Genome Size in Sphagnum (Peat Moss)

Abstract: Genome size was determined in thirty Austrian species of Sphagnum, using Feulgen absorbance photometry conducted on a video-based image analysis system (CIRES), and for comparison on a scanning cytophotometer (Leitz MPV II) with strongly correlated results. Pisum sativum (1C = 4.42pg DNA) was used for internal standardization. Between species, two levels of ploidy, haploid and diploid, could be unambiguously identified (although this identification remains, strictly speaking, hypothetical, as long as exact parallel chromosome counts are not available). Twenty-six haploid species yielded values from 0.392 pg to 0.506 pg DNA (1C), and four diploid species (including two varieties of S. palustre) from 0.814 pg to 0.952 pg. The average ratio between levels was 1:2.049. Variation between species within sections was lower than between sections. In some cases significant differences between accessions of one species were found. The genome size of Sphagnum palustre presented here strongly deviates from one estimate of this species in the literature.     

Cytophotometric evidence of variation in genome size of desmognathine salamanders

Summary The amount of DNA per haploid genome, the C-value, is often directly correlated with nuclear and cell volume, but inversely correlated with cell replication rate. Also, rates of cellular growth sometimes appear to be correlated with organismal developmental rates and life history patterns. Among vertebrates, salamanders exhibit the greatest variation in genome size. In the present study we have examined interspecific and intraspecific variation in blood cell DNA levels in the genus Desmognathus, which shows greater variation in life history traits than any other salamander genus. Specimens of Desmognathus quadramaculatus, D. Monticola, D. ochrophaeus and D. wrighti were collected from nature at two localities in the southern Appalachian Mountains. Estimates of genome size in pg of DNA were obtained from blood smears by DNA-Feulgen cytophotometry, using erythrocyte nuclei of Xenopus laevis as an internal reference standard of 6.35 pg DNA per cell. C-values of Desmognathus are the smallest in the order Caudata. Although significant variation in DNA levels was found among the four species, the differences were small, and do not support previously proposed relationships between C-value and life-history variation.     

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.     

DNA reassociation kinetics in relation to genome size in four amphibian species

DNA reassociation kinetics were studied, by means of the hydroxyapatite chromatography method, for four species of Amphibians with different nuclear DNA content: Xenopus laevis (3 pg DNA per haploid genome) and Bufo bufo (7 pg) of the Anura subclass and Trituras cristatus (23 pg) and Necturus maculosus (52 pg) of the Urodela subclass.Within each subclass the two species studied were found to have about the same absolute amount of unique DNA. The differences of total nuclear DNA can be accounted for by quantitative variations of the repetitive sequence classes, at least in part due to changes in the number of copies of the various sequences. On the contrary the great difference in nuclear DNA between the two subclasses, Anura and Urodela, involves all sequence classes in parallel; the slowly reassociating fraction appears to be unique in spite of a tenfold difference in absolute amount.The dependence of reassociation kinetics on DNA fragment length for the four species indicates for all of them an interspersed organization of the various sequence classes.     

Diploidization in megagametophyte-derived cultures of the gymnosperm Larix decidua

Tested haploid embryogenic lines (n=12) of Larix dedicua Mill, initiated from megagametophyte tissue were maintained on half-strength LM medium without growth regulators. The cultures were analyzed for ploidy level after 1–9 years. All lines tested were found to have doubled (2n=24) their chromosome number at the end of the experiment, though there were a few lines that still gave occasional haploid counts. Flow cytometric data of embryogenic tissue confirmed these results. Protoplasts were stained in ethidium bromide, and cultured human leucocytes and chicken erythrocytes were used as internal standards. Haploid megagametophytes from immature seeds of L. decidua and known diploid culture lines of a related hybrid (L. x eurolepis) were also analyzed by flow cytometry. Haploid reference material had 12.3–13.6 pg DNA per cell, whereas formerly haploid callus lines had an average of 25.0 pg DNA per cell. The one exception was a known, genetically unstable line of L. decidua (34.8 pg DNA per cell). The diploid cell line of L. x eurolepis had 27.6 pg DNA per cell. The results show that spontaneous diploidization of megagametophyte lines is relatively rapid and that both haploid and dihaploid lines are embryogenic in larch.     

The determination of genome size in male and female germ cells of Drosophila melanogaster by DNA-Feulgen cytophotometry

The amounts of DNA in haploid and diploid cells of Drosophila melanogaster have been determined by DNA-Feulgen cytophotometry, using Xenopus laevis erythrocyte nuclei as a reference standard. The haploid male genome is estimated to be 0.18 pg DNA and the haploid female genome, 0.20 pg DNA.     

Non xenopus-like DNA sequence organization in the Chironomus tentans genome

Summary We have examined the sequence organization of Chironomus tentans DNA by means of optical and hydroxyapatite renaturation kinetics of total DNA fragment sizes of 0.36, 2.6 and 13.5 kilobases (kb) as well as isolated middle repeat DNA at sizes of 0.36 and 13.5 kb. 90% of the DNA renatured as unique sequences of a genome of 0.20 pg with the balance of DNA renaturing as middle repetitive sequences present on average 90 times per haploid genome. At a DNA fragment length of 13.5 kb, 35% of the DNA was trapped on the hydroxyapatite as middle repetitive fraction. We concluded C. tentans DNA to have a mean repeat length of about 4.3 kb distributed through out at least 35% of the genome with an inter repeat spacing of at least 13.5 kb but possibly being distributed throughout the whole genome with an inter repeat spacing of 36 kb. This shows C. tentans DNA organization not to follow the almost ubiquitous Xenopus model but to be similar to the organization of Drosophila melanogaster DNA.     

Length and interspersion of repetitive and non repetitive DNA sequences in four Amphibian species with different genome sizes

The interspersion period of repetitive and unique sequences was analyzed by two different methods, electron microscopy and agarose gel electrophoresis, for four Amphibian species with different nuclear DNA content, namely the Anura Xenopus laevis (3 pg DNA per haploid genome) and Bufo bufo (7 pg) and the Urodela Triturus cristatus (23 pg) and Necturus maculosus (52 pg). Within each of the two subclasses it has been found that interspecific differences, in DNA content, due to variations in the amount of repetitive sequences, do not involve variations in length of the interspersed repetitive sequences. They remain about 380 base pairs. Furthermore, the unique sequences length has been found to be shorter in Bufo (760 base pairs) than in Xenopus (1600) and in Necturus (880) than in Triturus (1340). A study of the interspersion period has shown that the great difference in DNA content between Anura and Urodela, which had been previously shown not to have involved changes in the relative amounts of the various sequence classes, does not involve changes in the interspersion period.     

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.     

Lotus japonicus, an autogamous, diploid legume species for classical and molecular genetics

In the Leguminosae plant family, few of the individual plant species have been used for plant molecular biology research. Among the species investigated no obvious representative ‘model’ legume has emerged. Here a member of the tribe Loteae, Lotus japonicus (Regel) Larsen is proposed as a candidate. L. japonicus is a diploid, autogamous species, with a good seed set, and a generation time of approximately 3 months. The haploid genome consists of six chromosomes and the genome size was estimated to be relatively small (0.5 pg per haploid complement). L. japonicus is susceptible to Agrobacterium tumefaciens and transgenic plants can be regenerated after hygromycin or kanamycin selection. Tissue culture conditions and procedures for transformation and regeneration are described. Stable transformation is demonstrated by segregation of the hygromycin selectable marker after selfing of transgenic plants or test crosses. The possibility of mapping polymorphic DNA markers inbred lines of L. japonicus is also discussed.     

Nuclear DNA amounts in pacific Crustacea

Nuclear DNA amounts have been determined for 42 species of crustaceans bringing the total number of species with known nuclear DNA content to over 70. Genome size in Crustacea varies over a 25-fold range with a modal value of 2 to 3 pg haploid being common in many groups. Both average genome size and the amount of variability among species are characteristic for certain groups. A trend towards small genomes is evident in advanced and specialized crustacean groups. Somatic polyploidy is a very pronounced feature of the Crustacea. The data suggest that evolution by polyploidy may be more common in crustaceans than earlier data had indicated. These features and the presence of very characteristic satellite fractions in the nuclear DNA recommend the Crustacea for further studies in evolutionary genetics.     

Spatial and temporal expression of the two sucrose synthase genes in maize: immunohistological evidence

Summary The DNAs of two diploid species of Gossypium, G. herbaceum var. africanum (A₁ genome) and G. raimondii (D₅ genome), and the allotetraploid species, G. hirsutum (A_h and D_h genomes), were characterized by kinetic analyses of single copy and repetitive sequences. Estimated haploid genome sizes of A₁ and D₅ were 1.04 pg and 0.68 pg, respectively, in approximate agreement with cytological observations that A genome chromosomes are about twice the size of D genome chromosomes. This differences in genome size was accounted for entirely by differences in the major repetitive fraction (0.56 pg versus 0.20 pg), as single copy fractions of the two genomes were essentially identical (0.41 pg for A₁ and 0.43 pg for D₅). Kinetic analyses and thermal denaturation measurements of single copy duplexes from reciprocal intergenomic hybridizations showed considerable sequence similarity between A₁ and D₅ genomes (77% duplex formation with an average thermal depression of 6 °C). Moreover, little sequence divergence was detectable between diploid single copy sequences and their corresponding genomes in the allotetraploid, consistent with previous chromosome pairing observations in interspecific F₁ hybrids.Journal paper No. 4461 of the Arizona Agricultural Experiment Station     

Cytometric determination of genome size and base composition of tree species of three genera of Casuarinaceae

The genome size and base composition of diploid plant species from three genera of the Casuarinaceae family were determined by flow cytometry. Casuarina glauca Sieb. ex Spring. and Gymnostoma deplancheana (Miq.) L. Johnson showed a small genome with 2C = 0.70 pg, 58.6% AT, 40.5% GC for the first species and 2C = 0.75 pg, 58.7% AT, 40.5% GC for the second. Allocasuarina verticillata (Lam.) L. Johnson had a larger genome: 2C = 1.90 pg, 59.3% AT, 41.1% GC. One haploid genome of C. glauca is therefore about 340×10⁶ base pairs. In leaves, roots or bark of these three species, polysomaty was virtually absent: a maximum frequency of 4C nuclei of only 0.08 was found in bark of C. glauca. The genome sizes of C. glauca and G. deplancheana are among the smallest described for higher plants. Small genome size, diploidy and the absence of polysomaty are advantageous traits for facilitating molecular approaches to improvement of these actinorhizal plants and developing the study of their symbiotic interactions with Frankia. Received: 20 December 1997 / Revision received: 13 March 1998 / Accepted: 30 March 1998     

Genome Size Evaluation in Tetraodontiform Fishes from the Neotropical Region

Smooth pufferfish of the family Tetraodontidae had become pure genomic models because of the remarkable compaction of their genome. This trait seems to be the result of DNA loss following its divergence from the sister family Diodontidae, which possess larger genomes. In this study, flow cytometry was used for estimate the genome size of four pufferfish species from the Neotropical region. Cytogenetic data and confocal microscopy were also used attempting to confirm relationships between DNA content and cytological parameters. The haploid genome size was 0.71?±?0.03 pg for Sphoeroides greeleyi, 0.34?±?0.01 pg for Sphoeroides spengleri, 0.82?±?0.03 pg for Sphoeroides testudineus (all Tetraodontidae), and 1.00?±?0.03 pg for Chilomycterus spinosus (Diodontidae). These differences are not related with ploidy level, because 46 chromosomes are considered basal for both families. The value for S. spengleri represents the smallest vertebrate genome reported to date. Since erythrocyte cell and nuclear sizes are strongly correlated with genome size, the variation in this last is considered under both adaptive and evolutionary perspectives.     

Karyotype analysis of the Russian wheat aphid, <Emphasis Type="Italic">Diuraphis noxia</Emphasis> (Kurdjumov) (Hemiptera: Aphididae) reveals a large X chromosome with rRNA and histone gene families

The Russsian wheat aphid (RWA), Diuraphis noxia (Kurdjumov), is a worldwide pest of cereals. Despite its economic importance, little is known about its genome. Here we investigated physical genomic features in RWA by karyotype analysis using differential staining with AgNO₃, CMA₃, and DAPI, by chromosomal localization of ribosomal DNA (rDNA), H3 and H4 histone genes, and the “arthropod” telomeric sequence (TTAGG) _n using fluorescence in situ hybridization (FISH), and by measuring the RWA genome size using flow cytometry. The female karyotype, 2n = 10, is composed of four autosome pairs and a pair of X chromosomes, whereas the male karyotype, 2n = 9, has a single X. The X chromosome is the largest element in the karyotype. All three molecular markers used, i.e., 18S rRNA and both H3 and H4 probes are co-localized at one end of the X chromosome. The FISH probes revealed that the AgNO₃-positive bridge between two prometaphase X chromosomes of females, which is believed to be responsible for the elimination of one X chromosome in aphid oocytes determined to undergo male development, contains clusters of both histone genes, in addition to an rDNA cluster. Interestingly, RWA lacks the (TTAGG) _n telomeric sequence in its genome, in contrast to several previously investigated aphid species. Additionally, we compared female and male genome sizes. The female genome size is 2C = 0.86 pg, whereas the male genome size is 2C = 0.70 pg. The difference between the DNA content in the two genders suggests that the RWA X chromosome occupies about 35% of the female haploid genome (1C = 0.43 pg), which makes it one of the largest sex chromosomes in the animal kingdom.     

Flow cytometric measurement of nuclear DNA content inCapsicum (Solanaceae)

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

Purification and restriction endonuclease mapping of soybean 18 S and 25 S ribosomal RNA genes

The restriction endonuclease map of the 25 S and 18 S ribosomal RNA genes of a higher plant is presented. Soybean (Glycine max) rDNA was enriched by preparative buoyant density centrifugation in CsCl-actinomycin D gradients. The buoyant density of the rDNA was determined to be 1.6988 g cm^–3 by analytical centrifugation in CsCl. Saturation hybridization showed that 0.1% of the total DNA contains 25 S and 18 S rRNA coding sequences. This is equivalent to 800 rRNA genes per haploid genome (DNA content: 1.29 pg) or 3200 for the tetraploid genome. Restriction endonuclease mapping was performed with Bam H I, Hind III, Eco R I, and BstI. The repeating unit of the soybean ribosomal DNA has a molecular weight of 5.9·10⁶ or approximately 9,000 kb. The 25 S and 18 S rRNA coding sequences were localized within the restriction map of the repeating unit by specific hybridization with either [¹²⁵I]25 S or [¹²⁵I]18 S rRNA. It was demonstrated that there is no heterogeneity even in the spacer region of the soybean rDNA.     

Genome sizes for 71 species of Zamia (Cycadales: Zamiaceae) correspond with three different biogeographic regions

Nuclear DNA contents (2C‐value) are reported for 71 out of 76 accepted species of Zamia (Zamiaceae) using flow cytometry with propidium iodide. Nuclear DNA content in Zamia ranges between 33.7 and 45.7 pg. Despite this small range, the largest genome contains roughly 10¹⁰ more base pairs than the smallest genome. The results for Zamia point to two centers of biogeographic distribution: Mexico and Colombia. Nicaragua seems to be the biogeographic boundary for these two centres for Zamia. To the north, genome sizes of 33.7–38.0 pg (average 35.6 pg) are found and to the south (Costa Rica, Panama and South America) 41.2–45.7 pg (average 42.9 pg). Plants from the Caribbean islands (including Florida) have intermediate genome sizes with 37.3–40.9 pg (average 38.7 pg). Costa Rica and Panama are in a transition zone and its species can be divided into three subsections: four species with ‘Caribbean’ values of 38.4–39.5 pg (average 39.0 pg), six species with ‘South American’ values with 42.7–43.6 pg, (average 42.9 pg, and six species with intermediate values ranging between 40.1–41.0 pg (average 40.4 pg). The latter values are nearly absent in other areas, suggesting that they could be the products of (introgressive) hybridization. This study represents the first, nearly complete overview of the genome sizes of the genus Zamia and their relationship with biogeography.     

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.     

Intraspecific variation in nuclear DNA content among world populations of a mosquito,Aedes albopictus (Skuse)

Summary Aedes albopictus is commonly distributed in most parts of the Oriental region and on many islands in the Indian and the Pacific Oceans. The species was recently introduced into the United States and Brazil. Feulgen cytophotometric quantitation of haploid nuclear DNA content was carried out for 37 populations of Ae. albopictus to determine the extent of intraspecific variation in nuclear DNA content and whether the range expansion of the species has coincided with an increase in DNA content. The haploid nuclear DNA content varied nearly three-fold. The minimum DNA content was 0.62 pg in Koh Samui from Thailand, and the maximum DNA content was 1.66 pg in Houston-61 from the United States. Statistical comparisons of populations revealed significant differences in DNA contents. No geographic clustering of populations was noted with respect to DNA content. In general, populations from the United States and Brazil had higher DNA contents, but there was no indication that the range expansion had occurred hand in hand with an increase in DNA content. Each population had a specific amount of DNA that is probably imposed by the microenvironment.