Johann Greilhuber

Estimation of nuclear DNA content in plants using flow cytometry

Flow cytometry (FCM) using DNA-selective fluorochromes is now the prevailing method for the measurement of nuclear DNA content in plants. Ease of sample preparation and high sample throughput make it generally better suited than other methods such as Feulgen densitometry to estimate genome size, level of generative polyploidy, nuclear replication state and endopolyploidy (polysomaty). Here we present four protocols for sample preparation (suspensions of intact cell nuclei) and describe the analysis of nuclear DNA amounts using FCM. We consider the chemicals and equipment necessary, the measurement process, data analysis, and describe the most frequent problems encountered with plant material such as the interference of secondary metabolites. The purpose and requirement of internal and external standardization are discussed. The importance of using a correct terminology for DNA amounts and genome size is underlined, and its basic principles are explained.

Plant Genome Size Estimation by Flow Cytometry: Inter-laboratory Comparison

Flow cytometry is a convenient and rapid method that has been used extensively for estimation of nuclear genome size in plants. In contrast to general expectations, results obtained in different laboratories showed some striking discrepancies. The aim of this joint experiment was to test the reliability and reproducibility of methods. Care was taken to avoid a bias due to the quantity of DNA in the nucleus, the procedure for nuclei isolation or the type of instrument. Nuclear DNA content was estimated in nine plant species representing a typical range of genome size (2C = approx. 03-30 pg DNA). Each of the four laboratories involved in this study used a different buffer and/or procedure for nuclei isolation. Two laboratories used arc lamp-based instruments while the other two used laser-based instruments. The results obtained after nuclei staining with propidium iodide (a DNA intercalator) agreed well with those obtained using Feulgen densitometry. On the other hand, results obtained after staining with DAPI (binding preferentially to AT-rich regions) did not agree with those obtained using Feulgen densitometry. Small, but statistically significant, differences were found between data obtained with individual instruments. Differences between the same type of instruments were negligible, while larger differences were observed between lamp- and laserbased instruments. Ratios of fluorescence intensity obtained by laser instruments were higher than those obtained by lamp-based cytometers or by Feulgen densitometry. The results obtained in this study demonstrate that flow cytometry with DNA intercalators is a reliable method for estimation of nuclear genome size in plants. However, the study confirmed an urgent need for an agreement on standards. Given the small but systematic differences between different types of flow cytometers, analysis of very small differences in genome size should be made in the same laboratory and using the same instrument.

Flow Cytometry with Plant Cells

Overview 1 1.1 Origins 1 1.2 From Absorption to Fluorescence, from Imaging to Flow 2 1.2.1 Early Microspectrophotometry and Image Cytometry 3 1.2.2 Fluorescence Microscopy and the Fluorescent Antibody Technique 3 1.2.3 Computers Meet Cytometers: The Birth of Analytical Flow Cytometry 4 1.2.4 The Development of Cell Sorting 7 1.3 The Growth of Multiparameter Flow Cytometry 8 1.4 Bench-tops and Behemoths: Convergent Evolution 11 1.5 Image Cytometry: New Beginnings? 14 References 15

C-banded karyotypes in theScilla hohenackeri group,S. persica, andPuschkinia (Liliaceae)

Quantitatively evaluated C-banding karyograms and further observations on karyotype structure are presented forScilla persica, 7 species of theScilla hohenackeri group, andPuschkinia scilloides. Within theS. hohenackeri group very diverse and species-specific banding patterns are found. On the basis of karyology,S. bisotunensis andS. furseorum should be closely grouped together, whileS. persica andPuschkinia scilloides appear quite isolated.

Characterization of heterochromatin in different species of the Scilla siberica group (Liliaceae) by in situ hybridization of satellite DNAs and fluorochrome banding

Satellite DNAs have been isolated from the monocotyledonous plants Scilla siberica, S. amoena, S. ingridae (all are highly GC-rich), and S. mischtschenkoana by using the Ag+ −Cs2SO4 density centrifugation technique. Hybridization in situ has been performed with 3H-cRNA to these satellite DNAs in all four species. In each species, the endogenous satellite DNA is located mainly in intercalary and major heterochromatin bands associated with terminal regions and nucleolar organizer regions (NORs) but not in centromeric regions. Patterns observed after cross-species hybridization show a high degree of satellite DNA homology between S. siberica, S. amoena, and S. ingridae. By contrast, satellite DNA of S. mischtschenkoana consists largely of different, non homologous DNA sequences, with two exceptions: (i) the NORs of all four species contain similar satellite sequences, and (ii) a strong homology exists between the satellite DNA of S. mischtschenkoana and centromeric DNA of S. siberica but not with those of S. amoena and S. ingridae. — Heterochromatin has also been characterized by the AT-specific fluorochromes quinacrine (Q) and DAPI and the GC-specific agent chromomycin A3 (CMA3), in combination with two counterstaining techniques. While CMA3-fluorescence is largely in agreement with data on base composition and location of the specific satellite DNAs, the results with Q and DAPI are conflicting. Prolonged fixation has been found to change the fluorescence character in certain instances, indicating that other factors than the base sequence of the DNA also play a role in fluorochrome staining of chromosomes. The results are discussed in relation to the taxonomy and phylogeny of the four species.

Self-tanning — a new and important source of stoichiometric error in cytophotometric determination of nuclear DNA content in plants*

A Feulgen-densitometric comparison of nuclear DNA contents (C-values) was performed in various plant species (a fern, four gymnosperms, 16 woody and herbaceous angiosperms) after two types of fixation, additive (neutral formaldehyde) and non-additive (methanol-acetic acid, 3:1, MAA). Nuclei from tissues containing a significant amount of polyphenols (of the hydrolysable and non-hydrolysable tannin type) always showed reduced stainability and distorted spectral absorbance curves after MAA-fixation, while after formaldehyde-fixation no evidence for distorted staining was found. No fixation-dependent differences in Feulgen-DNA contents were stated in nuclei from tissues having no polyphenols. Distorted Feulgen-staining is a consequence of cellular self-tanning during fixation. Tanning is impaired by formaldehyde which binds to tannins and inactivates them. The rationale for using formaldehyde as a fixative in Feulgen-cytophotometry can be mainly seen in its capability of eliminating the “self-tanning error”. Standardization in plant DNA cytophotometry, and recent reports on unorthodox nuclear DNA variation in conifers are critically discussed.

Genome size and maturity group in Glycine max (soybean)

Recent literature data indicate a 1.15-fold difference in genome size between certain cultivars of soybean and a positive correlation of genome size and maturity group. The present analysis aims at a reinvestigation of these cultivars using DAPI and ethidium bromide flow cytometry and Feulgen densitometry. No reproducible genome size differences were found between these cultivars with either technique, and correlation with maturity group was not confirmed. The previously claimed statistical significance of such a correlation was found to result from only one exceedingly low DNA value of an early maturing cultivar, which, according to our data, is not different from the others.

Patterns, sources and ecological implications of clonal diversity in apomictic Ranunculus carpaticola (Ranunculus auricomus complex, Ranunculaceae)

Sources and implications of genetic diversity in agamic complexes are still under debate. Population studies (amplified fragment length polymorphisms, microsatellites) and karyological methods (Feulgen DNA image densitometry and flow cytometry) were employed for characterization of genetic diversity and ploidy levels of 10 populations of Ranunculus carpaticola in central Slovakia. Whereas two diploid populations showed high levels of genetic diversity, as expected for sexual reproduction, eight populations are hexaploid and harbour lower degrees of genotypic variation, but maintain high levels of heterozygosity at many loci, as is typical for apomicts. Polyploid populations consist either of a single AFLP genotype or of one dominant and a few deviating genotypes. genotype/genodive and character incompatibility analyses suggest that genotypic variation within apomictic populations is caused by mutations, but in one population probably also by recombination. This local facultative sexuality may have a great impact on regional genotypic diversity. Two microsatellite loci discriminated genotypes separated by the accumulation of few mutations (‘clone mates’) within each AFLP clone. Genetic diversity is partitioned mainly among apomictic populations and is not geographically structured, which may be due to facultative sexuality and/or multiple colonizations of sites by different clones. Habitat differentiation and a tendency to inhabit artificial meadows is more pronounced in apomictic than in sexual populations. We hypothesize that maintenance of genetic diversity and superior colonizing abilities of apomicts in temporally and spatially heterogeneous environments are important for their distributional success.

Nuclear genome size: Are we getting closer?

Correct information on genome size is important in many areas of research. For a long time, scientists have been struggling to understand the reason for the huge variation in eukaryotic genome size and its biological significance. More recently, the knowledge on genome size has become important to structure genome sequencing projects as their scale and cost depend on genome size. Despite the fact that the first estimates of genome size in eukaryotes were made more than 50 years ago, we are still not quite sure about the exact genome size in practically all animal and plant species. Moreover, different estimates continue to be published for the same species. These discrepancies compromise data comparison and interpretation and point to methodological problems, which include standardization. This article assesses the current state of DNA reference standards for flow cytometry and the issues related to their calibration.

Quantitative analyses of C-banded karyotypes, and systematics in the cultivated species of theScilla siberica group (Liliaceae)

Quantified C-band karyograms are presented for the related speciesScilla siberica, S. mordakiae, S. ingridae, S. amoena, andS. mischtschenkoana. Chromosome structure, banding style, and heterochromatin characters suggest a systematic grouping of two more closely related species pairs:S. siberica andS. mordakiae, S. ingridae andS. amoena; they are part of a larger aggregate, well separated fromS. mischtschenkoana. Four different heterochromatin fractions can be recognized inS. siberica and its relatives, but only two inS. mischtschenkoana. C-bands do not “replace”, but they are added to euchromatin. The particular origin and history of the cultivatedS. amoena and the triploidS. siberica cv. “Spring Beauty” appear to be responsible for their karyotype constancy, but chromosome conservatism obviously is genuine inS. mischtschenkoana. The banding data support the systematic grouping proposed on a morphological basis, and provide additional evolutionary evidence.