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.     

DNA content in nuclei of <Emphasis Type="Italic">Cyclops kolensis</Emphasis> and <Emphasis Type="Italic">Cyclops insignis</Emphasis> (Crustacea,Copepoda)

Chromatin diminution (CD) in two Cyclopoida species, Cyclops kolensis and Cyclops insignis, was studied by static digital Feulgen cytophotometry. DNA content (pg/cell) was evaluated with standard dependences constructed by amounts of DNA in the blood cells of five organisms with known DNA contents of 1.25–14.7 pg. It was found that the C. kolensis diploid genome had about 40 pg DNA before CD and 1.8–2.0 pg DNA after CD. These values are similar both for Moscow and Baikal population of C. kolensis and exceed previous estimates by six to ten times (Grishanin, 2008). Our data confirm that CD reaches 94–96% of DNA content in C. kolensis. In mitotic cells of C. insignis DNA content was about 7.5 pg in both early and late embryos; CD was not revealed for this species. The data obtained show that the DNA content in the C. kolensis genome before CD is highest among the examined Cyclopoides.     

Increased nuclear DNA content in developing cotton fiber cells

The nuclear DNA content of developing cotton fiber cells (Gossypium hirsutum, cv. MD51ne) increases ∼24% after 2 d postanthesis (dpa). The amount of nuclear DNA at 2 dpa is 5.4 ± 0.27 pg. At 3–4 dpa it increases to 6.7 ± 0.24 pg and by 5 dpa it is 6.8 ± 0.70 pg. These values were obtained by nuclear fluorescence after staining with Hoechst 33258. Human oral squamous cell nuclei were used as a DNA standard. Nuclear DNA content increases in fibers growing on either fertilized or unfertilized ovules. The increase also is detectable in Feulgen stained nuclei using two-wavelength cytospectrophotometry. All measurements were made on isolated fiber cell nuclei using a newly developed method tailored to cotton fiber cells. The results imply that during the early stages of development fiber cell nuclei either selectively amplify certain sequences or enter S-phase replicating a portion of their genome.     

Estimates of nuclear DNA content in bryophyte sperm cells: phylogenetic considerations

Nuclear DNA contents of developing sperm were estimated for 17 species of bryophytes by cytophotometry in squash preparations of antheridia after Feulgen staining. Genome sizes are in the lower end of the range for land plants. Two homwort C-values have the lowest recorded for bryophytes at 0.17 and 0.26 pg DNA per nucleus. In liverworts, C-values range from 0.49 pg in Blasia pusilla to 4.05 pg in Pellia epiphylla, while moss genome sizes are less variable, ranging from 0.38 pg in Takakia ceratophylla to 0.92 pg in Atrichum oerstedianum. DNA content is not correlated with chromosome number in these bryophytes, but sperm cell size and cellular complexity are directly related to C-value. Structural variations in the locomotory apparatus are viewed as evolutionary modifications associated with changes in genomic complexity, with a generalized increase in complexity of the motile assemblage accompanying increases in DNA content. Nuclear DNA values are not as variable in bryophytes as they are in pteridophytes and seed plants. We suggest that in plants producing biflagellated gametes, lower DNA contents afford a selective advantage. Comparisons with plants that produce multiflagellated or pollen-dispersed sperm indicate operation of a nucleotypic effect in archegoniates with biflagellated sperm. This effect may be on sperm cell functioning, which in turn influences reproductive success.     

Variable transmission rates of a B-chromosome in <Emphasis Type="Italic">Myrmeleotettix maculatus</Emphasis> (Thunb.) (<Emphasis Type="Italic">Acrididae: Orthoptera</Emphasis>)

Amounts of Feulgen staining in individual spermatid and primary spermatocyte nuclei ofTricholioproctia impatiens were measured by the two wavelength method of cytospectrophotometry and compared with Feulgen-DNA values found for bull sperm, taken as a presumed reference standard of 3.24×10^–12 g DNA per nucleus. The amount of DNA estimated for the haploid male genome ofTricholioproctia was 0.39×10^–12 g DNA. This value was used to determine the DNA content and degree of polyteny of Malpighian tubule nuclei sampled from the larval stages of development.     

Estimation of nuclear DNA content of various bamboo and rattan species

We determined the nuclear DNA content (genome size) of over 35 accessions each of bamboo and rattan species from Southeast Asia. The 2C DNA per nucleus was quantified by flow cytometry. The fluorescence of nuclei isolated from the leaves and stained with propidium iodide was measured. The genome size of the bamboo species examined was between 2.5 and 5.9 pg DNA per 2C nucleus. The genome size of the rattan species examined ranged from 1.8 to 10.5 pg DNA per 2C nucleus. This information will be useful for scientists working in diverse areas of plant biology such as biotechnology, biodiversity, genome analysis, plant breeding, physiology and molecular biology. Such data may be utilized to attempt to correlate the genome size with the ploidy status of bamboo species in cases where ploidy status has been reported.     

Absolute DNA values from Feulgen microspectrophotometric measurements and quantitative electron microscopy. A comparison of two species

Quantitative electron microscopy (QEM) and microspectrophotometry were used to correlate the Feulgen stain absorption values to the calculated picograms of DNA. Measurements were made in human lymphocytes, rainbow trout lymphocytes and nuclei of trout erythrocytes. The median dry weight of the nucleus, as determined by QEM, was 35.9 pg for a human lymphocyte and 30.5 pg for a trout lymphocyte. Using Salzman's value of 20% DNA per chromosome (i.e., chromatin), a human lymphocyte nucleus thus contains 7.18 pg of DNA and a trout lymphocyte nucleus 6.1 pg of DNA. The mean Feulgen absorption value of the nucleus, given in arbitrary units (AU), was 14.5 for a human lymphocyte, 12.7 for a trout lymphocyte and 12.0 for a trout erythrocyte. From these values, it was derived that each picogram of DNA of a human lymphocyte nucleus is represented by 2.02 arbitrary Feulgen units while the values for trout nuclei were 2.08 AU and 1.97 AU. On the average, we find that each picogram of DNA is represented by two arbitrary Feulgen units in our microspectrophotometric measurements.     

DNA contents in nuclei of Cyclops kolensis and C. insignis (Crustacea, Copepoda)

Chromatin diminution (CD) in two Cyclopoida species, Cyclops kolensis and C. insignis, was studied by static digital Feulgen cytophotometry. DNA content (pg/cell) was evaluated by standard curves builded up using blood cells of five organisms with known DNA content, which ranged from 1.25 to 14.70 pg. According to data obtained, diploid genome of C. kolensis has about 40 pg DNA before CD and 1.8-2.0 pg DNA after CD. These values are similar for both Moscow and Baikal populations of C. kolensis and 6-10 times exceed estimates made earlier (Grishanin, 2008), Our data confirm that CD in C. kolensis is 94-96% of DNA. In mitotic dividing cells of C. insignis, DNA content was about 7.5 pg both in early and late embryos, and CD was not revealed for this species. The data obtained show that, among Cyclopoida studied, the genome of C. kolensis before CD has a maximum content of DNA.     

DNA content in the genus Xenopus

Nuclear DNA amounts were determined by cytofluorometry for twelve species and subspecies of the genus Xenopus. Absolute values, in pg per nucleus, were obtained by direct comparison with human lymphocyte nuclei. The lowest DNA amount (3.55 pg) was found in X. tropicalis, which possess only 20 chromosomes, and the highest (16.25 pg), in the hexaploid X. ruwenzoriensis, with 108 chromosomes. The two recently discovered tetraploid species, X. sp.n. and X. vestitus have, respectively, 12.57 and 12.83 pg of DNA. Among the species and subspecies with 36 chromosomes, the DNA content ranges from 6.35 to 8.45 pg.     

Nuclear DNA content of Solanum species grown in vitro, as determined by flow cytometry

The DNA relative content in nuclei from several Solanum species, which were used as partners for somatic hybridization, were determined using a flow cytometry method. The nuclei were isolated mechanically or via protoplasts from leaves of in vitro grown plants. In the case of S. nigrum as well as S. tuberosum cv. Bzura and dihaploid clone H8105, the nuclei were also obtained from suspension cultured cells by lysis of protoplasts. The source and the method of nuclei isolation affected the pattern of nuclear DNA in the genotypes studied. The mesophyll nuclei showed two distinct peaks on the DNA histograms, whereas the DNA peaks produced by cell suspension nuclei were broad and less distinct. The DNA content in the nuclei, calculated from the DNA histograms of the samples and a DNA standard historgam (Trout Red Blood Cells, having DNA content of 5.05 pg per nucleus), were much lower in mesophyll nuclei than in those obtained from the cell suspension for the same genotypes. The results are discussed in respect of the genetic instability of Solanum genotypes. The usefulness of a flow cytometry approach in somatic hybridization research is also discussed.     

DNA-Feulgen cytophotometric determination of genome size for the freshwater-invading copepod Eurytemora affinis.

Variation in nuclear DNA content within some eukaryotic species is well documented, but causes and consequences of such variation remain unclear. Here we report genome size of an estuarine and salt-marsh calanoid copepod, Eurytemora affinis, which has recently invaded inland freshwater habitats independently and repeatedly in North America, Europe, and Asia. Adults and embryos of E. affinis from the St. Lawrence River drainage were examined for somatic cell DNA content and the presence or absence of embryonic chromatin diminution, using Feulgen-DNA cytophotometry to determine a diploid or 2C genome size of 0.6-0.7 pg DNA/cell. The majority of somatic cell nuclei, however, have twice this DNA content (1.3 pg/nucleus) in all of the adults examined and possibly represent a population of cells arrested at the G2 stage of the cell cycle or associated with some degree of endopolyploidy. Both suggestions contradict assumptions that DNA replication does not occur in adult tissues during the determinate growth characteristic of copepods. Absence of germ cell nuclei with markedly elevated DNA values, commonly found for species of cyclopoid copepods that show chromatin diminution, indicates that E. affinis lacks this trait. The small genome size and presumed absence of chromatin diminution increase the potential utility of E. affinis as a model for genomic studies on mechanisms of adaptation during freshwater invasions.     

Nuclear DNA content variation of three Miscanthus species in China

In order to estimate the variation in nuclear genome size in Miscanthus, flow cytometry of nuclei stained by propidium iodide was carried out using 36 populations of three Miscanthus species: M. lutarioriparius, M. sacchariflorus and M. sinensis, which were sampled from cold northern to warm and humid southern and central China, as well as near the sea level in eastern China to mountains in western China. The DNA content of diploid was 4.37 ± 0.02 pg/2C in M. lutarioriparius, 4.37 ± 0.01 pg/2C in M. sacchariflorus, and 5.37 ± 0.03 pg/2C in M. sinensis, respectively. There was no intraspecific variation in the three Miscanthus species at the diploid level, suggesting that the genome size was stable within species and the diverse environments did not induce variation in genome size at the diploid level. However, tetraploid populations were found in M. lutarioriparius and M. sacchariflorus, and their genome sizes were 8.56 and 8.54 pg, respectively, which are lower than expected values (8.74 pg), indicating the genome downsizing after polyploidization in the genus. Our results showed that the plant height of M. lutarioriparius was the highest one among the three species and the species was more closely related to M. sacchariflorus than M. sinensis. The intra-species genomic variation and inter-species differentiation in Miscanthus species provide important genetic and genomic information for the development of Miscanthus, especially for the endemic species, M. lutarioriparius, (together with Miscanthus × giganteus) which are now emerging as a key bio-energy crop because of their high yields and strong adaptability.     

Gene amplification in oocytes with 8 germinal vesicles from the tailed frog Ascaphus truei Stejneger

In the last 3 oogonial mitoses in Ascaphus truei all daughter nuclei remain in the same cell. The oocyte is 8-nucleate at the start of meiotic prophase and remains so until late in oogenesis when 7 of the nuclei disappear. All 8 nuclei in a single oocyte resemble one another with respect to size and chromatin distribution at all stages of meiotic prophase. Much of the Feulgen-positive material in pachytene nuclei is concentrated into one region of the nucleus. — All of the 8 germinal vesicles of yolky oocytes have a full set of lampbrush diplotene bivalents. Germinal vesicles from oocytes of up to 0.8 mm diameter have less than 100 nucleoli, some of which are multiple nucleoli in the sense that they have more than one core region. Each of the 8 nuclei in oocytes from one animal had about the same volume of nucleolar material. — Two values have been obtained for the amount of DNA in a diploid nucleus from Ascaphus. A biochemical estimate utilizing erythrocyte nuclei and the diphenylamine reaction yielded a value of 7.1 pg per nucleus. Microphotometry of erythrocyte nuclei stained with Feulgen's reagent gave a value of 8.2 pg per nucleus. — Microphotometric measurements of Feulgen-stained nuclei at various stages of meiotic prophase up to diplotene indicate that each nucleus synthesizes up to 5 pg of extrachromosomal DNA during and immediately after pachytene. This DNA is considered to be nucleolar. Autoradiography of nuclei from oocytes which had been incubated for 6h in ³H thymidine showed silver grains over pachytene and early diplotene nuclei only. In pachytene nuclei the silver grains overlaid that part of the nucleus where Feulgen-positive material was most concentrated. Most of the chromosomal material was unlabelled. — The significance of the 8-nucleate condition in Ascaphus oocytes is discussed, and the amount of nucleolar DNA synthesized at pachytene and of nucleolar material present in germinal vesicles is compared with corresponding situations in other amphibians.     

FEULGEN MICROSPECTROPHOTOMETRIC STUDIES OF PANDORINA MORUM AND OTHER VOLVOCALES (CHLOROPHYCEAE)12

The nuclear DNA content during normal vegetative growth and division has been examined in three species of Volvocales, Chlamydomonas reinhardtii Dangeard, Pandorina morum Bory, and Volvox carteri f. nagariensis Iyengar. The results are consistent with the nuclear cycle reported in the literature for Eudorina. Nuclear DNA content does not increase during the prolonged cell growth phase. At the time of colony formation, nuclear DNA doubles, the nucleus divides, and this alternation continues until the final 2ⁿ complement of progeny nuclei is formed. The 4- and 8-nucleate stages of dividing gonidia of V. carteri have a nuclear DNA content in the same range as the somatic cells; they are not polyploid or polytene. Four normal clones of Pandorina, having 2, 5 or 12 chromosomes, all had similar amounts of DNA per nucleus, suggesting that the species has a nuclear genome of fairly constant size rather than consisting of many strains representing a polyploid series. One unique clone, a hybrid with double the chromosome number of either its parents, had twice as much DNA as the normal clones. The Feulgen spectrophotometric method is sufficiently sensitive to detect 2-fold differences in DNA content at the level of 2 × 10^?13 g of DNA /nucleus, and its use avoids the complications associated with the presence of organelle DNA.     

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.     

Nuclear DNA content of some important plant species

Nuclear DNA contents of more than 100 important plant species were measured by flow cytometry of isolated nuclei stained with propidium iodide.Arabidopsis exhibits developmentally regulated multiploidy and has a 2C nuclear DNA content of 0.30 pg (145 Mbp/1C), twice the value usually cited. The 2C value for rice is only about three times that ofArabidopsis. Tomato has a 2C value of about 2.0 pg, larger than commonly cited. This survey identified several horticultural crops in a variety of families with genomes only two or three times as large asArabidopsis; these include several fruit trees (a pricot, cherry, mango, orange, papaya, and peach). The small genome sizes of rice and the horticultural plants should facilitate molecular studies of these crops.     

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).     

MICROFLUOROMETRIC MEASUREMENT OF DNA IN EUDORINA ELEGANS AND E. CALIFORNICA (CHOROPHYCEAE)1

The fluorescent dye 2,5-bis (4′-aminophenyl (1′))-1,3,4-oxidiwle (BAO) was compared to the Feulgen procedure as a method for quantitative comparison of DNA levels in two species of Eudorina. The BAO procedure proved to be specific and as quantitative as the Feulgen procedure, and was more convenient and rapid. The DNA level of the nuclei of gonidial cells of Eudorina elegans Ehrenberg and E. californica (Shaw) Goldstein doubled prior to each division. The DNA level of the somatic, usually non-dividing nuclei of E. californica did not vary from the IC (haploid) level.     

High DNA content of Sprekelia formosissima Herbert (Amaryllidaceae) and Ophioglossum petiolatum Hook. (Ophioglossaceae)

Ophioglossum petiolatum and Sprekelia formosissima root tips were chemically determined to have 170± 12 pg and 180±12 pg DNA/cell respectively, or 2.8 and 3.0 times the 60±4 pg DNA/ cell of Tradescantia sp. clone 02 root tips. These values were compared with those predicted from nuclear volume measurements. General qualifications of the nuclear volume-DNA content relationship are discussed. Microspectrophotometrically determined relative DNA values for Feulgen stained half-telophase root tip nuclei of O. petiolatum and S. formosissima were 2.8 and 2.6 the value for Tradescantia. The value for Sprekelia is among the highest in the angiosperms, and Ophioglossum probably has the highest nuclear DNA content of ferns. O. petiolatum has 131±3 pg DNA per dormant spore.     

Quantification of nuclear DNA and G-C content in marine macroalgae by flow cytometry of isolated nuclei

Summary The amounts of nuclear DNA in ten species of seaweeds belonging to the Rhodophyceae, Phaeophyceae, and Chlorophyceae were determined by flow cytometric analysis of nuclei isolated from protoplasts. Genome size was determined from the fluorescence of the nuclei stained with ethidium bromide. The size of the nuclear genome ranged from 0.13 pg per cell in the 1 C population ofUlva rigida to 3.40 pg per cell in the 2 C population ofSphacelaria sp. GC% analysis was based on staining with either Hoechst 33342 or mithramycin A, two fluorochromes specific for the bases A-T and G-C, respectively. Two models were used for the estimation of the proportion of guanine plus cytosine in the nuclear genome. The first one was based on the linear relationships mithramycin A fluorescence/G-C content and ethidium bromide fluorescence/total DNA content. The second model, based on the curvilinear relationships Hoechst 33342 fluorescence/A-T content and mithramycin A fluorescence/G-C content, resulted in comparatively more homogenous and consistent data and appears more accurate. Comparison with previous reports from other methods for the physical investigation of nuclear genomes shows that flow cytometry of nuclei isolated from protoplasts is an accurate, convenient and robust technique to assay for genome sizes and base pair composition in marine macroalgae.Abbreviations A-T nucleic bases adenine and thymine - CRBC chicken red blood cell - FALS forward-angle light scatter - G-C nucleic bases guanine and cytosine - SEIM sorbitol enzymatic incubation medium - SWIM sea water incubation medium - Tm thermal denaturation temperature of DNA