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     

Genome Size in Chinese Soybean Accessions--Stable or Variable?

Variation in genome size up to 1.12-fold has been recently reportedin 90 Chinese accessions ofGlycine max (soybean). Generallysuch results have to be viewed with reservation if rigorousinternal standardization and control tests for the repeatabilityof the results have not been done. Therefore, we reinvestigatedten accessions (five allegedly ranking high and five low) forgenome size using propidium iodide flow cytometry and Feulgendensitometry. Using flow cytometry, the maximum difference betweenaccessions was 1.018-fold (non-significant); the differencebetween the means of the high-ranking and low-ranking groupwas 1.002-fold (non-significant). With Feulgen densitometry,the maximum difference between accessions was 1.034-fold (non-significant).The present data suggest genome size constancy, in terms ofcytometric evidence, for the Chinese soybean accessions in question.Likewise, no reasonable evidence was obtained for a differencebetween Chinese and American soybeans. Copyright 1999 Annalsof Botany Company Glycine max, Chinese soybeans, U.S. soybeans, genome size variation, propidium iodide flow cytometry, Feulgen densitometry.     

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 Amounts in Pteridophytes

DNA amounts (C-value and genome size) are much-used biodiversitycharacters. A workshop held at Kew in 1997 identified majorgaps in our knowledge of plant DNA amounts, recommending targetsfor new work to fill them. Murray reviewed non-angiosperm plantsnoting that representation of pteridophyte species (approx.0.42%) was poor, while locating C-value data for them was verydifficult. The workshop confirmed the need to make data forother groups besides angiosperms accessible for reference purposes.This paper pools DNA C-values for 48 pteridophyte species fromeight original sources into one reference source, and fulfilsa key workshop recommendation for this group. Comparing thesedata shows that nuclear 1C-values in pteridophytes vary approx.1000-fold, from 0.055 pg in Selaginella species to about 55pg in Ophioglossum petiolatum. Genome size estimates for 25pteridophytes vary approx. 200-fold from 0.055 to 10.7 pg, andthe mean genome sizes in diploids and polyploids (5.15 and 4.59pg, respectively) are not significantly different. Wider comparisonsshow that ranges of genome sizes in the major groups of landplants are very different. Those in bryophytes and pteridophytesare narrow compared with those in gymnosperms and angiosperms.The data indicate that the origin of land plants possibly involveda first major increase in genome size in the evolution of vascularplants, while a second such increase occurred later in gymnosperms.C-values for pteridophytes remain very few, but conversely opportunitiesfor new work on them are many. Copyright 2001 Annals of BotanyCompany Pteridophyte DNA amounts, DNA C-values, nuclear genome sizes     

Relationships among Genome Size, Environmental Conditions and Geographical Distribution in Natural Populations of NW Patagonian Species of Berberis L. (Berberidaceae)

Variation in genome size of 24 populations belonging to 11 NWPatagonian species of Berberis was analysed as a function ofthe environment and geographical location. The variation showedthree levels of discontinuity, two of which corresponded todiploid species (2 n = 28) while the third corresponded to polyploidspecies (2 n = 56). Diploids with DNA content ranging from 1.463pg to 1.857 pg includedBerberis cabrerae , B. chillanensis,B. montana, B. serrato-dentata and B. bidentata. Diploids withDNA content ranging from 2.875 pg to 3.806 pg included B. linearifolia,B. darwinii, B. parodii and B. empetrifolia. The genome sizeof the polyploid species B. buxifolia and B. heterophylla rangedfrom 5.809 pg to 6.844 pg. Principal component analysis (PCA)was applied to represent the variability of environmental conditions.The eigenvectors of the principal component axes showed thatPC1 discriminates the populations according to rainfall, typesof vegetation and geomorphology; altitude and latitude, on theother hand, contribute to PC2 and PC3, respectively. From theseresults it is concluded: (1) that diploids with lower DNA contentgrow in high-elevation sites having greater rainfall but lowerwater availability; (2) diploids with higher DNA content areassociated with half-elevation forests where the vegetativeperiod is longer, the water availability is greater and thetemperatures are higher; and (3) the distribution pattern ofpolyploids is considerably wider than that of diploids, whichare geographically and ecologically restricted to forest areas.These results suggest that the C-value plays an important rolein the ability of the species to adapt to different growingconditions. Copyright 2000 Annals of Botany Company Berberis L., barberry, calafate, michay, genome size, DNA content, environmental correlation, Patagonia     

Genome size variation inCajanus cajan (Fabaceae): A reconsideration

A recent investigation of genome size in certain samples of the pigeonpea,Cajanus cajan, indicates values from 1.55 pg to 1.99 pg (1C level), which is 1.29-fold variation between accessions. In the present analysis those of these accessions which had particularly high or low DNA contents in that study were subjected to a reanalysis using propidium iodide and DAPI flow cytometry and Feulgen densitometry. Only minor differences in genome size, not more than 1.047-fold, were found with flow cytometry, and no significant differences were obtained with Feulgen densitometry. The previously reported genome size cannot be confirmed. It is about half as large and was determined in the present study as 0.825 pg (1C, propidium iodide flow cytometry,Glycine max as standard) and 0.853 pg (1C, Feulgen densitometry,Allium cepa andPisum sativum as standards), respectively.     

Cytophotometric determination of genome size in two species of Cyclops of Lake Baikal (Crustacea: Copepoda, Cyclopoida) in ontogenetic development

The genome size of Cyclops in cells at early stages of cleavage (up to the fifth division) and in somatic cells was estimated by static digital Feulgen cytophotometry in order to study quantitative changes in DNA content during chromatin diminution. Described here cytophotometric method was approbated on five different digital-imaging systems in blood cells of four vertebrate species. In all cases, we observed a direct correlation between the data obtained with known from the literature on genome size and high reproducibility, which will allow these systems to be used in future work. We also optimized the conditions for DNA hydrolysis of both blood smears and for two species of Cyclops from the Moscow population as 30 min in 5 N HCl at 24°C. Here, we first revealed chromatin diminution in two endemic Baikal species of Cyclopoida: Acanthocyclops incolotaenia and Diacyclops galbinus. We estimated the extent of chromatin diminution in Diacyclops galbinus as 95.5–96.2%. Cytometric analysis of the third species, Mesocyclops leuckarti, did not reveal obvious chromatin diminution.     

Karyotype and DNA-content evolution in ten species of Crepis (Asteraceae) distributed in Bulgaria

Ten Crepis species from Bulgaria—five perennials (C. viscidula, C. paludosa, C. coryzaefolia, C. bilhynica, C. schochtii) four annuals (C. pulchra, C. sancta, C. setosa, C. zacintha) and one biennial (C. biennis)—were analysed karyologically using haematoxyh staining, Feulgen cytophotometry (scanning densitometry and video-based image analysis), and DNA flow cytometry with propidium iodide. All taxa but the biennial are diploids with descending basic chromosome numbers, x=6, 5, 4, 3. Significant positive correlations were found between nuclear DNA content and karyotype length and nuclear DNA content and karyotypic asymmetry. Together with the results of previous authors our data suggest that evolutionary advancement could be correlated with more symmetrical karyotypes. Negative significant correlations were established between presumably advanced growth habit (from rhizomatous and tap-rooted perennials towards highly specialized annuals) and chromosome number and karyotype length. Nuclear DNA 1C-values on average were higher in perennials than in annuals, but the ranges were overlapping and the differences not significant. Crepis biennis (2n=c. 40, presumably 10x) had the highest DNA quantity, but calculated at its x-level ranked relatively low in the species sample.     

Variation of Nuclear DNA Content in the Biflorus Species of Lonchocarpus (Leguminosae)

This represents the first study of nuclear DNA content in alarge sample (135 spp.) from a tropical arboreal genus, in whicha large proportion of the species were examined (42 spp., 31.1%).Somatic chromosome numbers and 4C-DNA values for 51 taxa ofLonchocarpus are reported. All taxa were diploid with 2 n =22,but their DNA content ranged from 1.92 to 2.86 pg 4C nucleus,corresponding to a 48.95% variation in genome size. In the 74collections studied, no correlation was observed between DNAcontent and habitat altitude. Variation in nuclear DNA contentwas analysed at the level of genus, subgenus, section and subsection.Variation in genome size was also studied within some species,either among widely separated populations or among differentintraspecific taxa. Very little variation in genome size wasdetected between populations, subspecies, and varieties of thesame species. The taxonomic implications of variation in nuclearDNA content are discussed.Copyright 2000 Annals of Botany Company Lonchocarpus (Leguminosae), DNA content, chromosome number.     

Evolution of genome size inAllium (Alliaceae)

The 4C DNA amounts of 86 species fromAllium subgg.Allium, Rhizirideum, Bromatorrhiza, Melanocrommyum, Caloscordum andAmerallium show a 8.35-fold difference ranging from 35.60 pg (A. ledebourianum, 2n = 16) to 297.13 pg (A. validum 2n = 56). At diploid level the difference is 3.57-fold betweenA. ledebourianum (35.60 pg) andA. ursinum (127.14 pg). This shows that a significant loss and/or gain of DNA has occurred during evolution. On average subgg.Rhizirideum andAllium have less DNA amount than subgg.Melanocrommyum andAmerallium. The distribution of nuclear DNA amounts does not show discontinuous pattern and regular groups. The evolution of genome size has been discussed in relation to polyploidy and genomes, heterochromatin, adaptive changes in morphological characteristics, phenology and ecological factors, and infrageneric classification.     

From pixels to picograms: a beginners' guide to genome quantification by Feulgen image analysis densitometry.

The study of genome size variation is important from a number of practical and theoretical perspectives. For example, the long-standing "C-value enigma" relating to the more than 200,000-fold range in eukaryotic genome sizes is best studied from a broad comparative standpoint. Genome size data are also required in detailed analyses of genome structure and evolution. The choice of future genome sequencing projects will be dependent on knowledge regarding the sizes of genomes to be sequenced, and so on. To date, genome size data have been acquired primarily by Feulgen microdensitometry or flow cytometry. Each has several advantages but also important limitations. In this review, we provide a practical guide to the new technique of Feulgen image analysis densitometry. The review is designed for those interested in genome size measurements but not extensively experienced in histochemistry, densitometry, or microscopy. Therefore, relevant historical and technical background information is included. For easy reference, we provide recipes for required reagents, guidelines for cell staining, and a checklist of steps for successful image analysis. We hope that the accuracy, rapidity, and cost-effectiveness of Feulgen image analysis demonstrated here will stimulate further surveys of genome sizes in a variety of taxa.     

Nuclear DNA Amounts in Angiosperms

Bennett and Smith (Philosophical Transactions of the Royal Societyof London B274:227-274; B334: 309-345) and Bennett, Smith andHeslop-Harrison (Proceedings of the Royal Society of London,B216: 179-199) published lists of nuclear DNA amounts estimatedfor 1612 angiosperm species collected from 163 sources datedbetween 1951 and 1986. Subsequently, interest in genome sizein angiosperms and its significance has continued, and manynew DNA estimates were published during 1986-1994. Their inaccessibility,and the flow of enquiries for such information, shows that afurther compilation is needed. This paper presents a supplementarylist of nuclear DNA C-values for 105 sources for 899 angiospermspecies not listed in the above-mentioned compilations, plus284 additional estimates for 208 species already listed by them.The data are assembled primarily for reference purposes, withspecies listed in alphabetical order, rather than by any taxonomicscheme. Some advantages and limitations of flow cytometry, nowincreasingly used to quantify DNA C-values in plants, are reviewed.Recent reports regarding the occurrence and extent of intraspecificvariation in genome size are also discussed. While some examplesare real, others reflect technical shortcomings. Work has begunto combine the genome size data compiled in this and the above-mentionedpapers into a unified data base, and to present the informationin separate lists, with species in alphabetical and systematicorders, respectively. DNA C-values are now known for 1% of theworld's angiosperm flora, but improved representation of taxonomicgroups, geographical regions and plant life forms is urgentlyneeded.Copyright 1995, 1999 Academic Press Angiosperm DNA amounts, DNA C-values, nuclear genome sizes, intraspecific variation, flow cytometry, reference lists, genome size database     

Analysis of plant genomes. V. Comparative study of molecular properties of DNAs of seven Allium species

The genomes of seven plant species belonging to the genus Allium and exhibiting a threefold variation in their nuclear DNA content were analyzed by studying their reassociation kinetics, equilibrium centrifugation behavior in neutral CsCl gradients, and melting properties. The reassociation kinetics experiments revealed the presence of 44–65% repeated DNA sequences. A comparison between DNA contents and the proportion of repeated DNA sequences indicated that, in Allium, increase in the genome size is not exclusively due to variations in the proportions of repetitive DNA. The total DNA as well as the various repetitive DNA fractions in all the Allium species examined exhibited, in spite of a few differences, a gross similarity in their behavior in neutral CsCl gradients and in their melting properties.     

Genome size and chromosome number in velvet worms (Onychophora)

The Onychophora (velvet worms) represents a small group of invertebrates (~180 valid species), which is commonly united with Tardigrada and Arthropoda in a clade called Panarthropoda. As with the majority of invertebrate taxa, genome size data are very limited for the Onychophora, with only one previously published estimate. Here we use both flow cytometry and Feulgen image analysis densitometry to provide genome size estimates for seven species of velvet worms from both major subgroups, Peripatidae and Peripatopsidae, along with karyotype data for each species. Genome sizes in these species range from roughly 5–19 pg, with densitometric estimates being slightly larger than those obtained by flow cytometry for all species. Chromosome numbers range from 2n = 8 to 2n = 54. No relationship is evident between genome size, chromosome number, or reproductive mode. Various avenues for future genomic research are presented based on these results.     

Genome Size Determination in Peronosporales (Oomycota) by Feulgen Image Analysis

Genome size was determined, by nuclear Feulgen staining and image analysis, in 46 accessions of 31 species of Peronosporales (Oomycota), including important plant pathogens such asBremia lactucae, Plasmopara viticola, Pseudoperonospora cubensis,andPseudoperonospora humuli.The 1C DNA contents ranged from 0.046 (45.6 Mb) to 0.163 pg (159.9 Mb). This is 0.041- to 0.144-fold that ofGlycine max(soybean, 1C = 1.134 pg), which was used as an internal standard for genome size determination. The linearity of Feulgen absorbance photometry method over this range was demonstrated by calibration ofAspergillusspecies (1C = 31–38 Mb) againstGlycine,which revealed differences of less than 6% compared to the published CHEF data. The low coefficients of variation (usually between 5 and 10%), repeatability of the results, and compatibility with CHEF data prove the resolution power of Feulgen image analysis. The applicability and limitations of Feulgen photometry are discussed in relation to other methods of genome size determination (CHEF gel electrophoresis, reassociation kinetics, genomic reconstruction) that have been previously applied to Oomycota.     

Genome size in wild Pisum species

Genome size was measured in 75 samples of the wild pea species Pisum abyssinicum, P. elatius, P. fulvum and P. humile by ethidium-bromide (EB) flow cytometry (internal standard: Triticum monococcum) and Feulgen densitometry (internal standard: Pisum sativum Kleine Rheinländerin). Total variation of EB-DNA between samples covered 97.7% to 114.9% of the P. sativum value, and Feulgen DNA values were strongly correlated with EB-DNA values (r=0.9317, P < 0.001). Only P. fulvum was homogeneous in genome size (108.9% of P. sativum). Wide variation was observed between samples in P. abyssinicum (100.9–109.7%), P. elatius (97.7–114.9%) and P. humile (98.3–111.1% of P. sativum). In view of the world-wide genome size constancy in P. sativum, the present data are interpreted to show that the pea taxa with variable genome size are genetically inhomogeneous and that the current classification is not sufficient to describe the biological species groups adequately.     

Genome size in Dahlia Cav. (Asteraceae–Coreopsideae)

The genus Dahlia (Asteraceae–Coreopsideae) is monophyletic according to a recent DNA phylogeny (ETS and ITS of rDNA). Traditionally, the genus has been divided into sections, but these have been shown not to be monophyletic. We have studied variation in genome size (DNA C-values) in a sample of species to investigate the possible effects of secondary metabolites on flow cytometry and Feulgen densitometry, and to see whether genome size variation has any systematic or phylogenetic significance. Using a range of cultivars, secondary compounds from corollas were shown to have only minor effects on the Feulgen method; the floral pigments were found to be relatively inert and seemed to have been extracted on fixation with acetic methanol. Freshly expanded corollas showed apparent apoptotic DNA decay in epidermal cells, so need to be used with caution. Flow cytometric measurements with propidium iodide in some cultivars resulted in a very similar average genome size (2C = 8.62 pg) as compared with Feulgen densitometry (2C = 8.84 pg). Leaf cytosol of D. variabilis has a demonstrable inhibitory effect on propidium iodide fluorescence, which may explain some of the intraspecific variation of C-values observed. DNA 2C-values ranged from 3.30 pg in D. dissecta (2n = 34) to 9.62 pg in a D. variabilis cultivar (2n = 64). The D. variabilis cultivars had broadly similar C-values showing a 1.16-fold range between cultivars. Some of this variation probably results from technical variables and the extent of genuine variation is uncertain. The highest 2Cx-value occurred in one D. coccinea accession (2.47 pg, 2n = 32; x = 8). D. coccinea with 2n = 64 showed slightly reduced Cx-values compared to D. coccinea with 2n = 32. Artificially produced interspecific hybrids had C-values that corresponded closely with expectations from the measured values obtained from their parents.     

Genome size and endonuclear DNA replication in spiders

Although genome sizes (C-values) are now available for 115 arachnid species (Gregory and Shorthouse [2003] J Hered 94:285-290), the extent of genome amplification (endonuclear DNA replication or polyploidization) accompanying tissue differentiation in this diverse and abundant class of invertebrates remains unknown. To explore this aspect of arachnid development, samples of hemolymph and other tissues were taken from wild-caught specimens as air-dried smears, stained with the Feulgen reaction for DNA, and assayed using both scanning and image analysis densitometry. Cells from midgut diverticula and Malpighian tubules of Argiope and Lycosa (=Pardosa) often showed giant nuclei with 50-100 pg of DNA per nucleus, reflecting at least four cycles of endonuclear DNA replication when compared to the DNA content of hemocytes or sperm from the same specimen. Nuclei with markedly elevated DNA levels also appeared, but far less frequently, in tissue samples from several other arachnid species (Antrodiaetus, Hypochilus, Latrodectus, Liphistus and Loxosceles), but revealed no correlation with differences in somatic cell (2C) genome sizes. Our data show that several DNA classes of polysomatic nuclei regularly arise during tissue differentiation in some species of spiders and may provide an interesting model system for further study of patterns of tissue-specific variation in DNA endoreduplication during development.     

Nuclear DNA Amounts in Angiosperms--583 New Estimates

The amount of DNA in the unreplicated haploid nuclear genome(itsC-value) varies over 600-fold between angiosperm species.Information regarding this character is used in a strikinglywide variety of plant biological fields. Moreover, recent studieshave noted a significant need for more information about thisimportant aspect of genome biodiversity. Bennett and co-authorshave published four collected lists of nuclear DNA amounts inangiosperm species, compiled primarily for reference purposes,including the most recent in 1995 (Annals of Botany76: 113–176).Together they list estimates for over 2500 species which representabout 1% of the global angiosperm flora. Interest in angiospermgenome size has remained high, as shown by the recent publicationof many new estimates, creating a need for a fifth compilation.This paper presents a supplementary list of nuclear DNAC-valuesfrom 37 sources for 471 angiosperm species not listed in theaforementioned compilations, plus additional estimates for 113species already listed by them. It contains estimates for palm,orchid and tropical hardwood species which significantly improvesrepresentation of the global flora. Work is in hand to combinethe genome size data compiled in this and the aforementionedpapers into a unified database, and to present the informationin separate lists, with species in alphabetical and systematicorders, respectively. Meanwhile, the availability of DNAC-valuesfor angiosperm species can be checked on the World Wide Web(http://www.rbgkew.org.uk/cval/database1.html). Angiosperm DNA amounts; DNAC-values; nuclear genome sizes; reference lists; plant DNA database     

Cytogenetic studies in the genus Zea

Summary The nuclear DNA amount and the heterochromatin content in species and hybrids of Zea were analyzed in telophase nuclei (2C) of the root apex of germinating seeds. The results revealed significant differences among taxa and also among lines and races of maize. The hybrids between Z. mays ssp. mays x Z. mays ssp. mexicana (2n=20), Z. diploperennis x Z. perennis (2n=30), and Z. diploperennis x Z.perennis (2n=40) showed DNA content intermediate between that of the parents. The number of chromosomal C-bands and the proportion of the genome comprising C-band heterochromatin were positively related to genome size. In the different lines and races of maize studied, there was a positive correlation between genome size and the interval from germination to flowering. Octoploid Z. perennis (2n=40) showed the smallest DNA content per basic genome and the smallest heterochromatic blocks, suggesting that the DNA lost by this species consisted mainly of repetitive sequences. Considering that the extant species of Zea are tetraploid (2n=20) and octoploid (2n=40) and that the ancestral diploids are extinct, any consideration of the direction (increase or decrease) of the DNA change would be entirely speculative. The extant species could be the product of natural and artificial selection acting on different genotypic and nucleotypical constitutions at the diploid and/or tetraploid levels.