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

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

Genome size variation in Arachis hypogaea and A. monticola re-evaluated.

Genome size variation within species is a frequently reported, but still a controversial problem. In the present study, we re-evaluated recently published Feulgen densitometric data on genome size and its infraspecific variation in Arachis hypogaea, and also conducted measurements in one accession of its wild relative A. monticola. The methods applied were propidium iodide flow cytometry and Feulgen densitometry using Pisum sativum as an internal standard. The 2C DNA contents previously published cannot be confirmed, but values obtained in this study are about half as large. Additionally, we could not reproduce the previously reported 1.15-fold variation within A. hypogaea; our data indicate genome size stability between respective accessions of this species. Based on 8.84 pg (2C) for Pisum sativum the DNA amounts (2C) were: 5.914 pg in A. hypogaea, and 5.979 pg in A. monticola.     

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.     

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).     

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.     

Does genome size in Dasypyrum villosum vary with fruit colour?

Dasypyrum villosum (2n=14), a Mediterranean grass species of the Triticeae, exhibits intraindividual fruit colour polymorphism from pale yellow to almost black. Several studies have reported differences between the plants emerging from pale and dark fruits. They include histone content in root meristem nuclei, cell cycle duration, heterochromatin banding pattern, frequency of a tandemly repeated sequence, and nuclear genome size. In the present study, we examine whether the reports of genome size being up to 1.24-fold larger in seedlings from the lighter caryopses are reproducible. In all, 29 accessions from various countries, totaling 186 plants, were investigated for genome size using flow cytometry with propidium iodide as the DNA stain. Individuals differed 1.12-fold at most and accessions 1.07-fold. The mean genome size (1C-value) was 5.07 pg or 4954 Mbp. Within-accession comparisons of seedlings derived from light and dark caryopses were insignificant (P>0.100). Thus, we found no evidence for a modificatory genome size plasticity in D. villosum. In the light of our data, the previously reported genome size variation, up to 1.66-fold within populations and 1.67-fold between populations, appears unrealistically high. Suboptimal technical procedures for quantitative Feulgen staining are probably responsible for these earlier observations.     

Genome size in Bulgarian Centaurea s.l. (Asteraceae)

Thirty-nine species and subspecies of the genera Centaurea, Colymbada, Psephellus and Cyanus (all included in Centaurea s.l.) including many rare and endemic taxa of preponderantly Bulgarian distribution have been investigated with Feulgen DNA image densitometry for holoploid and monoploid genome size (C- and Cx-values). Cyanus varies gradually 2.17-fold between 0.74 pg and 1.56 pg (1Cx). In the remaining taxa two major genome size groups are found, which differ about 1.8-fold in Cx-value. Low values occur in Centaurea subgenera Acrolophus, Solstitiaria, Phalolepis (0.77 pg to 0.90 pg, 1Cx) and Jacea (0.95 pg to 1.09 pg, 1Cx), high values in the genera Colymbada (1.65 pg to 1.93 pg, 1Cx) and Psephellus (1.79 pg, 1Cx, in P. marschallianus). Cx-values support a distinction of Colymbada from Centaurea. Genome size variation is discussed with regard to phylogeny, life form (annual versus perennial), polyploidy, chromosome basic numbers, altitude of occurrence and climate, endemism, and rarity.     

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 in Chenopodium quinoa (Chenopodiaceae)

The extent and significance of intraspecific genome size variation were analysed in quinoa (Chenopodium quinoa Willd.), a pseudocereal important for human consumption in the Andean region of South America. Flow cytometry, with propidium iodide as the DNA stain, was used to estimate the genome size of 20 quinoa accessions from Ecuador, Peru, Bolivia, Argentina, Chile and the USA. Limited genome size variation was found among the analysed accessions. The differences between the accessions were statistically significant but the maximum inter-accession difference between the populations with the largest and the smallest genome reached only 5.9%. The largest genome was found in population C4 from Chile (mean 3.077 pg/2C) and the smallest in the Peruvian population P2 (mean 2.905 pg/2C). The variation was not correlated with collection site; however, the quinoa accessions analysed in this study belonged to three distinct geographical groups: northern highland, southern highland and lowland.     

Chromosome banding and genome size differentiation inProspero (Hyacinthaceae): Diploids

Prospero is a Mediterranean autumn-flowering genus ofHyacinthaceae commonly classified inScilla asS. autumnalis andS. obtusifolia. Extensive dysploid and polyploid variation has been reported. In the present study 77 diploid accessions from the western to the eastern part of the area of distribution, the major part being from continental Greece and Crete, have been analysed for karyotype structure and, in part, for genome size. Methods employed were acetocarmine staining, Giemsa C-banding, fluorochrome staining mainly with chromomycin A₃/DAPI, silver impregnation, and Feulgen densitometry. Banded idiograms were established with a computer assisted karyotype analysis procedure. Chromosome numbers were 2n = 8 inP. obtusifolium, and 2n = 12 and 14 inP. autumnale s. l. Dispensable euchromatic chromosome segments and different types of B chromosomes occurred. Among the cytotypes with 2n = 14 two karyotypes from Turkey differed from each other and from the rest in form, position of the nucleolar constriction, and in genome size. The remaining accessions were similar in karyotype shape but three levels of genome size could be discerned, the highest (1C = 7.50 pg) being found on the Iberian Peninsula, an intermediate one on Corsica and Malta, and the lowest (4.27 pg) in the Aegean. The karyotype with 2n = 12 had an intermediate genome size, and that ofP. obtusifolium a relatively low one. Heterochromatin amount was generally low, but some karyotypes showed characteristic banding patterns. The relationship between the chromosome complements with 2n = 14, 12 and 8 is discussed on the basis of idiograms and DNA amounts.The authors respectfully dedicate this papers to emer. o. Prof. Dr.Elisabeth Tschermak-Woess on the occasion of her 80th birthday.     

Nuclear DNA content of Pinus sylvestris (L.) as determined by laser flow cytometry

Nuclear DNA content was determined in nuclei isolated from needles, stems and roots of in vitro grown seedlings and from megagametophytes and embryo of mature seeds in three accessions of Pinus sylvestris L. One accession was from Inari, northern Finland at timber line, and two accessions were from the Alpine region in Italy. Nuclei were mechanically isolated by a chopping method, stained with propidium iodide, and DNA content was determined using an EPICS PROFILE laser flow cytometer. Nuclei isolated from leaves of barley (Hordeum vulgare L. cv. Sultan; 2C=11.12 pg) were used as an internal standard for measurement of pine nuclei. Mean 1C nuclear DNA content of P. sylvestris was 27.88 pg as determined from megagametophyte tissue. Mean 2C value was 52.25 pg as determined from stem and root tissue, and 55.58 pg as determined from embryo tissue. The ratio of 2C to 1C value was 1.87 and 1.99, respectively. Extracts of nuclei from needles contained propidium iodide-absorbing debris which may have interfered with measurements and resulted in lower 2C values than those obtained from stem and root.     

Genome size in Dahlia Cav. (Asteraceae–Coreopsideae)

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

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.     

Genome size in Arachis duranensis: a critical study.

Arachis duranensis is a diploid wild relative of the tetraploid cultivated peanut Arachis hypogaea. The literature indicates two 2C genomic DNA mean values (genome size) for A. duranensis, 4.92 and 5.64 pg, and intraspecific variation of up to 11% negatively correlated with altitude above sea level of the collection sites has been reported. Our recent investigations of Arachis species have shown that unrecognized technical problems with peanut material may have influenced previous genome-size data and rendered them open to critical comments. In the present study, 20 accessions of A. duranensis were investigated by means of DNA flow cytometry (propidium iodide staining) and several of these also by Feulgen DNA image analysis. Pisum sativum was used as the internal standard (2C = 8.84 pg). 2C values in A. duranensis were about half those described previously and varied between 2.49 and 2.87 pg (flow cytometry). This variation was statistically significant and reproducible. There was a negative correlation of genome size with latitude and altitude above sea level of the collection sites. Such a correlation had been already found in one of the previous studies. However, the incongruences between the absolute DNA content values obtained in the present investigation and those in the literature point to the importance of carrying out methodological studies on best practice in DNA-content determinations in plants.     

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 and Feulgen densitometric analysis of genome size variation in Pisum

A DAPI and ethidium bromide flow cytometric and Feulgen densitometric analysis of genome size variation in Pisum was conducted. The material included 38 accessions of P. sativum of widely different geographic origin and altogether 14 samples of P. elatius, P. abyssinicum, P. humile and P. fulvum. The relative genome size values obtained with the three staining methods were strongly correlated. No evidence for genome size variation was found among P. sativum cultivars. In particular, certain Italian cultivars, for which strongly deviating C-values have been reported, proved to be invariant. The only occasion when ambiguous evidence for marginal genome size variation was found was when all 38 accessions taxonomically affiliated with P. sativum were considered. Pisum abyssinicum and P. fulvum differed from P. sativum by about 1.066-and 1.070-fold, respectively; 1 accession of P. humile differed by 1.089-fold, and 2 of P. elatius by 1.122- and 1.195-fold, respectively (ethidiumbromide comparison), while the other accessions of these taxa were not different from P. sativum. This variation may indicate taxonomic inhomogeneity and demands further investigation. Cultivated P. sativum has long been suspected of not being constant with respect to genome size. As shown here, these findings were not based on genuine differences, but rather were technical in origin.     

Genetic variation and conservation of Changnienia amoena, an endangered orchid endemic to China

Nuclear DNA content (2C) is used as a new criterion to investigate nearly all species of the genus Nerine Herb. The species have the same chromosome number (2n = 2x = 22), with the exception of three triploid plants found. The nuclear DNA content of the diploids, as measured by flow cytometry with propidium iodide, is demonstrated to range from 18.0–35.3 pg. This implies that the largest genome contains roughly 2 × 10¹⁰ more base pairs than the smallest. The species, arranged according to increasing genome size, fell apart in three groups if growth cycle and leaf width were also considered. A narrow-leafed, evergreen group with a DNA content between 18.0 and 24.6 pg contains thirteen species, a broad-leaved winter growing group with four species has a DNA content from 25.3–26.2 pg and a broad-leafed summer growing group has a DNA content of 26.8–35.3 pg and contains six species. If the presence of filament appendages and hairiness of the pedicels were also considered, the thirteen evergreen species could be further divided into a group without filament appendages or hairy pedicels with a DNA content of 18.0–18.7 pg. A second group without filament appendages but with hairy pedicels had a DNA content of 19.7–22.3 pg. And a third group with both filament appendages and hairy pedicels had a DNA content of 22.0–24.6 pg. The exception is N. marincowitzii that, despite a low DNA content and narrow leaves is summer growing. The broad-leafed group is further characterised by the absence of filament appendages and the absence of strongly hairy pedicels. The exception here is N. pusilla that, despite a high DNA content, has narrow leaves and minutely hairy pedicels. Nuclear DNA content as measured by flow cytometry is shown to be relevant to throw new light on the relationships between Nerine species.     

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.     

Flow cytometric analysis of genome size variation in cultivated and wildPisum sativum (Fabaceae)

Fifteen cultivars, landraces, and wild accessions ofPisum sativum subspecies, and one accession ofP. abyssinicum were analysed with flow cytometry (DAPI staining) usingP. sativum Kleine Rheinländerin as internal standard. Applying the method of jointly isolating, staining, and measuring nuclei of individual seedlings of test and standard material, it was found that in allP. sativum comparisons G 1 and G 2 peaks were invariably unimodal and symmetric at coefficients of variation mostly less than 2%. This is strong evidence for absence of significant genome size variation in theP. sativum strains analysed. These data are markedly at variance to results of other authors reporting considerable genome size variation withinP. sativum. However, inP. abyssinicum flow cytograms and Feulgen densitometric measurements indicate 4–8% more DNA, at same chromosome number (2n = 14), than inP. sativum. This result demonstrates that genome size variation is indeed existent in the genus and requires further examination.     

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.