Quantitative nuclear DNA differences associated with genome evolution in Guizotia (Compositae)

The present communication deals with 2C nuclear genome size variation in a fairly small genus Guizotia. Twenty-four accessions belonging to six species, out of seven known, were analysed in order to elucidate the extent of DNA variation both at an intra—as well as interspecific level. At the intraspecific level none of the species exhibited significant differences in their genome size. Between the species, the 2C DNA amounts ranged from 3.61 pg in G. reptans to 11.37 pg in G. zavattarii; over three-fold DNA variation is evident. Apparently these interspecific DNA differences have been achieved independent of the numerical chromosomal change(s), as all the Guizotias share a common chromosome number 2n=2x=30. The cultivated oilseed crop, G. abyssinica (7.57 pg), has accommodated nearly 78% extra DNA in its chromosome complement during the evolutionary time scale of its origin and domestication from the wild progenitor G. schimperi (4.25 pg). The extent of genomic DNA difference(s) between the species has been discussed in the light of their interrelationships and diversity.     

Nuclear DNA content of Vitis species,cultivars, and other genera of the Vitaceae

The nuclear DNA content was analyzed in Vitis species, hybrid cultivars, and genera of the Vitaceae using flow cytometry. Significant variation was found among Vitis species, hybrids, and other genera of the Vitaceae (Ampelopsis and Parthenocissus). DNA content was estimated to range from 0.98 to 1.05 pg/2C within V. labrusca (ns) and 0.86 to 1.00 pg/2C within V. vinifera (ns). Genotypes from Vitis and Parthenocissus were similar in nuclear DNA content (approximately 1.00 pg/2C) whereas they differed significantly from Ampelopsis (1.39 pg/2C). No correlation between DNA content and the center of origin of genotypes of the Vitaceae was noted. Based on the present study, the Vitis genome size is 475 Mbp, 96% of which is non-coding. Knowledge of DNA content is useful in order to understand the complexity of the Vitis genome and to establish a relationship between the genetic and physical map for map-based cloning.     

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.     

Quantitative nuclear DNA changes inEleusine (Gramineae)

2C nuclear DNA amounts were determined in 30 collections belonging to 10 species ofEleusine. About a 2.5-fold variation in genome size is evident in the genus. The 2C DNA amount in the diploid species ranged from 2.50 pg inE. verticillata to 3.35 pg inE. intermedia. In contrast, the tetraploid species showed a range from 4.95 pg inE. africana to 6.13 pg inE. floccifolia. At intraspecific level 10 collections ofE. coracana, 6 ofE. indica, 4 ofE. africana, 2 ofE. tristachya, and 2 ofE. kigeziensis did not show any significant variation. However, 2 collections ofE. floccifolia, connected with polyploidy, displayed about 90% variation. Polyploid species showed approximately double the genome size of that of their corresponding diploids. An evolutionary increase in DNA amount is evident inE. coracana during the course of its origin and domestication fromE. africana.     

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

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

Changes in DNA-methylation during zygotic embryogenesis in interspecific hybrids of beans (<Emphasis Type="Italic">Phaseolus</Emphasis> ssp.)

Hybrid embryos resulting from crosses between Phaseolus species often fail to reach maturity and some combinations frequently abort at early developmental stages. The genetic or molecular basis for these consistent developmental defects is at present not clear. However, an extremely complex genetic system, thought to be caused by major epigenetic changes associated with gene expression changes, has been shown to be active in plant species. We have investigated DNA methylation in two interspecific hybrids, Phaseolus vulgaris × Phaseolus coccineus and its reciprocal crosses, using methylation sensitive amplification polymorphism (MSAP). The potential use of MSAP for detecting methylation variation during embryogenesis in interspecific hybrids is discussed. Significant differences in the DNA methylation patterns were observed in abortive (interspecific hybrids) and non abortive (parental) genotypes. Taken together, our results strongly suggest that generalized alterations in DNA methylation profiles could play a causative role in early interspecific embryo abortion in vivo. A considerable change in the methylation pattern during embryogenesis could be involved in the disruption of the regulation or maintenance of the embryogenesis process of Phaseolus interspecific hybrids. The results also support the earlier hypothesis that DNA methylation is critical for the regulation of plant embryogenesis and gene expression.     

Genome size and polyploidy variation in the tropical hardwood genusTerminalia (Combretaceae)

Chromosome complements and 2C DNA amounts of six species ofTerminalia have been studied.Terminalia oliveri, T. myriocarpa andT. arjuna are diploid (2n = 24),T. chebula andT. bellirica are tetraploid (2n = 48),T. muelleri shows a triploid number (2n = 36). Two well demarcated groups of species are recognizable on the basis of chromosome length and 2C DNA values which range from 3.60 pg (T. oliveri) to 12.80 pg (T. bellirica) showing a 3.5-fold difference. Differences of DNA per basic genome or per chromosome are greatest (1.97-fold) betweenT. oliveri andT. arjuna. Two species groups (1)T. oliveri andT. chebula, and (2)T. myriocarpa, T. arjuna, T. muelleri, T. bellirica, therefore are well differentiated by DNA per basic genome, irrespective of polyploidy. The mean values of the two groups are 1.81 pg and 3.34 pg, respectively, showing a 1.84-fold difference. Within diploids and tetraploids there is 1.97-fold and 1.76-fold variation, respectively.     

Light-dependent modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in the genus Phaseolus

Modulation of the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in low light and darkness was measured in A) 25 genotypes from the four cultivated species of Phaseolus (P. vulgaris, P. acutifolius, P. lunatus and P. coccineus), B) 8 non-cultivated Phaseolus species, and C) the related species Macroptileum atropurpureum. The activity ratio of Rubisco (the ratio of initial and total Rubisco activities, which reflects Rubisco carbamylation), and the molar activity of fully-activated Rubisco (which primarily reflects the inhibition of Rubisco activity by carboxyarabinitol 1-phosphate, CA1P) were assayed in leaves from the cultivated species sampled at midday in full sunlight, in low light at dusk (60 to 100 mol photons m^-2s^-1), and after at least 4 h in darkness. Dark inhibition of Rubisco molar activity was compared in both cultivated and non-cultivated species. In all cultivated genotypes, a significant reduction of the activity ratio of Rubisco was measured in leaves sampled at low light; however, the molar activity of fully activated Rubisco was not greatly reduced in these low light samples. In darkened leaves, molar activities substantially declined in most Phaseolus species with 11 of 13 exhibiting greater than 60% reduction. In P. vulgaris, the reduction of molar activity was extensive (greater than 69%) in all genotypes studied, which included wild progenitors as well as ancient and advanced cultivars. These results indicate that at low light late in the day, modulation of Rubisco activity is primarily through changes in carbamylation state, with CA1P playing a more limited role. By contrast in the dark, binding of CA1P dominates the modulation of Rubisco activity in Phaseolus in a pattern that appears to be conserved within a species, but can vary significantly between species within a genus. The degree of CA1P inhibition in Phaseolus was associated with phylogenetic affinities within the genus, as the species with extensive dark-inhibition of Rubisco activity tended to be more closely related to each other than to species with reduced inhibition of Rubisco activity.Abbreviations CA1P carboxyarabinitol 1-phosphate - CABP carboxyarabinitol bisphosphate - PFD photon flux density between 400 and 700 nm - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase     

Genome size in gymnosperms

The DNA 2C and per chromosome values of 57 species belonging to 22 genera of gymnosperms have been analysed. The overall range is 12-fold with a modal value of about 30.0 pg.Cycadales exhibit a 2-fold difference. AmongConiferales with a 4-fold variation, thePinaceae have higher mean DNA contents as well as a greater range and diversity than other families. Remarkable interspecific differences are found inCycas, Picea, Larix, Pinus, Callitris, Cupressus, andChamaecyparis. Despite this, there is a constancy of basikaryotypes within these genera.Gnetum shows a distinctly low DNA value.     

Quantitative DNA variation between and within chromosome complements of Vigna species (Fabaceae)

Cytogenetical investigations, so far, on the organisation and evolution of the genomes of Vigna species have proved difficult due to small chromosome size, large chromosome number and uniformity in chromosome shape and size within and between the complements. In this investigation the nature and extent of DNA variation between thirteen diploid and one polyploid species have been estimated. The DNA variation between diploid species was small and species clustered around a mean value of 2.7 pg. The polyploid species had a greater DNA value of 4.95 pg. No significant variation in 2C DNA content was found between accessions of V. radiata. A comparison of the distribution of DNA among the chromosomes within complements has shown that the excess DNA acquired in evolution was distributed evenly in all chromosomes despite significant differences in chromosome size. The relative changes in chromatin area and DNA density which accompany evolutionary DNA variation was also compared.     

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.     

Taxonomic implications of genome size for all species of the genus Gasteria Duval (Aloaceae)

Nuclear DNA content (2C) is used as a new criterion to investigate all species of the genus Gasteria Duval including the three recently described species Gasteria polita van Jaarsv., G. pendulifolia van Jaarsv. and G. glauca van Jaarsv.. The 122 accessions investigated have the same chromosome number (2n=2x=14), with exception of three tetraploid plants found. The nuclear DNA content of the diploids, as measured by flow cytometry with Propidium Iodide, is demonstrated to range from 32.8–43.2 pg. This implies that the largest genome contains roughly 10¹⁰ more base pairs than the smallest. Based on DNA content the species could be divided in five groups: G. rawlinsonii Oberm. with 32.8 pg, 13 mostly inland species with 34.3–36.0 pg, five coastal species with 36.5–39.0 pg and Gasteria batesiana Rowley with 43.2 pg. The thirteen species with 34.3–36.0 pg could be divided further, in a group of eight species occupying mainly very restricted areas with 34.3–35.1 pg and a second group of five species with 35.2–36.0 pg mainly occupying large areas. These five groups did not coincide very well with the two sections and four series of Gasteria based on a cladistic analysis by van Jaarsveld et al. (1994). Based on its long leafy branches, location in the centre of Gasteria species distribution and its by far lowest DNA content, G. rawlinsonii might be the most primitive member of the genus. Nuclear DNA content as measured by flow cytometry is shown to be relevant to provide additional information on the relationships between Gasteria species.     

Conifer genome sizes of 172 species,covering 64 of 67 genera,range from 8 to 72 picogram

Nuclear genome size of conifers as measured by flow cytometry with propidium iodide was investigated, striving to collect at least a single species from each genus. 64 out of 67 genera and 172 species were measured. Of the 67 genera, 21 are reported here for the first time and the same is true for 76 species. This nearly doubles the number of measured genera and adds 50% to the number of analyzed species. Conifers have chromosome numbers in the range of n = (7)10–12(19). However, the nuclear DNA content (2C‐value) is shown here to range from 8.3 to 71.6 picogram. The largest genome contains roughly 6 × 10¹⁰ more base pairs than the smallest genome. Genome sizes are evaluated and compared with available taxonomic treatments. For the mainly (sub)tropical Podocarpaceae small genome sizes were found with a 2C‐value of only 8–28 pg, with 13.5 pg on average. For the Taxaceae 2C‐values from 23–60 pg were determined. Not surprisingly, the genus Pinus with 97 species (39 species measured here) has a broad range with 2C = 38–72 pg. A factor of 2 difference is also found in the Cupressaceae (136 species) with nuclear DNA contents in the range 18–35 pg. Apart from the allohexaploid Sequoia, ploidy plays a role only in Juniperus and some new polyploids are found. The data on genome size support conclusions on phylogenetic relationships obtained by DNA sequencing. Flow cytometry is applicable even to young plants or seeds for the monitoring of trade in endangered species.     

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.     

Reference standards for determination of DNA content of plant nuclei

Flow cytometry was used to compare 14 potential reference standards for plant DNA content determination. Both chicken and plant internal standards were used, as were propidium iodide (PI) and 4'-6-diamidino-2-phenylindole (DAPI) as fluorochromes. Means and standard errors of the means are presented for the 14 potential reference standards, and the means are compared to those obtained by Feulgen densitometry. Five species are recommended as an initial set of international standards for future plant DNA content determinations: Sorghum bicolor cv. Pioneer 8695 (2C = 1.74 pg), Pisum sativum cv. Minerva Maple (2C = 9.56 pg), Hordeum vulgare cv. Sultan (2C = 11.12 pg), Vicia faba (2C = 26.66 pg), and Allium cepa cv. Ailsa Craig (2C = 33.55 pg). It is recommended that the reference standard of choice be one with 2C and 4C nuclear DNA content peaks similar to, but not overlapping, the 2C and 4C peaks of the target species. We recommend PI as the fluorochrome of choice for flow cytometric determination of plant DNA content. DAPI should be used only if the estimated DNA value is corroborated by using a second stain that has no bias for AT- or GC-rich sequences within genomes.     

FLOW-CYTOMETRIC ANALYSES OF NUCLEAR DNA CONTENT IN FOUR FAMILIES OF NEOTROPICAL BATS

Flow-cytometric analyses of 29 species of microchiropteran bats representing four families and 20 genera revealed that bats possess only 79% (5.43 pg) of the DNA content of a “typical” mammal (e.g., Mus musculus strain C57BL; 7 pg). Chiroptera, the second largest order of mammals, is thus an exception to the prevailing view that mammals possess a minimum nuclear DNA content of 7 pg. Limitations on cell size resulting from a high metabolic rate may have constrained evolution of DNA content and could explain why the extensive heterochromatic additions that are common in some groups of mammals are absent in bats. Chromosomes of bats have been well studied; detailed chromosomal banding data are available for nearly all the species used in this investigation. However, no significant correlations were found between DNA content and karyotypic characteristics such as 2n, fundamental number, and rate or pattern of chromosomal evolution.     

Flow cytometric analysis reveals different nuclear DNA contents in cultivated Fonio (Digitaria spp.) and some wild relatives from West-Africa

Nuclear DNA amounts of 118 cultivated fonio accessions representing 94 landraces collected from the major growing areas of West-Africa (Benin, Burkina Faso, Guinea, Mali and Togo) and eight accessions of four wild relatives were investigated by Laser flow cytometry. In cultivated species, average 2C-values ranged from 1.848 ± 0.031 pg for Digitaria iburua to 1.956 ± 0.004 pg for D. exilis. In D. exilis landraces the chromosome number was determined at 2n = 36. The closely related wild species D. longiflora and D. ternata showed similar 2C DNA contents of 1.869 ± 0.035 pg and 1.775 ± 0.070 pg, respectively. Distinctly larger genomes were identified for more distant species D. lecardii and D. ciliaris with 2.660 ± 0.070 pg and 2.576 ± 0.030 pg per 2C nucleus, respectively. Intra-specific variations were found to be slight and insignificant, suggesting genome size stability mainly within the cultivated gene pool. These results support the distance of cultivated fonio species D. exilis and D. iburua from D. lecardii and D. ciliaris as well as their close relationships with D. longiflora and D. ternata. Relevance of the results for ploidy level considerations in fonio millets is discussed.     

Karyology and nuclear DNA quantification of four species ofChaetomorpha (Cladophorales,Chlorophyta) from the western Atlantic

Chromosome numbers are given for four species ofChaetomorpha from the warm temperate and tropical western Atlantic. The basic chromosome number is six, with three median and three submedian chromosomes.Chaetomorpha species represent a polyploid series, with numbers of 12, 18 and 24 found in the present study. Microspectrophotometry data for each species were quantified by reference to standards with known DNA contents. Results indicate similar 2X =1C=12 genome sizes forC. aerea (0.20 pg) andC. brachygona (0.26 pg), and forC. antennina (0.53 pg) andC. melagonium (0.58 pg). These findings are compared with karyological features ofCladophora species to characterize the karyology of the cladophoralean genome.     

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.     

Quantitative karyology of some species ofLuzula

The dimensions of metaphase chromosomes and nuclear DNA contents were measured in eight species ofLuzula. The 2 C DNA contents ranged from 8.51 pg inL. purpurea to 0.55 pg inL. pilosa. Total chromosome volume shows a linear relationship with DNA content; however, the total chromosome length of the complement of the different species is approximately constant. Nucleolar volume and the number of chromocentres in the different species also show a relationship with DNA content. Taken together, these data suggest that while chromosome fragmentation could have generated the present-day range of chromosome numbers in the genus, there have also been changes in the total quantity of DNA with the result that species with similar chromosome numbers have different DNA contents. The relationships of DNA content with chromosome volume inLuzula and other genera are compared and the differences discussed.