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.     

Genome size variation in Pisum sativum.

Pisum sativum L. is one of the plant species where infraspecific genome size variation, up to 1.29-fold between cultivars, has been reported. The present investigation deals with a Feulgen cytophotometric analysis of this phenomenon in 25 wild accessions, landraces, and cultivars of widely different geographic origin. Differences between accessions were maximally 1.054-fold in single experiments but proved to be nonreproducible upon repeated measurements. Seedlings of the same accession often differed significantly, up to 1.056-fold, but values from root and shoot tips in one individual were not significantly correlated, indicating the absence of true genome size variation between plants. Upon calibration against Allium cepa a 1C value of 4.42 pg is estimated for Pisum sativum. Altogether the data suggest that, contrary to the divergence in the literature data and recent reports on DNA content variation, the pea has a stable genome size.     

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.     

Variation in chromosomal DNA associated with the evolution of Arachis species.

The 2C nuclear DNA amounts were determined for 99 accessions, representing 23 Arachis species from 8 of 9 taxonomic sections, and two synthetic amphidiploids. Mean 2C DNA amounts varied by 15.20%, ranging from 10.26 to 11.82 pg, between accessions of Arachis hypogaea (2n = 4x = 40). Nuclear DNA content variation (5.33-5.91 pg) was also detected among Arachis duranensis (2n = 2x = 20) accessions. The intraspecific variation in the two species may have resulted from indirect selection for favourable genome sizes in particular environmental conditions. The accessions belonging to A. hypogaea ssp. hypogaea (mean value 11.27 pg) with longer life cycle had significantly larger mean DNA content than the accessions of A. hypogaea ssp. fastigiata (mean value 10.97 pg). For 20 diploid (2n = 2x = 20) species of the genus, 2C nuclear DNA amounts ranged from approximately 3 to 7 pg. The diploid perennial species of section Arachis have about 12% more DNA than the annual species. Comparisons of DNA amounts show that evolutionary rating is not a reliable guide to DNA amounts in generic sections of the genus; lower DNA values with evolutionary advancement were found in sections Heteranthae and Triseminatae, but the same was not true for sections Arachis and Caulorrhizae. Similarly, there is evidence of significant differences in DNA content between 4 ancient sections (Procumbentes, Erectoides, Rhizomatosae, and Extranervosae) of the genus. The occurrence of genome size plasticity in both A. duranensis and A. hypogaea provides evidence that A. duranensis could be one of the diploid progenitors of A. hypogaea. The DNA content in the two synthetic amphidiploids corresponded to the sum value estimated for parental species. Key words : Arachis species, genome size, Arachis hypogaea, Arachis duranensis, intraspecific variation.     

A survey of Penstemon's genome size

Penstemon is the largest genus in North America with more than 270 reported species. However, little is known about its genome size. This information may be useful in developing hybrids for landscape use and for gaining insight into its current taxonomy. Using flow cytometry, we estimated the genome size of approximately 40% of the genus (115 accessions from 105 different species). Genome sizes for both reported and probable diploids range from P. dissectus 2C = 0.94 pg (1C = 462 Mbp) to P. pachyphyllus var. mucronatus 2C = 1.88 pg (1C = 919 Mbp), and the polyploids range from P. attenuatus var. attenuatus 2C = 2.35 pg (1C = 1148 Mbp) to P. digitalis 2C = 6.45 pg (1C = 3152 Mbp). Chromosome counts were done for ten previously published and four previously unreported Penstemon species (P. dissectus, P. navajoa, P. caespitosus var. desertipicti, and P. ramaleyi). These counts were compiled with all previously published chromosome data and compared with the flow cytometry results. Ploidy within this study ranged from diploid to dodecaploid. These data were compared and contrasted with the current taxonomy of Penstemon and previously published internal transcribed spacer and chloroplast DNA phylogenetic work. Based on genome size and previous studies, reassigning P. montanus to the subgenus Penstemon and P. personatus to the subgenus Dasanthera, would better reflect the phylogeny of the genus. Furthermore, our data concur with previous studies suggesting that the subgenus Habroanthus be included in the subgenus Penstemon. The DNA content of subgenus Penstemon exhibits high plasticity and spans a sixfold increase from the smallest to the largest genome (P. linarioides subsp. sileri and P. digitalis, respectively). Our study found flow cytometry to be useful in species identification and verification.     

Intraspecific variation in genome size in angiosperms: identifying its existence

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

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.     

Genomic relationships between the cultivated peanut (Arachis hypogaea, Leguminosae) and its close relatives revealed by double GISH

Arachis hypogaea is a natural, well-established allotetraploid (AABB) with 2n = 40. However, researchers disagree on the diploid genome donor species and on whether peanut originated by a single or multiple events of polyploidization. Here we provide evidence on the genetic origin of peanut and on the involved wild relatives using double GISH (genomic in situ hybridization). Seven wild diploid species (2n = 20), harboring either the A or B genome, were tested. Of all genomic DNA probe combinations assayed, A. duranensis (A genome) and A. ipaensis (B genome) appeared to be the best candidates for the genome donors because they yielded the most intense and uniform hybridization pattern when tested against the corresponding chromosome subsets of A. hypogaea. A similar GISH pattern was observed for all varieties of the cultigen and also for A. monticola. These results suggest that all presently known subspecies and varieties of A. hypogaea have arisen from a unique allotetraploid plant population, or alternatively, from different allotetraploid populations that originated from the same two diploid species. Furthermore, the bulk of the data demonstrated a close genomic relationship between both tetraploids and strongly supports the hypothesis that A. monticola is the immediate wild antecessor of A. hypogaea.     

Patterns of genome size in the copepoda

Adult somatic nuclear DNA contents are reported for eleven cyclopoid species (Megacyclops latipes, Mesocyclops edax, M. longisetus, M. ruttneri, M. leuckarti, M. woutersi, Macrocyclops albidus, Cyclops strenuus, Acanthocyclops robustus, Diothona oculata, Thermocyclops crassus) and for the harpacticoid Tigriopus californicus and range from 0.50 to 4.1 pg DNA per nucleus. These diploid genome sizes are consistent with previously published values for four Cyclops species (0.28–1.8 pg DNA per nucleus), but are strikingly smaller than those reported for marine calanoids (4.32–24.92 pg DNA per nucleus). We discuss three explanations, none of them exclusive of another, to account for the smaller size and range of cyclopoid genome sizes relative to calanoid genome sizes: (1) higher prevalence of chromatin diminution in the Cyclopoida, (2) phylogenetic structure or older age of the Calanoida relative to Cyclopoida and (3) nucleotypic selection that may influence life history variation and fitness. Measurements of genome size were made on Feulgen stained, somatic cell nuclei, using scanning microdensitometry which is well suited to the sparse and heterogeneous populations of copepod nuclei. The importance of measuring large numbers of nuclei per specimen, possible sources of variation associated with cytophotometric measurements, and appropriate use of internal reference standards and stoichiometry of the Feulgen stained nuclei are discussed.     

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

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

Genome Size 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.     

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 variation in the genus Carthamus (Asteraceae, Cardueae): systematic implications and additive changes during allopolyploidization

BACKGROUND AND AIMS: Plant genome size is an important biological characteristic, with relationships to systematics, ecology and distribution. Currently, there is no information regarding nuclear DNA content for any Carthamus species. In addition to improving the knowledge base, this research focuses on interspecific variation and its implications for the infrageneric classification of this genus. Genome size variation in the process of allopolyploid formation is also addressed. METHODS: Nuclear DNA samples from 34 populations of 16 species of the genus Carthamus were assessed by flow cytometry using propidium iodide. KEY RESULTS: The 2C values ranged from 2.26 pg for C. leucocaulos to 7.46 pg for C. turkestanicus, and monoploid genome size (1Cx-value) ranged from 1.13 pg in C. leucocaulos to 1.53 pg in C. alexandrinus. Mean genome sizes differed significantly, based on sectional classification. Both allopolyploid species (C. creticus and C. turkestanicus) exhibited nuclear DNA contents in accordance with the sum of the putative parental C-values (in one case with a slight reduction, frequent in polyploids), supporting their hybrid origin. CONCLUSIONS: Genome size represents a useful tool in elucidating systematic relationships between closely related species. A considerable reduction in monoploid genome size, possibly due to the hybrid formation, is also reported within these taxa.     

Genome size and karyotype evolution in the slipper orchids (Cypripedioideae: Orchidaceae)

Nuclear DNA contents (4C) were estimated by Feulgen microdensitometry in 27 species of slipper orchids. These data and recent information concerning the molecular systematics of Cypripedioideae allow an interesting re-evaluation of karyotype and genome size variation among slipper orchids in a phylogenetic context. DNA amounts differed 5.7-fold, from 24.4 pg in Phragmipedium longifolium to 138.1 pg in Paphiopedilum wardii. The most derived clades of the conduplicate-leaved slipper orchids have undergone a radical process of genome fragmentation that is most parsimoniously explained by Robertsonian changes involving centric fission. This process seems to have occurred independently of genome size variation. However, it may reflect environmental or selective pressures favoring higher numbers of linkage groups in the karyotype.     

Cytophotometric evidence of variation in genome size of desmognathine salamanders

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.     

Karyotype and genome size variation in Ajuga L. (Ajugoideae–Lamiaceae)

Chromosome number changes and karyotype evolution play an important role in plant genome diversification and eventually in speciation. The genus Ajuga L. (Lamiaceae) has approximately 50 species distributed in temperate to subtropical regions. Four of these species are currently recognized in Korea (A. decumbens Thunb., A. multiflora Bunge, A. nipponensis Makino and A. spectabilis Nakai). Understanding the karyotype evolution in Ajuga has been hampered by the small size of their chromosomes and symmetrical karyotypes. Here we used classic Feulgen staining to establish chromosome numbers and construct karyotypes of the four species of Ajuga recognized in Korea and flow cytometry was used to study their variation in genome. The chromosome number of all investigated plants was 2n = 32. Still, the 2C DNA content ranged from 2.18 pg (A. decumbens) to 4.53 pg (A. multiflora). While the chromosome numbers were the same for all investigated species, the genome size variation could potentially be used as a taxonomic marker.     

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 inAllium: In Quest of Reproducible Data

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

The phylogenetic relationship of possible progenitors of the cultivated peanut

The cultivated peanut (Arachis hypogaea L.) is an allotetraploid composed of A and B genomes. The phylogenetic relationship among the cultivated peanut, wild diploid, and tetraploid species in the section Arachis was studied based on sequence comparison of stearoyl-ACP desaturase and oleoyl-PC desaturase. The topology of the trees for both fatty acid desaturases displayed two clusters; one cluster with A genome diploid species and the other with B genome diploid species. The two homeologous genes obtained for each of the two fatty acid desaturases from the tetraploid species A. hypogaea and A. monticola were separated into the A and B genome clusters, respectively. The gene phylogenetic trees showed that A. hypogaea is more closely related to the diploid species A. duranensis and A. ipaensis than to the wild tetraploid species A. monticola, suggesting that A. monticola is not a progenitor of the cultivated peanut. In addition, for the stearoyl-ACP desaturase, the A. duranensis sequence was identical with one of the sequences of A. hypogaea and the A. ipaensis sequence was identical with the other. These results support the hypothesis that A. duranensis and A. ipaensis are the most likely diploid progenitors of the cultivated tetraploid A. hypogaea.     

Karyotypes, constitutive heterochromatin, and genomic DNA values in the blowfly genera Chrysomya, Lucilia, and Protophormia (Diptera: Calliphoridae).

The karyotypes and C-banding patterns of Chrysomya species C. marginalis, C. phaonis, C. pinguis, C. saffranea, C. megacephala (New Guinean strain), Lucilia sericata, and Protophormia terraenovae are described. All species are amphogenic and have similar chromosome complements (2n = 12), including an XY-XX sex-chromosome pair varying in size and morphology between species. Additionally, the C-banding pattern of the monogenic species Chrysomya albiceps is presented. The DNA contents of these and of further species Chrysomya rufifacies, Chrysomya varipes, and Chrysomya putoria were assessed on mitotic metaphases by Feulgen cytophotometry. The average 2C DNA value of the male genomes ranged from 1.04 pg in C. varipes to 2.31 pg in C. pinguis. The DNA content of metaphase X chromosomes varied from 0.013 pg (= 1.23% of the total genome) in C. varipes to 0.277 pg (12.20%) in L. sericata; that of Y chromosomes ranged from 0.003 pg (0.27%) in C. varipes to 0.104 pg (5.59%) in L. sericata. In most species, the corresponding 5 large chromosome pairs showed similar relative DNA contents. The data suggest that the interspecific DNA differences in most species are mainly due to quantitative variation of (repetitive) sequences lying outside the centromeric heterochromatin blocks of the large chromosomes. The results are also discussed with regard to phylogenetic relationships of some species.