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Departments of 2Entomology and 6Soil and Crop Sciences, Texas A&M University, College Station, Texas 77843; 3Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK; 4Department of Agronomy, University of Illinois, Urbana, Illinois 61821; and 5Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721
Received for publication June 30, 1998. Accepted for publication October 8, 1998.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: DAPI • DNA content • flow cytometry • propidium iodide • reference standards
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Dhillon, Berlyn, and Miksche, 1977
; Price et al., 1980
). However, a significant source of error affecting estimates of DNA amount in plant nuclei continues to be the lack of proper calibration of target species against reliable standards of known DNA amount. Most of the published DNA values for plants have been calibrated directly or indirectly (Bennett and Smith, 1976
, 1991
; Bennett and Leitch, 1995
) to Allium cepa (2C DNA content = 33.55 pg; Van't Hof, 1965
), but some have been calibrated against chicken erythrocytes or other animal nuclear standards (e.g., Dhillon, Berlyn, and Miksche, 1977
; Arumuganathan and Earle, 1991
; Costich et al., 1993
; Kankanpaa, Mannonen, and Schulman, 1996
; and Keeler, 1992
). Many such animal DNA contents directly or indirectly go back to the chemically determined value of 2.52 pg per erythrocyte nucleus for the chicken (Rasch, Barr, and Rasch, 1971
). In some reports, e.g., Arumuganathan and Earle (1991)
, Kankanpaa, Mannonen, and Schulman (1996)
, and Keeler (1992)
, the DNA content of the chicken used was unknown and an assumed DNA content was used. This may result in significant error in estimated DNA contents, as there is considerable reported variation in DNA content among chicken lines (Bennett and Leitch, 1995
). Although animal nuclei are the standards of choice in animal studies, Price et al. (1980)
suggested that for technical reasons a plant standard is better for estimating the DNA content of plants. To help resolve the problem of calibration of DNA values, Bennett and colleagues (Bennett and Smith, 1976
, 1991
; Bennett and Leitch, 1995
) calibrated a series of plants against Allium cepa cv. Ailsa Craig (33.5 pg) and recommended these as DNA content standards.
Price and Johnston (1996)
considered the choice of a standard as critical for flow cytometric determination of DNA contents, and recommended that the standard(s) used should have DNA values close to, but not overlapping the 2C and 4C peaks of the target species. They further recommended that species from the list of Bennett and Leitch (1995)
undergo further scrutiny to see which are suitable as standards for both flow and Feulgen cytometry. In this paper, we tested the suitability of a set of species, including several from the list of Bennett and Leitch (1995)
, as standards for flow cytometry of plant nuclei. We also recommend the use of propidium iodide as the fluorochrome of choice for flow cytometric determination of plant nuclear DNA contents.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1 to 34 pg. This included several from the list of plants recommended as standards by Bennett and Leitch (1995)
. A complete list of plants and their sources are listed in Table 1. All plants were grown from seed in growth chambers adjusted to a 16/8 h, 28°/20°C day/night cycle.
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and Price and Johnston (1996)
. The chopped leaves were filtered through a 53-µm mesh nylon filter or 60-µm and 15-µm mesh nylon filters, and centrifuged at 1000 x g for 3–5 min at 4°C. The pellet was resuspended in 500 µL stain (50 ppm PI in chopping buffer). The stain solution was replaced at 5 min, after centrifugation, by drawing off the original stain solution and adding 300 µL of new stain solution. The standard (Hordeum vulgare cv. Sultan, and/or chicken erythrocytes) and experimental samples were mixed at this point, stored in a dark refrigerator, and analyzed after 1–2 h by flow cytometry. The flow cytometers were Coulter Elite (Hialeah, FL) models operated at 514 nm and an output of 500 mW (Texas A&M), and at 488 nm and an output of 20 mW (Arizona). Fluorescence emission was detected using a photomultiplier screened by a long pass filter permitting passage of light of wavelength above 615 nm.
Flow cytometry using 4'-6-diamidino-2-phenylindole (DAPI)
Nuclei were isolated from seedlings following procedures of Rayburn et al. (1989
, 1993
). Sorghum bicolor cv. Pioneer 8695, calibrated against Hordeum vulgare cv. Sultan, is routinely used as a standard by ALR and, therefore, was used as the standard for the DAPI-based flow cytometry of this study. Seedlings of the target species and S. bicolor were ground together in a small homogenizer for 30 s in 10 mL of extraction buffer consisting of 1.0 mol/m3 hexelene glycol, 10 mol/m3 Tris (pH = 8.0), 10 mol/m3 Mg2Cl2, and 0.5% Triton X-100. The homogenized tissues were filtered through 250 µm, 53 µm, and 20 µm mesh and centrifuged at 500 x g for 15 min. The pellet was resuspended in 70% ethanol for 10 min, centrifuged at 500 x g for 10 min, and resuspended in 250 µL extraction buffer. DAPI (4 µg) was added and the samples were kept in a dark refrigerator for 1 h prior to analysis. Samples of nuclei were analyzed with a Coulter EPICS 751 flow cytometer cell sorter system. The excitation beam was provided by a 5-W argon-ion laser tuned to a wavelength of 351 nm and operating at 250 mW. Fluorescence emission was detected using a photomultiplier screened by a band pass filter permitting passage of light of wavelength 480 ± 20 nm.
Comparisons of chicken erythrocytes
Chicken (Gallus domesticus L.) red blood cells (CRBCs) were compared from three sources: (1) a White Leghorn from the University of Arizona collected in heparinized tubes and stored several years at -80°C prior to use; (2) an inbred pathogen-free White Leghorn line from the College of Veterinary Medicine, Texas A&M University (TAMU), and (3) a Rhode Island Red line (Biosure CEN Cytometry Control, Product Number 1006, Reese Enterprises, Inc., Grass Valley, California) prepared within 1 wk of use and held at -20°C until preparation. One sample of CRBCs from a TAMU White Leghorn had been stored in heparinized tubes for several years at -80°C, and a second sample from a recent collection from the same populations was kept at -20°C for <1 wk prior to use in these studies. CRBCs from these sources were prepared for flow cytometric analysis individually and in paired combinations (six total), following the detergent and proteolytic enzyme-based technique of Vindelov, Christensen, and Nisson (1983)
.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Although most of the plants possessed little variation in DNA content among repeated runs, the coefficient of variation of the G1 peak was high for some of the cereals, i.e., Oryza sativa, Secale cereale cv. Petkus spring, Triticum turgicum, and T. aestivum. Table 3 presents a similar set of plants for which the DNA contents were determined at the University of Arizona by flow cytometry of PI-stained nuclei using male White Leghorn CRBCs as the internal standard. The plant genome values determined by flow cytometry at the two locations are in close agreement (see Table 6), if a value for the chicken standard at the University of Arizona is taken to be 3.01 pg. This value was obtained following transport of heparinized samples on ice from the University of Arizona to TAMU and involved calibration against recently collected CRBCs from a White Leghorn rooster (2.54 pg) and Tetraodes sp. (1.0 pg) (Table 4). We also compared these to CRBCs from a rooster of the same TAMU population, which had been stored in heparinized tubes at -80°C for several years and to CRBCs from a recent commercial preparation from a Rhode Island Red hen. These had mean fluorescence equivalent to that expected for DNA contents of 2.98 pg and 2.48 pg, respectively (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Choice of standards
Animal nuclei have been used by some investigators for standards in estimating plant DNA by Feulgen microspectrophotometry (Dhillon, Berlyn, and Miksche, 1977
; Berlyn, Berlyn, and Beck, 1986
) and by flow cytometry (Galbraith et al., 1983
; Arumuganathan and Earle, 1991
; also see Bennett and Leitch, 1995
). An advantage of a chicken standard is that a single chicken can supply an easily extracted source of cells for numerous experiments. Dhillon, Berlyn, and Miksche (1977)
and Berlyn, Berlyn, and Beck (1986)
were strong proponents of chicken erythrocytes as standards for plant DNA contents determined by Feulgen microspectrophotometry. However, Price et al. (1980)
recommended plant nuclei for internal standards. This was based in part on differences in the hydrolysis rates of chicken and plant nuclei. Since the peak in the maximum absorbancy of chicken erythrocytes following acid hydrolysis occurs at a much shorter time than that of most plants, chicken and plant nuclei must be hydrolyzed separately and combined on a common slide after hydrolysis (Dhillon, Berlyn, and Miksche, 1977
). Therefore, the chicken does not provide a true internal standard for Feulgen microspectrophotometry. A further disadvantage to using chicken as a standard in both microspectrophotometry and flow cytometry is its low nuclear DNA content relative to many plant species. Since DNA contents are more accurate when the standard and sample have nuclear DNA contents of similar size, the chicken is not preferable for use as a standard for plant species of high DNA content. This problem is exemplified in Table 6, which indicates a divergence in the estimated DNA contents of plants with larger genomes when comparing those determined with chicken (3.01 pg) and H. vulgare (11.12 pg) standards to Feulgen-derived values. Another potential problem with a chicken standard is that mean fluorescence of samples may not be the same among different preparations, even though the DNA content is apparently the same (Table 4)
The reason for the different mean fluorescence values among CRBC samples is not known, but the values at 3 pg have in common that they were collected in heparinized tubes and stored at -80°C for several years prior to use in this study. Heparin can induce swelling of nuclei and depletion of histones from interphase chromatin of CRBCs (Adolph, Cheng, and Laemmli, 1977
) and hence may be expected to increase accessibility of PI. Therefore, the observed differences in fluorescence likely represent differential PI intercalation by CRBCs rather than real differences in DNA content.
Choice of fluorochromes (PI vs. DAPI)
The choice of fluorochromes is primarily determined by the excitation source available. PI is excited by visible light with an absorbancy maximum at 490 nm, while DAPI is excited by UV light at 350 nm. However, the two dyes have quite different stain reactions. PI intercalates between base pairs of double-stranded DNA and RNA with little or no base specificity (Properi, Giangare, and Bottiroli, 1991
), while DAPI is a nonintercalating stain that binds preferentially and in a complex manner to A-T base regions (Godelle et al., 1993
). We show here that PI-based flow cytometry produces results very consistent with those based on Feulgen microspectrophotometry. Moreover, the results were consistent for different laboratories using different excitation wavelengths, different excitation energy, and different internal standards.
With few exceptions, DAPI-based flow cytometry gave values that agreed well with Feulgen and PI results. The largest exception involved DAPI staining of A. cepa nuclei, where the estimated value was almost twice that found for measurements using Feulgen or PI. This major discrepancy was detected using different standards in different laboratories and indicates that DAPI should not be used as the sole basis for DNA content comparisons. Dolezel, Sgorbati, and Lucretti (1992)
detected highly significant differences in plant nuclear DNA contents obtained using DAPI and PI and questioned the reliability of base preference fluorochromes. Therefore any initial genome size report based upon DAPI should be verified using a base ratio independent stain. For UV excitation, ethidium bromide, which is excited at 350 nm, has been successfully used to measure genome size (Baranyi, Greilhuber, and Swiecicki, 1996
).
Recommended plant standards
For flow cytometry it is preferable to select an internal standard with 2C and 4C peaks close to those for the target species, so that large errors are not introduced by relatively small random errors in the estimated mean of the standard itself. Also, differences in nuclear size and amounts of heterochromatin influence light scattering properties of the measured object and therefore the measurement itself. If the standard and target nuclei have similar chromatin/DNA properties, this source of error is minimized. At the same time, the peaks of the standard should not overlap those of the target species. Therefore, a set of standards should be available for use with plants of different C values. After considering agreement between Feulgen and PI determined values, ease of preparation, clean peaks with low coefficients of variation, and representation of a wide range of nuclear DNA contents, the following plants are recommended as excellent DNA content standards for flow cytometry using PI and Feulgen microspectrophotometry: Sorghum bicolor cv. Pioneer 8695 (2C = 1.74 pg); Pisum sativum cv. Minerva Maple (2C = 9.56 pg); Hordeum vulgare cv. Sultan (2C = 11.12 pg); Vicia faba cv. GS011 (2C = 26.66 pg); and Allium cepa cv. Ailsa Craig (2C = 33.55 pg). The above values were obtained by averaging the DNA contents calculated by flow cytometry (PI, barley standard) and Feulgen microspectrophotometry, with the exception of Allium cepa, which is a well-established plant standard, determined by chemical means (Van't Hof, 1965
).
We have found P. sativum cv. Minerva Maple to be an extremely good standard in terms of availability of plant material, ease of preparation, and stability within and among runs. Greilhuber and Ebert (1994)
observed from a sample of 25 acquisitions, including Minerva Maple, representing a large geographic area that nuclear DNA content was essentially identical among the reported P. sativum genotypes. The same conclusion was drawn from subsequent studies involving flow cytometric determination of genome size using ethidium bromide as the fluorochrome (Baranyi and Greilhuber, 1995
, 1996
). However, the 2C DNA content of 8.84 pg for P. sativum reported by Greilhuber and Ebert (1994)
is lower than the 9.73 pg estimated using Feulgen cytometry by Bennett and Smith (1976)
and the 9.39 pg estimated by PI-based flow cytometry reported herein. Murray, Cuellar, and Thompson (1978)
, using the very different technique of reassociation kinetics, estimated the 2C DNA content of P. sativum to be 9.2 pg. Although there are some laboratory to laboratory incongruities, it is very important that a common set of plant calibration standards be agreed upon and used throughout the world. Further internationally coordinated research should lead to the regularization of differences in assumed nuclear DNA amounts of the standards and allow the subsequent adjustments in DNA amounts of target species. It should also lead to the addition of other species to the list of recommended standards.
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Arumuganathan, K., and E. D. Earle. 1991 Estimation of nuclear DNA content of plants by flow cytometry. Plant Molecular Biology Reporter 9: 229–233.
Baranyi, M., and J. Greilhuber. 1995 Flow cytometric analysis of genome size variation in cultivated and wild Pisum sativum (Fabaceae). Plant Systematics and Evolution 194: 231–239.
———, and ———. 1996 Flow cytometric and Feulgen densitometric analysis of genome size variation in Pisum. Theoretical and Applied Genetics 92: 297–307.[CrossRef][ISI]
———, ———, and W. K. Swiecicki. 1996 Genome size in wild Pisum species. Theoretical and Applied Genetics 93:717–721.
Bennett, M.D., and I. Leitch. 1995 Nuclear DNA amounts in angiosperms. Annals of Botany 76: 113–176.
———, and J. B. Smith. 1976 Nuclear DNA amounts in angiosperms. Philosophical Transactions of the Royal Society of London B 274: 227–274.[CrossRef]
——— and ———. 1991 Nuclear DNA amounts in angiosperms. Philosophical Transactions of the Royal Society of London B 334: 309–345.[CrossRef]
Berlyn, G. P., M. K. B. Berlyn, and R. C. Beck. 1986 A comparison of internal standards for plant cytophotometry. Stain Technology 61: 297–302.[ISI][Medline]
Costich, D. E., R. Ortiz, T. R. Meagher, L. P. Bruederle, and N. Vorsa. 1993 Determination of ploidy level and nuclear DNA content in blueberry by flow cytometry. Theoretical and Applied Genetics 86: 1001–1006.[ISI]
Dhillon, S. S., G. P. Berlyn, and J. P. Miksche. 1977 Requirement of an internal standard for microspectrophotometric measurement of DNA. American Journal of Botany 64: 117–121.[CrossRef][ISI]
Dolezel, J., S. Sgorbati, and S. Lucretti. 1992 Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. Physiological Plantarum 85: 625–631.[CrossRef]
Galbraith, D. W., K. R. Harkins, J. M. Maddox, N. M Ayres, D. P. Sharma, and E. Firoozabady. 1983 Rapid flow cytophotometric analysis of the cell cycle in intact plant tissues. Science 220: 1049–1051.
Godelle, B., D. Cartier, D. Marie, S. C. Brown, and S. Siljak-Yakovlev. 1993 Heterochromatin study demonstrating the non-linearity of fluorometry useful for calculating genomic base composition. Cytometry 14: 618–626.[CrossRef][ISI][Medline]
Greilhuber, J., and I. Ebert. 1994 Genome size variation in Pisum sativum. Genome 37: 646–655.
Kankanpaa, J., L. Mannonen, and A. H. Schulman. 1996 The genome sizes of Hordeum species show considerable variation. Genome 39: 730–735.
Keeler, K. H. 1992 Local polyploid variation in the native prairie grass Andropogon gerardii. American Journal of Botany 79: 1229–1232.
Laurie, D. A., and M. D. Bennett. 1985 Nuclear DNA content in the genera Zea and Sorghum: intergeneric, interspecific, and intraspecific variation. Heredity 55: 307–313.[ISI]
Michaelson, M. J., H. J. Price, J. R. Ellison, and J. S. Johnston. 1991 Comparison of plant DNA contents determined by feulgen microspectrophotometry and laser flow cytometry. American Journal of Botany 78: 183- 188.
Murray, M. G., R. E. Cuellar, and W. F. Thompson. 1978 DNA sequence organization in the pea genome. Biochemistry 17: 5781–5790.[CrossRef][Medline]
Price, H. J., K. Bachmann, K. L. Chambers, and J. Riggs. 1980 Detection of intraspecific variation in nuclear DNA content in Microseris douglasii. Botanical Gazette 141: 195–198.[CrossRef]
———, and J. S. Johnston. 1996 Analysis of plant DNA content by feulgen microspectrophotometry and flow cytometry. In P. P. Jauhar [ed.], Methods of genome analysis in plants, 115–132. CRC Press, Boca Raton, FL.
Properi, E., M. C. Giangare, and G. Bottiroli. 1991 Nuclease induced DNA structural changes assessed by flow cytometry with the intercalating dye propidium iodide. Cytometry 12: 323–329.[CrossRef][ISI][Medline]
Rasch, E. M., H. J. Barr, and R. W. Rasch. 1971 The DNA content of sperm of Drosophila melanogaster. Chromosoma 33: 1–18.[CrossRef][ISI][Medline]
Rayburn, A. L., J. A. Auger, E. A. Benzinger, and A. G. Hepburn. 1989 Detection of intraspecific DNA content in Zea mays L. by flow cytometry. Journal of Experimental Botany 40: 1179–1183.
———, D. P. Biradar, D. G. Bullock, and L. M. McMurphy. 1993 Nuclear DNA content in F1 hybrids of maize. Heredity 70: 294–300.[ISI]
Van't Hof, J. 1965 Relationships between mitotic duration, S period duration and the average rate of DNA synthesis in the root meristem cells of several plants. Experimental Cell Research 39: 48–58.
Vindelov, L. L., I. J. Christensen, and N. I. Nisson. 1983 Standardization of high-resolution flow cytometric DNA analysis by the simultaneous use of chicken and trout red blood cells as internal reference standards. Cytometry 3: 328–331.[CrossRef][ISI][Medline]
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