Genome size estimates for two important freshwater molluscs, the zebra mussel (Dreissena polymorpha) and the schistosomiasis vector snail (Biomphalaria glabrata).

The haploid genome sizes of two important molluscs were assessed by Feulgen image analysis densitometry. The genome size of the zebra mussel (Dreissena polymorpha), a prolific invader of North American lakes, was estimated to be 1C = 1.70 +/- 0.03 pg, and that of the freshwater snail Biomphalaria glabrata, the predominant intermediate vector of the human parasite Schistosoma mansoni, was estimated at 0.95 +/- 0.01 pg. These estimates will be important in future efforts in molluscan genomics, which at present lags far behind work being carried out with vertebrate and arthropod models. B. glabrata in particular, which has one of the smallest known gastropod genomes, is recommended as a highly suitable target for future genome sequencing.     

The first genome size estimates for six species of krill (Malacostraca, Euphausiidae): large genomes at the north and south poles

Krill (family Euphausiidae) represent some of the most abundant organisms in the both northern and southern oceanic environments and provide food for various animals including humans. Despite their importance, little is known about krill from a genomic standpoint, even with regard to basic properties such as total genome size. This study provides genome size estimates for six species of krill from both the North Atlantic and Southern Oceans which are the first such estimates for any species of euphausiid. Genome size estimates were obtained using both flow cytometry and Feulgen image analysis densitometry with chicken and trout blood as internal standards. Haploid genome sizes ranged from 12.77 to 48.53 pg, providing roughly fourfold variation within these six species alone. With such large estimates, sequencing of a krill genome will currently be costly and laborious, but further studies should be conducted to determine the composition of these exceptionally large genomes.     

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.     

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 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.     

Karyotype constancy and genome size variation in BulgarianCrepis foetida s. l. (Asteraceae)

Ten populations ofCrepis foetida from Bulgaria belonging to the three subspeciesfoetida, rhoeadifolia, andcommutata were analyzed karyologically using haematoxylin staining, Giemsa C-banding, fluorochrome banding, Ag-NOR staining, Feulgen cytophotometry (scanning densitometry and video-based image analysis), and propidium iodide flow cytometry. The quantitatively-evaluated karyotype structure was similar among all populations, with minor variation in a few intercalary sites only and in the amount of NOR-associated heterochromatin (satellites). In contrast to the karyotypic constancy the genome size ofC. foetida subsp.commutata was about 10% lower than those of the other two subspecies, which had similar genome sizes. The genome size measurements using three different methods resulted in highly correlated data. The genome size difference adds some weight to previous taxonomic opinions treatingC. foetida subsp.commutata at species level, asC. commutata. Prof. Dr. Stefan Kozhuharov (4 January 1933–24 August 1997).     

Cytochemistry and C-values: the less-well-known world of nuclear DNA amounts

BACKGROUND: 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. SCOPE: 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. CONCLUSIONS: 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.     

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.     

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 inAllium: In Quest of Reproducible Data

ComparingAlliumgenome size measurements of different authors,we noticed that the estimates for certain species diverge morestrongly than one would have expected in view of the methodologicaladvantages of the material. As the matter has theoretical significancefor explaining the biological role of genome size variation,we measured, by Feulgen densitometry, 28 species and altogether57 accessions or cultivars. Flow cytometric measurements supplementedthese data. The current hypothesis of a discontinuous and step-wisedistribution of DNA amounts inAlliumseems questionable, as mostof our DNA values did not appear in the corresponding DNA groupas proposed previously. On the other hand, we can confirm thatthere is a significant negative correlation between genome sizeof a species and its first month of flowering, but only in diploids,or in diploids and polyploids if only the basic genome size(2Cxlevel) is considered. We compared our results with thoseof nine other publications. Only 29 of 60 2C values publishedpreviously deviate less than 10% from our data, the others deviatemore strongly, from 0.44- to 1.44-fold. The more comprehensivedata sets of various authors were compared by correlation analysiswith our data. Positive and mostly significant correlationswere seen in all tests, but nevertheless the degree of incongruencebetween studies was unsatisfactory in view of the much betterintra-laboratory reproducibility of the present data. The presentwork highlights the need for generally agreed improvements instandardization and preparative procedures of cytophotometricgenome size determination.Copyright 1999 Annals of Botany Company Allium, genome size, Feulgen densitometry, flow cytometry, discontinuous DNA content variation, nucleotype hypothesis, flowering time, data reproducibility, correlation analysis.     

DNA content of heterochromatin and euchromatin in tomato (Lycopersicon esculentum) pachytene chromosomes.

Lycopersicon esculentum (tomato) has a small genome (2C = 1.90 pg of DNA) packaged in 2n = 2x = 24 small acrocentric to metacentric chromosomes. Like the chromosomes of other members of the family Solanaceae, tomato chromosomes have pericentromeric heterochromatin. To determine the fraction of the tomato genome found in euchromatin versus heterochromatin, we stained pachytene chromosomes from primary microsporocytes with Feulgen and analyzed them by densitometry and image analysis. In association with previously published synaptonemal complex karyotype data for tomato, our results indicate that 77% of the tomato microsporocyte genome is located in heterochromatin and 23% is found in euchromatin. If heterochromatin is assumed to contain few active genes, then the functional genes of the tomato must be concentrated in an effective genome of only 0.22 pg of DNA (1C = 0.95 pg x 0.23 = 0.22 pg). The physical segregation of euchromatin and heterochromatin in tomato chromosomes coupled with the small effective genome size suggests that tomato may be a more useful subject for chromosome walking and gene mapping studies than would be predicted based on its genome size alone. Key words : tomato, Lycopersicon esculentum, genome size, heterochromatin, euchromatin, pachytene chromosomes, synaptonemal complex.     

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 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.     

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.     

Genome size of 14 species of fireflies (Insecta,Coleoptera, Lampyridae)

Eukaryotic genome size data are important both as the basis for comparative research into genome evolution and as estimators of the cost and difficulty of genome sequencing programs for non-model organisms.In this study,the genome size of 14 species of fireflies (Lampyridae) (two genera in Lampyrinae,three genera in Luciolinae,and one genus in subfamily incertae sedis) were estimated by propidium iodide (PI)-based flow cytometry.The haploid genome sizes of Lampyridae ranged from 0.42 to 1.31 pg,a 3.1-fold span.Genome sizes of the fireflies varied within the tested subfamilies and genera.Lamprigera and Pyrocoelia species had large and small genome sizes,respectively.No correlation was found between genome size and morphological traits such as body length,body width,eye width,and antennal length.Our data provide additional information on genome size estimation of the firefly family Lampyridae.Furthermore,this study will help clarify the cost and difficulty of genome sequencing programs for non-model organisms and will help promote studies on firefly genome evolution.     

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 sizes and chromosomes in the basal metazoan Hydra

Hydras belong to one of the earliest eumetazoan animal groups, but to date very little is known about their genome sizes, gene numbers, and chromosomes. Here we provide genome size estimates and corresponding karyotypes for five Hydra species. Nuclear DNA contents were assessed by slide-based Feulgen microphotometry. Hydra oligactis possesses the largest genome of 1450 Mbp, followed by similar 1 C capacities in H. carnea (1350 Mbp), H. vulgaris (1250 Mpb) and H. circumcincta (1150 Mbp). The smallest genome of 380 Mbp was determined in H. viridissima. While the number of chromosomes is identical in all five Hydra species (2n = 30), the size of the chromosomes is strictly correlated to the size of the genome, with H. viridissima having conspicuously small chromosomes. The taxonomic and evolutionary significance of the C-value and chromosomal size variation in this ancient group of metazoans as well as its impact on genomic organization and forthcoming genome projects are discussed.     

Intraspecific variation in genome size in angiosperms: identifying its existence

BACKGROUND: The 6 years since the last Angiosperm Genome Size Discussion Meeting in 1997 have experienced the decline of the then widely held idea of the 'plastic' genome. Several published cases of intra-specific variation in cultivated plants have been questioned on re-investigation with an improved technical approach. At the same time, technical problems caused by staining inhibitors present in the plant material have been recognized. In the accumulation of genome size data more critical methods and rules for best practice are urgently needed. INFRA-SPECIFIC VARIATION RE-VISITED: This review is about (a) the basic requirement for repeatability of results and the need for self-criticism on the part of the investigator and (b) the critical points in the technical procedure, particularly the quantitative Feulgen reaction. Case studies are presented on Dasypyrum villosum (refuting a previously reported 'plastic genome' phenomenon), on Glycine max (refuting previously claimed intraspecific variation) and on Arachis hypogaea and A. duranensis, in which reported C-values are too high by roughly two-fold. In A. hypogaea the reported intraspecific genome size variation could not be confirmed. Furthermore, a claimed negative correlation between altitude and genome size in A. duranensis was shown to be based on an arbitrary omission of data points that did not fit the correlation (although a correlation was found). BEST PRACTICE METHODOLOGY: The finding of previously published questionable studies was the incentive for a re-consideration of the quantitative Feulgen procedure with regard to best practice in genome size studies. Clarification here of the critical steps of the method should help to improve the data in the literature. It must be stressed that the most important requirement is the need for a self-critical attitude of researchers to their data.     

Preparatory methods for DNA hydrolysis, cytochemistry, immunocytochemistry and ploidy analysis. Their application to automated and routine diagnostic cytopathology

A review is presented of some methods used to prepare cytologic specimens for analytical and/or automated studies, with the steps of the procedures detailed in appendices. The preparation of the cell monolayers required for optimal automated cell image analysis and classification, e.g., by the Cytoscan 110, is discussed, as is the preparation of poly-L-lysine-coated slides used in the production of monolayered specimens. These monolayers, which can be prepared from a variety of specimens, are also useful for cytochemical and immunocytochemical studies and DNA ploidy analysis. For DNA analysis, a modified gallocyanin chrome alum staining procedure is described as a stoichiometric alternative to the time-consuming Feulgen reaction. The hydrolysis technique required by the latter method is also detailed. The freeze-fracturing technique for the enhancement of monoclonal antibody immunocytochemical staining of detectable antigens is described, along with an indirect immunoalkaline phosphatase staining method. The use of enzyme cytochemical reactions for glucose 6 phosphate dehydrogenase and lysosomal naphthylamidase is also presented.     

Evolutionary analysis identifies multiple genome expansions and contractions in Sepsidae (Diptera) and suggests targets for future genomic research

We here argue that data from comparative studies of genome size and karyotypes provide important information for planning comparative research on genome evolution. We document for 39 species of sepsids that there is a four‐fold difference in genome size (151–618 Mbp). Mapping genome sizes onto a phylogenetic hypothesis identifies that this range is the result of five genome expansions and four genome contractions that we here define as changes in genome size of more than 50 Mbp. We then generate karyotype data for 10 species and find no changes in chromosome number. The study reveals that the “Oriental” clade of sepsids is a promising system for studying genome evolution because it has experienced three genome expansion events. These events can be compared with an expansion in the “Neotropical” clade in order to reveal the mechanisms that underlie genome expansion in Sepsidae. A review of the literature on genome sizes and karyotypes reveals that they have been poorly documented in Metazoa. This means that researchers interested in the evolution of genome expansions and contractions are currently not being able to identify appropriate target taxa for genome sequencing. We thus argue for more comparative research on genome sizes and karyotypes and point out that historically species were chosen for genome sequencing for reasons not related to genome evolution (e.g. small genome size, model species status, phylogenetic position, interesting phenotypes). We believe that it is now time to use a more genome‐centric selection criterion, where species for whole genome sequencing are selected based on their importance for understanding genome evolution.