ABSTRACT: The collection and appropriate storage of tissues will determine the future course of comparative genome biology, and for this reason biodiversity repositories are of great importance to genetics. While many efforts are being taken to ensure that genome sequencing, and even protein or RNA analyses, remain feasible with stored specimens, little attention has yet been paid to the important question of genome size diversity. Genome sizes vary more than 200,000-fold among eukaryotes, and correlate with many important cytological and organismal traits. Nuclear DNA content is also a primary consideration in the choice of future large-scale sequencing projects. Here we outline the case for including genome size quantifications as part of standard repository protocols, and describe some methods by which this can be accomplished efficiently. If implemented, such a program will allow the collection of genome size data on an unprecedented scale, and will permit the utility of tissue repositories to genome biology to be fully realized.

How much DNA? Genome size quantification as animportant application of biodiversity repositories

BACKGROUNDThe genome size (or "C-value") of an organism is defined as the total amount of DNA contained

within a single copy of its chromosomes. Among eukaryotes, genome sizes vary by more than 200,000-fold, and the amount of DNA in the genome bears no relationship to the complexity of the organism or the number of coding genes (Fig. 1). In fact, the vast majority of eukaryotic DNA is non-coding and has no known function (Fig. 2).

T. Ryan Gregory1 and Robert Hanner2

1American Museum of Natural History, New York, New York, USA2 Coriell Institute for Medical Research, Camden, New Jersey, USA

Figure 1. Ranges in haploid genome sizes (“C-values”, in picograms of DNA) displayed by the various groups of organisms that have been studied so far.

The reasons for the presence of such large quantities of non-coding DNA has remained an unsolved puzzle in biology for more than 50 years (known as the “C-value paradox” since the 1970s). Several distinct questions remain regarding the evolution of genome size, including the mechanisms of DNA gain and loss, the effects of non-coding DNA on the organism, and the reasons for the wide variation in genome size among species.

In order to address these questions, it is important to have genome size data from a broad array of species. Recent advances in technology have greatly facilitated the measurement of genome sizes, and will allow a mutually beneficial integration of genome size analysis into the standard protocols of tissue collection and storage.

RELEVANCE OFGENOME SIZE DATA

Genome size data are important from a variety of practical perspectives. Genome size directly impacts cell size (Fig. 3) and cell division rate, which results in correlations between DNA content and body size, metabolic rate, and developmental rate. As such, genome size bears on areas of study as different as physiology, developmental biology, and ecology.

The most important pragmatic consequence of genome size variation is in the area of molecular biology and genomics. Any work involving the assembly of genetic libraries, gene mapping, and other such manipulations of the genome is directly influenced by the quantity of DNA present in the chromosomes. Knowledge of genome size can greatly facilitate studies such as these, which will undoubtedly represent a major use of biodiversity repositories in the future.

Finally, it is notable that all of the current complete genome sequencing models have been chosen in part because of their small (and therefore tractable) genome sizes (Fig. 4). As whole-genome sequencing becomes a more common practice, and as the list of organisms with obvious medical and agricultural importance is exhausted, genome size data will become a major guide for the direction of future comparative genomics research. Again, this is an area in which biodiversity repositories are likely to play a major role, and it is therefore prudent to include a component of genome sizing in tissue collection protocols.

Figure 3. The relationship between genome size and cell size, as shown in photomicrographs of Feulgen-stained erythrocytes taken from various species. Dark areas are the nuclei containing the stained DNA. All cells were photographed at the same magnification (40x). (Species included are as follows: A) Siamese fighting fish, Betta splendens (0.64pg); B) Chicken, Gallus domesticus (1.25pg); C) Rainbow trout, Oncorhynchus mykiss (2.6pg); D) African clawed toad, Xenopus laevis (3.15pg); E) Leopard frog, Rana pipiens (2C = 6.7pg); F) Yellow-spotted salamander, Ambystoma maculatum (30pg); G) Red-spotted newt, Notophthalmus viridescens (40pg);H) Australian lungfish, Neoceratodus forsteri (52pg)).

Figure 2. Summary of the different components of the human genome. Only about 1.5% of the genome consists of protein-coding sequences, whereas about 45% of it is composed of transposable elements of various types.

TECHNIQUESSince the 1950s, most genome size estimates have been carried out by measuring the amount of

stain bound to DNA using either densitometry or fluorescence. In both cases, genome size is calculated by comparing the quantity of stain in an unknown specimen with that in a known standard.

Image analysis densitometry The most common staining method used in densitometry is the Feulgen reaction, in which free

aldehyde groups are generated in the DNA by treatment with strong acid and then stained with Schiff reagent. The amount of stain is proportional to the total DNA content, but because the Feulgen-DNA in the DNA is heterogenous and nuclei vary in size, a single density measurement is insufficient. Instead, it is necessary to take a series of "point densities" (Fig. 5), the sum of which is the "integrated optical density" (IOD).

1pg = 10-12

g = 0.9869 x 109

bp(Roughly, 1pg = 1 billion base pairs)

In image analysis densitometry, an image of the nuclei is captured using a CCD or digital camera connected to a computer, and then individual pixels are counted as point densities (Fig. 6). This allows the instantaneous measurement of all nuclei present in the microscope field (Fig. 7). Image analysis equipment is becoming increasingly cost-effective, is easy to use, and could be integrated into repository labs with minimal effort. Feulgen-stained slides can also be kept indefinitely as part of the specimen collection.

Flow cytometry Fluorometry is the opposite of densitometry, in that it involves the measurement of emitted rather

than absorbed light. In this case, the DNA is stained with a fluorochrome and stimulated to emit light by a laser. This process is generally automated by the use of a "flow cytometer", which can measure several thousand individual nuclei in a relatively short period of time.

Flow cytometry is very versatile in terms of the different tissues it can analyze, and it provides measurements on large numbers of nuclei, but the equipment is also large and expensive. In addition, flow cytometry does not provide permanent specimens in the form of stained slides. Both flow cytometry and image analysis densitometry have benefits and shortcomings, but either method can be used to provide large amounts of genome size data from repository specimens.

Figure 5. Because nuclei vary in size, and because the DNA-stain within them is heterogeneous (A), it is necessary to take a series of individual point densities throughout the entire nucleus (B) and to sum these to give an "integrated optical density". This has traditionally been done by slowly moving the nucleus through a fixed beam of light or by scanning the light beam through the nucleus (C), but with modern image analysis methods the process is much more efficient (see Figs. 6 and 7).

Figure 4. Summary of the eukaryotes whose genome sequences have been published to date. Genome size is – and will continue to be – a major consideration in choosing subjects for sequencing, as shown by the very small genomes of most of these species. (From left to right, beginning with the top row: Brewer's yeast, Saccharomyces cerevisiae (0.008pg), in May 1997; Roundworm, Caenorhabditis elegans (0.09pg), in Dec. 1998; Fruit fly, Drosophila melanogaster (0.18pg), in Mar. 2000; Mustard weed, Arabidopsis thaliana (0.13pg), in Dec. 2000; Human, Homo sapiens (3.5pg), in Feb. 2001; Rice, Oryza sativa (0.47pg) in Apr. 2002; Pufferfish, Takifugu rubripes (0.4pg) in Aug. 2002; Malaria parasite, Plasmodium falciparum (0.025pg) in Oct. 2002; Mosquito, Anopheles gambiae, (0.27pg) in Oct. 2002; Sea squirt, Ciona intestinalis (0.16pg) in Dec. 2002; Mouse, Mus musculus (3.3pg), in Dec. 2002).

Figure 6. Diagram of a generalized image analysis system. Images of Feulgen-stained nuclei are captured using a CCD (charge-coupled device) or digital camera connected to a computer. Each pixel in the image is taken as an individual point density measurement.

Figure 7. Screen capture of an image analysis program. Nuclei are automatically outlined by the software, the image pixels are taken as individual point density measurements, and then integrated optical densities (IODs) are calculated for all nuclei in the image simultaneously and instantly. Other parameters such as nucleus size can also be measured concurrently.

THE ROLE OF BIODIVERSITY REPOSITORIES

Existing specimens Some biodiversity repositories, such as the Ambrose Monell Cryo Collection at the AMNH,

already include tissues from several thousand species, most of which have not been analyzed for genome size. Existing collections therefore represent a major resource for genome sizing projects, since the specimens have already been collected, identified, and catalogued.

Some tissues should be readily amenable to both Feulgen image analysis and flow cytometry; for example, blood and liver from vertebrates, and hemolymph and sperm from insects and other invertebrates (Fig. 8). Other tissues that cannot easily be prepared as monolayers on microscope slides (e.g., muscle, skin) can be used in a flow cytometry protocol provided that enough tissue is available.

Genome size as part of future collections One of the most promising roles for biodiversity

repositories is as a hub for future genome size measurements. Specifically, the preparation of specimens for genome sizing could become a standard part of the collection and accessioning protocol of all new material being deposited in the repository. For example, for Feulgen densitometry this could include preparing blood smears in the field (Fig. 9) or dissecting insect sperm in the lab before freezing the remaining specimen. When dealing with fresh specimens, many tissues can be used for Feulgen densitometry (Fig. 8) and flow cytometry. Specimens that have been flash-frozen in the field should also be suitable.

Flow cytometry can be used with frozen tissue, and slides prepared for image analysis densitometry can be stored indefinitely both before and after staining. In the case of the latter, slides can be prepared as tissues become available but measured en masse at a later date; some Feulgen staining protocols allow up to 300 slides to be processed simultaneously (see Hardie et al. 2002).

Figure 9. A simple technique for preparing undamaged monolayers of vertebrate blood cells. A) A small drop of blood is placed centrally, near one end of the slide. B) A second slide is brought toward the drop at a 45 degree angle to the first slide. The second slide is backed into the drop, causing the blood to fill the space between the two slides. C) The second slide is run gently across the first slide, pulling the blood with it and creating a thin smear. This method is greatly preferred over "pushing" the blood, which can damage the cells. The blood smear is allowed to air-dry, and can be stored long-term both before and after Feulgen staining.

FUTURE DIRECTIONS

Four major objectives are apparent if genome size measurements are to be integrated into biodiversity repository procedures:

1) Determine the effect of different freezing regimes and storage buffers on the suitability of tissues for Feulgen image analysis and flow cytometry.

2) Refine tissue preparation methods to adapt them to existing frozen tissues.

3) Optimize sample preparation techniques for inclusion in future field collection and tissue storage protocols.

4) Target specific groups of organisms of particular interest from a genome size perspective.

Clearly, including genome size measurements as part of standard repository procedures will generate an unprecedented and much-needed expansion of the existing genome size dataset.

Figure 8. Photomicrographs of Feulgen-stained nuclei, showing the diversity of cell types that can be analyzed by the image analysis densitometry method. Because of the differences in staining characteristics of different cells, is important to use a standard of the same cell type for calculating genome size. (Cells shown include: A) Chicken red blood cells; B) Human white blood cells (mammalian red blood cells do not contain nuclei); C) Mouse liver; D) Beetle hemocytes; E) Beetle sperm; F) Earthworm coelomocytes and sperm. Photos were taken at 100x magnification).

ADDITIONAL INFORMATION

Arumuganathan, K. and E.D. Earle (1991). Estimation of nuclear DNA content of plants by flow cytometry. Plant Molecular Biology Reporter 9: 229-241.

Bennett, M.D. and I.J. Leitch (2001). Plant DNA C-values Database. Royal Botanic Gardens, Kew, UK. http://www.rbgkew.org.uk/cval/homepage.html.

Gregory, T.R. (2001a). Animal Genome Size Database. http://www.genomesize.com.

Gregory, T.R. (2001b). Coincidence, coevolution, or causation? DNA content, cell size, and the C-value enigma. Biological Reviews 76: 65-101.

Hardie, D.C., T.R. Gregory, and P.D.N. Hebert (2002). From pixels to picograms: a beginners' guide to genome quantification by Feulgen image analysis densitometry. Journal of Histochemistry and Cytochemistry 50: 735-749.

Murphy, R.W., L.A. Lowcock, C. Smith, I.S. Darevsky, N. Orlov, R.D. MacCulloch, and D.E. Upton (1997). Flow cytometry in biodiversity surveys: methods, utility, and constraints. Amphibia-Reptilia 18: 1-13.

Vilhar, B., J. Greilhuber, J.D. Koce, E.M. Temsch, and M. Dermastia (2001). Plant genome size measurement with DNA image cytometry. Annals of Botany 87: 719-728.

e-mail: rgregory@genomesize.com