First nuclear DNA C-values for 28 angiosperm genera

This paper reports first DNA C-values for 28 angiosperm genera. These include first DNA C-values for 25 families, of which 16 are monocots. Overall familial representation is 47.2 % for angiosperms, but is now much higher for monocots (75 %) and basal angiosperms (73.1 %) than for eudicots (38.7 %). Chromosome counts are reported for 22 taxa, including first records for six genera plus seven species. Unrepresented families will become increasingly enriched for monotypic taxa from obscure locations that are harder to access. Thus, completing familial representation for genome size for angiosperms may prove impossible in any short period, and progress towards this goal will become slower.     

First nuclear DNA C-values for 18 eudicot families

BACKGROUND AND AIMS: A key target set at the second Plant Genome Size Workshop, held at the Royal Botanic Gardens, Kew in 2003, was to produce first DNA C-value data for an additional 1 % of angiosperm species, and, within this, to achieve 75 % familial coverage overall (up from approx. 50 %) by 2009. The present study targeted eudicot families for which representation in 2003 (42.5 %) was much lower than monocot (72.8 %) and basal angiosperm (69.0 %) families. METHODS: Flow cytometry or Feulgen microdensitometry were used to estimate nuclear DNA C-values, and chromosome counts were obtained where possible. KEY RESULTS: First nuclear DNA C-values are reported for 20 angiosperm families, including 18 eudicots. This substantially increases familial representation to 55.2 % for angiosperms and 48.5 % for eudicots. CONCLUSIONS: The importance of targeting specific plant families to improve familial nuclear DNA C-value representation is reconfirmed. International collaboration will be increasingly essential to locate and obtain material of unsampled plant families, if the target set by the second Plant Genome Size Workshop is to be met.     

Nuclear DNA C-values Complete Familial Representation in Gymnosperms

The gymnosperms are a monophyletic yet diverse group of woodytrees with approx. 730 extant species in 17 families. A recentsurvey showed that DNA C-values were available for approx. 16%of species, but for only 12 of the 17 families. This paper completesfamilial representation reporting first C-values for the fiveremaining families: Boweniaceae, Stangeriaceae, Welwitschiaceae,Cephalotaxaceae and Sciadopityaceae. C-values for nine Ephedraand two Gnetum species are also reported. C-values are now availablefor 152 (21%) species. Analysis confirms that gymnosperms arecharacterized by larger C-values than angiosperms (modal 1Cof gymnosperms = 15.8 pg compared with 0.6 pg in angiosperms)although the range (1C = 2.25–32.20 pg) is smaller thanthat in angiosperms (1C = 0.05–127.4 pg). Given completefamilial coverage for C-values and increasing consensus in gymnospermphylogeny, the phylogenetic component of C-value variation wasalso investigated by comparing the two datasets. This analysisrevealed that ancestral gymnosperms (represented by cycads and/orGinkgo; mean genome size = 14.71 pg) probably had larger genomes thanancestral angiosperms. Copyright 2001 Annals of Botany Company Gymnosperm DNA amounts, C-values, phylogeny, ancestral genome size, Cycadales, Ginkgo, Gnetales, conifers, Pinaceae     

Nuclear DNA C-values in 30 species double the familial representation in pteridophytes

Nuclear DNA C-values and genome size are important biodiversity characters with fundamental biological significance. Yet C-value data for pteridophytes, a diverse group of vascular plants with approx. 9000 extant species, remain scarce. A recent survey by Bennett and Leitch (2001, Annals of Botany 87: 335-345) found that C-values were reported for only 48 pteridophyte species. To improve phylogenetic representation in this group and to check previously reported estimates, C-values for 30 taxa in 17 families were measured using flow cytometry for all but one species. This technique proved generally applicable, but the ease with which C-value data were generated varied greatly between materials. Comparing the new data with those previously published revealed several large discrepancies. After discounting doubtful data, C-values for 62 pteridophyte species remained acceptable for analysis. The present work has increased the number of such species' C-values by 93 %, and more than doubled the number of families represented (from 10 to 21). Analysis shows that pteridophyte C-values vary approx. 450-fold, from 0-16 pg in Selaginella kraussiana to 72.7 pg in Psilotum nudum var. gasa. Superimposing C-value data onto a robust phylogeny of pteridophytes suggests some possible trends in C-value evolution and highlights areas for future work.     

Distribution of the Dimorphic Hypodermis of Roots in Angiosperm Families

The dimorphic hypodermis is composed of two lands of cells differingin size and staining properties, often alternating in a regularpattern. The roots of 98 of 358 genera examined for the presenceof a dimorphic hypodermis had one and 97 additional genera witha dimorphic hypodermis were compiled from the literature. Thehypodermal type of the root was found to be a fairly constantcharacteristic at the family level. Forty-one angiosperm familieshad members with a distinct dimorphic hypodermis. A dimorphichypodermis was found both in families with many specializedcharacteristics such as the Orchidaceae and in families whichhad many unspecialized characteristics such as the Magnoliaceae. Angiosperms, hypodermis, roots     

Applying DNA C-values to evaluate invasiveness of angiosperms: validity and limitation

DNA C-values might be one of important indices in the evaluation of angiospermous invasiveness. To test this viewpoint, we compared DNA 1C-values and basic genome sizes in 3,676 angiosperms. The results suggest that: (1) the two nuclear values vary greatly among different groups, e.g., significantly higher in herbs than in trees, in monocots than in dicots, in perennials than in non-perennials; (2) the two nuclear values both have significant effects on plant invasiveness, especially in herbs, dicots, monocots, perennials, non-perennials, diploids, polyploids, Compositae, and Poaceae, while not significant in trees and Fabaceae. Similar to weeds, the two values in crops are low, which suggests that crops may commonly hold higher invasive potential; (3) to evaluate the invasiveness of a given species, the information about ploidy level is necessary, and for polyploids, basic genome sizes are more reliable. For the results suggest that the selection favors polyploids in weeds at least partly because of the downsizing of basic genome size along with polyploidization.     

Cytochemistry and C-values: the less-well-known world of nuclear DNA amounts

BACKGROUND: In the plant sciences there are two widely applied technologies for measuring nuclear DNA content: Feulgen absorbance cytophotometry and flow cytometry (FCM). While FCM is, with good reasons, increasingly popular among plant scientists, absorbance-cytophotometric techniques lose ground. This results in a narrowing of the methodological repertoire, which is neither desirable nor beneficial. Both approaches have their advantages, but static cytophotometry seems to pose more instrumental difficulties and material-based problems than FCM, so that Feulgen-based data in the literature are often less reliable than one would expect. SCOPE: The purpose of this article is to present a selective overview of the field of nuclear DNA content measurement, and C-values in particular, with a focus on the technical difficulties imposed by the characteristics of the biological material and with some comments on the photometrical aspects of the work. For over 20 years it has been known that plant polyphenols cause problems in Feulgen DNA cytophotometry, since they act as major staining inhibitors leading to unreliable results. However, little information is available about the chemical classes of plant metabolites capable of DNA staining interference and the mechanisms of their inhibition. Plant slimes are another source of concern. CONCLUSIONS: In FCM research to uncover the effects of secondary metabolites on measurement results has begun only recently. In particular, the analysis of intraspecific genome size variation demands a stringent methodology which accounts for inhibitors. FCM tests for inhibitory effects of endogenous metabolites should become obligatory. The use of dry seeds for harvesting embryo and endosperm nuclei for FCM and Feulgen densitometry may often provide a means of circumventing staining inhibitors. The importance of internal standardization is highlighted. Our goal is a better understanding of phytochemical/cytochemical interactions in plant DNA photometry for the benefit of an ever-growing list of plant genome sizes.     

Nuclear exclusion of Cdc25 is not required for the DNA damage checkpoint in fission yeast

Maintenance of genome integrity requires a checkpoint that restrains mitosis in response to DNA damage [1]. This checkpoint is enforced by Chk1, a protein kinase that targets Cdc25 [2--7]. Phosphorylated Cdc25 associates with 14-3-3 proteins, which appear to occlude a nuclear localization signal (NLS) and thereby inhibit Cdc25 nuclear import [6, 8--14]. Proficient checkpoint arrest is thought to require Cdc25 nuclear exclusion, although definitive evidence for this model is lacking. We have tested this hypothesis in fission yeast. We show that elimination of an NLS in Cdc25 causes Cdc25 nuclear exclusion and a mitotic delay, as predicted by the model. Attachment of an exogenous NLS forces nuclear inclusion of Cdc25 in damaged cells. However, forced nuclear localization of Cdc25 fails to override the damage checkpoint. Thus, nuclear exclusion of Cdc25 is unnecessary for checkpoint enforcement. We propose that direct inhibition of Cdc25 phosphatase activity by Chk1, as demonstrated in vitro with fission yeast and human Chk1 [15, 16], is sufficient for proficient checkpoint regulation of Cdc25 and may be the primary mechanism of checkpoint enforcement in fission yeast.     

Rates of Nucleotide Substitution in Angiosperm Mitochondrial DNA Sequences and Dates of Divergence Between Brassica and Other Angiosperm Lineages

We obtained 16 nucleotide sequences (∼1400 bp each) of the first intron of the mitochondrial (mt) gene for NADH subunit 4 (nad4) from 10 species of Brassicaceae. Using these new sequences and five published sequences from GenBank, we constructed a phylogenetic tree of the Brassicaceae species under study and showed that the rate of nucleotide substitution in the first intron of nad4 is very low, about 0.16–0.23 × 10⁻⁹ substitution per site per year, which is about half of the silent rate in exons of nad4. The ratios of substitution rates in this intron, ITS, and IGS are approximately 1:23:73, where ITS is the nuclear intergenic spacer between 18S and 25S rRNA genes and IGS is the intergenic spacer of 5S rRNA genes. A segment (335 bp) in the first intron of nad4 in Brassicaceae species that is absent in wheat was considered as a nonfunctional sequence and used to estimate the neutral rate (the rate of mutation) in mtDNA to be 0.5–0.7 × 10⁻⁹ substitution per site per year, which is about three times higher than the substitution rate in the rest of the first intron of nad4. We estimated that the dates of divergence are 170–235 million years (Myr) for the monocot–dicot split, 112–156 Myr for the Brassicaceae–Lettuce split, 14.5–20.4 Myr for the Brassica–Arabidopsis split, and 14.5–20.4 Myr for the Arabidopsis–Arabideae split. Received: 14 July 1998 / Accepted: 1 October 1998     

Angiosperm DNA contamination by endophytic fungi: Detection and methods of avoidance

PCR primers with broad applicability are useful in many molecular-based studies; however, their universality can compromise results when DNA contaminants also are amplified. Eighty-one templates ofDahlia (Asteraceae), primarily extracted from native Mexican populations, were tested for the presence of fungal contaminants; out of these, almost 1 in 7 templates (13.6%) was contaminated. In a second survey across 12 angiosperm families using material collected in Illinois, fungal DNA contaminated over 60% of the templates analyzed. Endophytic fungi often are symptomless symbionts living within the above-ground tissues of their angiosperm hosts and are not affected by surface sterilization techniques. Recent studies have revealed their widespread occurrence and broad host range. We also present field strategies for obtaining plant material to reduce the possibility of collecting infected leaves and a simple screening test for detecting fungal DNA in angiosperm templates.     

A Model for DNA Sequence Evolution within Transposable Element Families

A quantitative model is proposed for the expected degree of relationship between copies of a family of transposable elements in a finite population of hosts. Special cases of the model (in which the process of homogenization of element copies either is or is not limited by transposition rate) are presented and illustrated, using data on mobile sequences from different species. It is shown that transposition will be expected, in large populations, to result in only a rather distant relationship between transposable elements at different genomic sites. Possible inadequacies of the model are suggested and quantified.     

Nuclear export signal in CDC25B

CDC25B is a dual-specificity phosphatase that activates CDK1/cyclin B. The nuclear exclusion of CDC25B is controlled by the binding of 14-3-3 to the nuclear export signal (NES) of CDC25B, which was reported to be amino acids H28 to L40 in the N-terminal region of CDC25B. In studying the subcellular localization of CDC25B, we found a functional NES at V52 to L65, the sequence of which is VTTLTQTMHDLAGL, where bold letters are leucine or hydrophobic amino acids frequently seen in an NES. The deletion of this NES sequence caused the mutant protein to locate exclusively in nuclei, while NES-fused GFP was detected in the cytoplasm. Moreover, the introduction of point mutations at some of the critical amino acids impaired cytoplasmic localization. Treatment with leptomycin B, a potent inhibitor of CRM1/exportin1, disrupted the cytoplasmic localization of both Flag-tagged CDC25B and NES-fused GFP. From these results, we concluded that the sequence we found is a bona fide NES of CDC25B.     

Nuclear matrix-associated DNA methylase

Previous procedures for the extraction of DNA methylase (EC 2.1.1.37) from nuclei of mouse ascites cells have involved the use of buffers containing 0.2M NaCl. Whilst such 'soluble' methylase accounts for the bulk (70-80%) of DNA methylase activity a further portion of activity is detectable in a 'bound' form firmly associated with 2 M NaCl-resistant nuclear matrix-like structures. This association, which in part requires continuing DNA replication and protein synthesis, can, however, be disrupted in vitro with high concentrations of ammonium sulphate, and the enzymic properties of the 'bound' form of DNA methylase are similar to those described for the 'soluble' form.     

Comparison of Nonparametric Statistics for Detection of Linkage in Nuclear Families: Single-Marker Evaluation

We have evaluated 23 different statistics, from a total of 10 popular software packages for model-free linkage analysis of nuclear-family data, by applying them to single-marker data simulated under several two-locus disease models. The statistics that we examined fall into two broad categories: (1) those that test directly for increased identity-by-state or identity-by-descent sharing (by use of the programs APM, Genetic Analysis System [GAS] SIBSTATE and SIBDES, SAGE SIBPAL, ERPA, SimIBD, and Genehunter NPL) and (2) those that are based on likelihood-ratio tests and that report LOD scores (by use of the programs Splink, SIBPAIR, Mapmaker/Sibs, ASPEX, and GAS SIBMLS). For each of eight two-locus disease models, we analyzed six data sets; the first three data sets consisted of two-child families with both sibs affected and zero, one, or both parents typed, whereas the other three data sets consisted of four-child families with at least two affected sibs and zero, one, or both parents typed. We report false-positive rates, overall rank by power, and the power for each statistic. We give rough recommendations regarding which programs provide the most powerful tests for linkage, as well as the programs to be avoided under certain conditions. For the likelihood-ratio-based statistics, we examined the effects of various treatments of sibships with multiple affected individuals. Finally, we explored the use of some simple two-of-three composite statistics and found that such tests are of only marginal benefit over the most powerful single statistic.