical, amount of nuclear DNA of, on average, 31.9?pg. This corroborates the identical DNA rbcL sequences found for the New Caledonian araucarias. It suggests that the species from New Caledonia diversified more recently and it questions their status as separate species. Compared with this 31.9?pg a strong increase seems to have occurred in the genome size of the “mainland” araucarias. Genome sizes are evaluated and compared with available taxonomic treatments and extant geographic spreading. The nuclear DNA contents found within the sections are close, making it possible to assign an unknown plant to a section. A difference of 1?pg, which amounts to a difference of 978?Mbp, far exceeds a single character. Nuclear DNA content, as measured by flow cytometry, may conveniently be used to produce systematic data. It is applicable even with young plants or seeds for monitoring the trade in endangered species.     

Genome size and genome evolution in diploid Triticeae species.

One of the intriguing issues concerning the dynamics of plant genomes is the occurrence of intraspecific variation in nuclear DNA amount. The aim of this work was to assess the ranges of intraspecific, interspecific, and intergeneric variation in nuclear DNA content of diploid species of the tribe Triticeae (Poaceae) and to examine the relation between life form or habitat and genome size. Altogether, 438 plants representing 272 lines that belong to 22 species were analyzed. Nuclear DNA content was estimated by flow cytometry. Very small intraspecific variation in DNA amount was found between lines of Triticeae diploid species collected from different habitats or between different morphs. In contrast to the constancy in nuclear DNA amount at the intraspecific level, there are significant differences in genome size between the various diploid species. Within the genus Aegilops, the 1C DNA amount ranged from 4.84 pg in A. caudata to 7.52 pg in A. sharonensis; among genera, the 1C DNA amount ranged from 4.18 pg in Heteranthelium piliferum to 9.45 pg in Secale montanum. No evidence was found for a smaller genome size in annual, self-pollinating species relative to perennial, cross-pollinating ones. Diploids that grow in the southern part of the group's distribution have larger genomes than those growing in other parts of the distribution. The contrast between the low variation at the intraspecific level and the high variation at the interspecific one suggests that changes in genome size originated in close temporal proximity to the speciation event, i.e., before, during, or immediately after it. The possible effects of sudden changes in genome size on speciation processes are discussed.     

COMPARISON OF PLANT DNA CONTENTS DETERMINED BY FEULGEN MICROSPECTROPHOTOMETRY AND LASER FLOW CYTOMETRY

An improved procedure is reported for determining DNA amounts of plant nuclei. Nuclei stained with propidium iodide, isolated from chopped plant leaves, were passed through an Ortho Cytofluorograph with a Lexel model 95 argon laser (514 nm) and the fluorescence measured, integrated, and recorded using an Ortho 2140 Data Acquisition computer. All nuclear samples were mixed with nuclei of Sultan barley (2C DNA content = 11.12 pg [picogram]) as an internal standard. DNA contents of ten plant species, ranging from 2C = 1.7 pg to 36.1 pg measured by flow cytometry, correlated strongly (r = 0.99, slope = + 1.00) with DNA contents determined from Feulgen-stained nuclei of the same species using microspectrophotometry. The flow cytometric procedures were sufficiently sensitive to detect differences in DNA content between inbred lines of corn and their F1 hybrids. Our results obtained with improved procedures, specifically using propidium iodide as a fluorochrome and plant nuclei instead of chicken erythrocytes as an internal standard, demonstrate that laser flow cytometry can be a precise, rapid, and reliable method for determining nuclear DNA content of plants.