Published in Crop Sci 39:1518-1521 (1999)
© 1999 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
Crop Science 39:1518-1521 (1999)
© 1999 Crop Science Society of America
NOTES
Nuclear dna content of thirteen turfgrass species by flow cytometry
K. Arumuganathan1,a,
S.P. Tallury1,b,
M.L. Fraserc,
A.H. Bruneaub and
R. Qub
a Center for Biotechnology, Univ. of Nebraska, Lincoln, NE 68588 USA
b Dep. of Crop Science, North Carolina State Univ., Raleigh, NC 27695 USA
c Pure Seed Testing, Inc., Rolesville, NC 27571 USA
rongda_qu{at}ncsu.edu
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ABSTRACT
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Knowledge of nuclear DNA content will facilitate future molecular studies of turfgrasses. The objective of this study was to determine genome size of 12 turfgrass species and an interspecific hybrid by means of flow cytometry and to compare genome size of warm and cool-season grasses. The seven species and an interspecific hybrid of warm-season turfgrasses had genome sizes ranging from 0.86 to 1.95 pg/2C, while the genome sizes of five cool-season grasses ranged from 5.65 to 15.59 pg/2C. Ploidy levels of the samples were also determined. The observation of the distinct genome sizes of the warm and cool-season turfgrasses agrees with previous reports regarding genome sizes of tropical and temperate species in certain angiosperm families including Gramineae.
Abbreviations: C, DNA content of the non-replicated haploid chromosome complement
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INTRODUCTION
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NUCLEAR DNA content is a characteristic of a species. Knowledge of genome size of a species is essential for assessing the coverage of a genomic library, estimating the copy number of a gene in the genome, and developing strategies for gene cloning based on genome mapping. Such information also helps in determining the ploidy level of plants, and is relevant to studies of plant physiology, ecology, and evolution (see review by Heslop-Harrison, 1995). Bennett and Smith (1991) and Bennett and Leitch (1995) compiled the information of nuclear DNA content of over 2000 angiosperm species. Most of the DNA estimates in their survey were determined by Feulgen densitometry. In recent years, flow cytometry has become the preferred technique for estimating the nuclear DNA content because of its ease and accuracy (Rayburn et al., 1989; Heslop-Harrison, 1995). Nuclear DNA contents of more than 100 major crop plant species were measured by flow cytometry (Arumuganathan and Earle, 1991a). Flow cytometry estimation of nuclear DNA content has recently been employed in turfgrass species to help determine genetic origins of aberrant progeny from facultative apomictic Kentucky bluegrass (Huff and Bara, 1993), to identify and to separate fine fescue species (Huff and Palazzo, 1998), and to determine ploidy level in buffalograss (Riordan et al., 1998). However, for a majority of turfgrass species, nuclear DNA content has not been reported.
Concomitant with the rapid development of the turfgrass industry in recent years is an accelerated interest in molecular studies and biotechnology of turfgrass species. A compilation of genome sizes of major turfgrasses will provide a useful tool for future molecular studies of these species. Here, we report the nuclear DNA content of 13 turfgrass species estimated by flow cytometry, and the observation that cool season turfgrasses have larger genome than warm season turfgrass species. Because of the variation in ploidy levels in certain turfgrass species, chromosome number of each sample was also determined.
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Materials and Methods
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Healthy leaf blades of 12 turfgrass species and an interspecific hybrid, one variety for each species in most cases, were collected from various field locations in North Carolina during summer of 1997 and 1998 (Table 1)
. For the convenience of compilation, hybrid bermudagrass was treated as a species.
Flow cytometry procedures described by Arumuganathan and Earle (1991b) were used to determine nuclear DNA content. The prepared material was analyzed at the University of Nebraska Flow Cytometry Core Research Facilities with a standard FACscan flow cytometer (Becton Dickinson Immunocytometry System, San Jose, CA) with a 15 mW, 488 nm laser. Leaf samples were kept moist at room temperature for no longer than 3 d before further preparation. Fifty milligrams of healthy leaf tissue was excised and placed on ice in a 60- by 10-mm plastic petri dish. Twenty milligrams of leaf tissue from a DNA standard species were added to the petri dish. The tissue was sliced into thin strips (0.25–0.5 mm wide) in 1 mL of propidium iodide-MgSO4 buffer solution (10 mM MgSO4·7H20, 50 mM KCl, 5 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], pH 8.0) with 1.5 mg mL-1 dithiothreitol, 0.1 mg mL-1 propidium iodide and 0.25% (v/v) Triton X-100. Suspended nuclei were withdrawn using a pipettor, filtered through 30-µm nylon mesh into a 1.5-mL microfuge tube, and centrifuged at 13 000 g for 15 s. The pellet was resuspended in 200 µL propidium iodide-MgSO4 buffer solution with 1.25 mg mL-1 DNase-free RNase (Boehringer Mannheim, Indianapolis, IN). Samples were incubated at 37°C for 15 min before flow cytometric analysis. For each measurement, the propidium iodide fluorescence area signals (FL2-A) from 1000 nuclei were collected and analyzed by CellQuest software (Becton Dickinson Immunocytometry System, San Jose, CA). The mean position of the G0/G1 (nuclei) peak of the sample and the internal standard were determined by CellQuest software. The mean nuclear DNA content of each plant sample, measured in picograms, was based on 1000 scanned nuclei. Standards used for comparison included chicken red blood cells (2.33 pg/2C), diploid barley (Hordeum vulgare L. `Stark', 10.68 pg/2C), hexaploid wheat (Triticum aestivum L. `Chinese Spring', 34.68 pg/2C), and tobacco (Nicotiana tabacum L. `Samsun', 9.07 pg/2C) in various assays. At least four measurements were obtained from each sample, and the mean values and standard deviations are presented. The formula used for converting fluorescence values to DNA content was as follows: nuclear DNA content = [(mean position of sample peak) / (mean position of the peak of standard)] DNA content of the standard.
Cytological investigations were performed on all the species on the same samples used for flow cytometry. For buffalograss, creeping bentgrass, hard fescue, and Kentucky bluegrass, mitotic cells from root tips were examined. For samples of other nine species, meiotic pollen mother cells were observed for chromosome counts. Rapidly growing root tips were collected between 1000 and 1030 h and pretreated in 0.03% 8-hydroxyquinoline at 4°C for 3 h, rinsed in distilled water and fixed in a freshly prepared fixation solution containing absolute ethanol, chloroform, and glacial acetic acid in a ratio of 6:3:1 for 24 h at room temperature. The samples were then stored in 70% (v/v) ethanol at 4°C until further use. For mitotic analysis, root tips were hydrolyzed in 1 M HCl at 60°C for 10 min, rinsed in distilled water and transferred to Feulgen stain for 30 min in dark. Deeply stained root tips were squashed in a drop of acetocarmine stain and cells observed under a light microscope to determine the chromosome number. For meiotic analysis, young inflorescences were collected between 0800 to 0900 h and fixed as mentioned above. Young anthers were squashed in acetocarmine stain and the cells were viewed under a light microscope. At least 10 cells at metaphase or early anaphase were observed from each sample to determine the chromosome number.
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Results and Discussion
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The nuclear DNA contents of the turfgrass species surveyed are presented in Table 2
. The standard deviations of DNA content measurements were lower than 1% in most cases, indicating that the flow cytometry technique was very precise. The chromosome numbers of the samples are presented in Table 2. The observed chromosome numbers agreed with those previously reported (see table for citations).
Data presented in Table 2 indicate that the nuclear DNA content of turfgrass species varies considerably, ranging from 0.86 pg/2C for Japanese lawngrass to 15.59 pg/2C for tall fescue with a difference of approximately 18 fold. It is interesting to note that all warm-season turfgrass species assayed had lower nuclear DNA content than the cool-season grasses. The nuclear genome size of some warm-season turfgrass species, including Japanese lawngrass, African bermudagrass and St. Augustinegrass, are among the smallest in Monocotyledoneae, similar to that of rice (Oryza sativa L.), (Arumuganathan and Earle, 1991a), which is considered a model species for monocot molecular biology studies, partially due to its small genome size. It is notable that Japanese lawngrass is a tetraploid and still has the lowest nuclear DNA content. The genome sizes of bahiagrass, centipedegrass, buffalograss, common bermudagrass, and hybrid bermudagrass, with the latter three samples being triploid or tetraploid, are not much larger. Small genome size indicates a low amount of repetitive DNA in the genome and may facilitate future molecular and genetic studies of these species.
The nuclear DNA content of the cool-season grasses surveyed was larger, with the smallest ones (creeping bentgrass and perennial ryegrass) being approximately three fold higher than the largest ones in warm-season grasses, regardless of ploidy level. The hexaploidy cool-season species, hard fescue and tall fescue, had the highest nuclear DNA content.
In certain angiosperm families, including Gramineae, the tropical species are reported to have smaller genomes than the temperate ones (Bennett, 1976; Levin and Funderburg, 1979). Our data on genome sizes of warm- and cool-season turfgrasses agree with this observation. Levin and Funderburg (1979) indicated that the increase of the genome size in temperate species is mostly due to the increase of repetitive DNA rather than an increase of ploidy level. A similar conclusion can be drawn for the turfgrass species examined in the present study, although ploidy level also accounts substantially for the large genome size of certain cool-season turfgrass species. Levin and Funderburg (1979) also demonstrated that genome size is positively correlated with cell size, DNA synthesis and minimum cell cycle time, duration of meiosis, pollen development, and minimum generation time and therefore aids adptation to a specific environment. Price (1988) concluded that the geographical distributions of DNA content are not random. They represent results from natural selection.
Of the 13 turfgrass species surveyed in this study, nuclear DNA contents of three species determined by a similar approach were reported recently. Our data for buffalograss and hard fescue are in good agreement with those previously reported (Riordan et al., 1998; Huff and Palazzo, 1998). The nuclear DNA content of Kentucky bluegrass reported here is within the wide range of the previously reported ones in this species (Huff and Bara, 1993). As pointed out by these authors, the flow cytometry data accurately reflected the ploidy level in Kentucky bluegrass, which varied because of the facultative apomictic nature of the species.
Nuclear DNA content estimates based on Feulgen densitometry have been reported for bahiagrass, perennial ryegrass, and tall fescue. They were 1.2 pg/2C for bahiagrass (Bennett, 1976), 4.16 pg/2C (Rees et al., 1982), and 9.9 pg/2C (Bennett, 1976) for perennial ryegrass, and 11.66 pg /2C (Seal, 1983) and 17.9 pg/2C (Bennett and Leitch, 1995) for tall fescue. The nuclear DNA content of bahiagrass determined by our study is similar to the previously reported one while the value of perennial ryegrass we obtained is close to the one reported by Rees et al. (1982), but half of what Bennett (1976) reported. In tall fescue, the nuclear DNA content value we determined is different from both reported but closer to the one reported by Bennett and Leitch (1995). Although in many cases the nuclear DNA content estimates determined by flow cytometry agreed with those determined by microdensitometry (Rayburn et al., 1989; Arumuganathan and Earle, 1991a), varied nuclear DNA content values for the same species determined by different methods have been reported (Arumuganathan and Earle, 1991a). In addition to the possible differences of nuclear DNA content among the cultivars and selections used for the assay (Rayburn et al., 1989; Bennett and Leitch, 1995), the sensitivity of the technique, the careful exclusion of the G2 stage nuclei, and the accuracy of the knowledge of the reference DNA standard used in the experiments may also contribute to the observed differences.
It seems that sample age and growing condition are not an important factor in the estimate of turfgrass nuclear DNA content. In this study, we collected samples at different ages growing at either greenhouse or field conditions for tall fescue and perennial ryegrass, two cultivars of each, and the values determined (unpublished) were quite similar to each other.
A turfgrass species often has more than one ploidy level among its cultivars and/or accessions. Nuclear DNA content would vary on the basis of ploidy level within a species. Thus it is very important to obtain the ploidy level information when determining the nuclear DNA content of a sample. On the other hand, once certain correlation has been established, flow cytometry is an easy way to help estimate ploidy level in a particular turgrass species (Riordan et al., 1998).Forbes GW. Burton. 1961
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ACKNOWLEDGMENTS
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The authors are grateful to R. Uriarte, L. Privette, C. Rose-Fricker, and Drs. E. Elsner and W.W. Hanna for providing materials in the experiment. The authors thank Drs. D. Bowman, R. Cooper, and R. Dewey for their critical reading of the manuscript. This work was supported by the start-up funds provided by the North Carolina Agricultural Research Service and the Turfgrass Council of North Carolina to R.Q.
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NOTES
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1 Both authors contributed equally and should be considered co-first authors. 
Received for publication November 18, 1998.
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REFERENCES
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ABSTRACT
INTRODUCTION
