Estimation of the Nuclear DNA Content of Strawberries (Fragaria Spp.) Compared with Arabidopsis Thaliana by Using Dual-Step Flow Cytometry

C2001 The Japan Mendel Society Cytologia 66: 431-436, 2001

Estimation of the Nuclear DNA Content of Strawberries (Fragaria spp.) Compared with Arabidopsis thaliana by Using Dual-step Flow Cytometry

Yukio Akiyama1,2, Yoshihisa Yamamoto3, Nobuko Ohmido2, Masahiro Ohshima2 and Kiichi Fukui"

1Japan Science and Technology Corporation, Kawaguchi 332-0012, Japan 2Laboratory of Rice Genetic Engineering, Hokuriku National Agricultural Experiment Station, Joetsu 943-0193, Japan 3Hyo90 Prefectural Agricultural Research Institute, Kasai 679-0198, Japan 4Department of Biotechnology, Faculty of Engineering, Graduate School cf Osaka University, Suita 565-0871, Japan

Accepted November 12, 2001

Summary Genus Fragaria consists of several species, ranging from diploid to octoploid, and in- cluding various types of sex determination. The genome size of Fragaria species is not known cor- rectly, mainly because preparation of samples suitable for genome size estimation has been difficult. Moreover, it is difficult to choose a suitable standard material for estimation given the small genome size of Fragaria species. Our experimental data showed that the flower petal is a more suitable tissue for flow cytometry. We developed a new procedure, which prevents contamination of tissue samples with debris, and thus allows more accurate estimation of the DNA content during flow cytometry. Arabidopsis thaliana has been sequenced nearly complete, and the estimated genome size from the sequence data is 125 Mbp. We chose Arabidopsis thaliana as standard material. Using this approach, we successfully estimated the genome size of the diploid Fragaria species to be around 164 Mbp/C . Key words Strawberry, Fragaria species, Flow cytometry, Flow sorting, Genome size, Petal.

Genome size among plants varies widely ranging from 125 Mbp/C in Arabidopsis thaliana (Genome Sequencing Group et al. 2000) to 123 Gbp/C in Fritillaria assyriaca (Bennett and Smith 1976). Genome size represents an important piece of information for genetic-related research. For example, it can be used to estimate the number of clones necessary to construct a representative DNA library for the respective species, or to find the appropriate number of molecular markers, such as AFLP, to construct physical and genetic maps. Moreover, genome size is essential in studying diversity and plant evolution (Lan et al. 2000, Grant et al. 2000, O'Neill and Bancroft 2000). Therefore, several studies were aimed at estimating the DNA content of many plant species in the past (Bennet and Smith 1976, Bennet and Leitch 1997, Bennett et al. 1982, 2000). Flow cytometry is a powerful tool for estimating the DNA content (Arumuganathan and Earle 1991, Martinez et al. 1994, Sakamoto et al. 1998, Ohmido et al. 2000), because the method is high- ly sensitive, rapid, easy and convenient. Flow sorting is commonly used for isolation of either entire cells or individual chromosomes (Dolezel et al. 1994). The method was also effective in purifica- tion of nuclei to analyze nuclear matrix proteins by 2D PAGE (Dynlacht et al. 1996). Strawberry (Fragaria species) is one of the most important fruit crops. Genus Fragaria in- cludes diploid (2n =2x=14), tetraploid (2n =4x=28), hexaploid (2n =6x=42) and octoploid (2n =8x=56) species, with the basic number being 7. The bisexual, dioecious and trioecious behav- iors seem to be closely related with the ploidy level in those species (Ahmadi and Bringhurst 1991). As a consequence, Fragaria species are useful in studying the relationship between evolution and * Corresponding author, e-mail: [email protected] 432 Yukio Akiyama et al. Cytologia 66

polyploidy. Although the DNA content of a few Fragaria species was reported previously (Anto- nius and Ahokas 1996), the DNA content of Fragaria species is largely unknown, mainly because it is difficult to prepare the samples required for the estimation of DNA content. For example, nuclei isolated from cells are very often associated with complex macromolecules, such as polysaccha- rides. Flow sorting of nuclei lacking polysaccharide contamination was considered an effective procedure for DNA content estimation by flow cytometry. It is important to choose appropriate standard material for accurate plant DNA content estimation. Chicken Red Blood Cell (CRBC) used to be standard material, but it is known in recent years that it is not suitable for plant C-values estimation (Johnston et al. 1999, Bennett et al. 2000). The

DNA content is more accurately determined by comparing the sample with a standard with similar DNA content (Johnston et al. 1999). Arabidopsis thaliana, which has small DNA content and endopolyploidy, is considered as appropriate standard for plant DNA content estimation. Here, we describe some advantages of A. thaliana as standard material and a procedure for nuclear sample preparation, which allows subsequent flow-sorting analysis of DNA content in species from genus Fragaria.

Materials and methods

Plant materials

Fragaria vesca (wild strawberry) strain U5, Fragaria ananassa (garden strawberry) cv. Hokowase and cv. Toyonoka were used as plant materials. Arabidopsis thaliana ecotype Columbia was used as a control system for nuclear DNA content estimation. Fragaria species were grown in a greenhouse. A. thaliana was grown in pots, in an incubator set at 21°C. Mature leaves, young leaves, runners and petals of F. ananassa cv. Hokowase were used as starting material for nuclei isolation. Young leaves were used for isolation of nuclei in A. thaliana.

Isolation of the nuclei and flow sorting Nuclei were isolated from each tissue, and their DNA content was estimated using a protocol previously described by Arumuganathan and Earle (1991). Each tissue was placed in plastic Petri dishes with an isolation buffer (50 ƒÊg/ml propidium iodide (PI), 10 mM MgSO4, 50 mM KCl, 5 mM HEPES, pH 8.0, 0.25% Triton X-100, 6 mM dithiothreitol). Then, the tissue was chopped using a sharp razor blade on an ice bath. The cut tissue was centrifuged at 100 G for 2 min after filtration through a 50 ƒÊm nylon mesh. The sediment was re-suspended in isolation buffer with 1.25ƒÊg/ml RNase A, and incubated for 15 min at 37°C. The isolated nuclear sample was incubated for more than 30 min on an ice bath. Then, the sample was analyzed with a flow sorter (Altra, Beckman Coulter, USA), equipped with a water-cooled argon ion laser (Coherent, CA) operating at 488 nm emission wave length. Fluorescence emission was detected using a photomultiplier screened by a band path filter, permitting the passage of light with a wavelength around 610 nm.

Results and discussion

When nuclei from various tissues from F. ananassa cv. Hokowase were subjected to flow cytometry, a peak of fluorescent intensity could not be obtained. Rather, a vast amount of noise ap- peared in all samples, except in the ones originating from petals (Fig. 1). Although the nuclei from petals could be measured, the flourescent signal was unstable. When nuclei from each tissue were examined at the fluorescent microscope, large amount of tissue debris was visible, probably from the cutting step, especially in samples of mature leaves, young leaves and runners. At the same time, in the nuclear fraction of petals, there were many intact cells still present (Fig. 2a). This may indicate that cell junctions in the petal tissue are loose, and tissue chopping does not disrupt the 2001 Nuclear DNA Content of Strawberries 433

petal cells. The unstableness of the flourescent signal in the tissue of cut petals is likely to be the re-

sult of either insufficient fluorescence staining due to cytoplasm interference, or due to re-

maining live cells after chopping. PI does not stain the nuclei of living cells. The tissue from

cut petals was subjected to flow sorting, isolat- ing particles based on their fluorescent intensi-

ty. The isolated fractions were observed by fluorescent microscope, to confirm whether

the nuclei were completely separated (Fig. 2b). The nuclei became naked when subjected Fig. 1. Nuclear DNA content of the leaves of F. ananassa to high pressure of flow sorting, and were col- cv. Hokowase by flow cytometry. lected without cytosol and polysaccharide

residues. We decided to subject these clean, isolated nuclei to flow sorting again, so as to

improve on the instability of the fluorescent signal. As a consequence, all isolated nuclear

fractions from the petals of F. ananassa cv. Hokowase, cv. Toyonoka and F. vesca strain

U5 were flow-sorted for the second time, based on their fluorescent intensity. This was

done by subjecting the initial nuclear fractions to centrifugation, 2 min at 100 g. The sediment

was re-suspended again in isolation buffer with 1.25 ƒÊg/ml RNase A, and incubated for more

than 30 min on an ice bath. Subsequently, fluorescent intensities were measured again by a passing the nuclei through flow cytometry. As a result, the fluorescent intensities measured became stable (Fig. 3). Johnston et al. (1999) indicated that the

DNA content is more accurately determined by comparing the sample with a standard with

similar DNA content. The Fragaria species were thought to have small genome sizes,

since the chromosomes were as small as rice chromosomes (Fukui and Niizeki 1983, Iwa- tsubo and Naruhashi 1989). Recently, A.

thaliana has been sequenced nearly complete. The genome size of A. thaliana is estimated at

125 Mbp from their sequence data (Genome Sequencing Groups et al. 2000), it is consid- b ered to be more accurate. The estimation of Fig. 2. Image of cells and nuclei from petals of F. the DNA content has been carried out by the ananassa cv. Hokowase stained with PI. Arrow heads ratio of the fluorescent intensity of sample show the nuclei of the isolated petal cells. Bar indicates against standard by a simple formula, DNA 10 ,um. a) Cells isolated using the chopping method. b) Sorted nuclei. content =(fluorescent intensity of sample/fluo- 434 Yukio Akiyama et al. Cytologia 66

a b

C d

Fig. 3. Fluorescent intensity of Fragaria species by flow cytometry. a) A. thaliana ecotype Columbia as standard, b) F. vesca strain U5, c) F. ananassa cv. Hokowase, d) F. ananassa cv. Toyonoka.

rescent intensity of standard) XDNA content of standard, was used to estimate the DNA content. However, it is expected that the relation between DNA content and fluorescent intensity is not proportion of one to one. The endopolyploidy is considered an effective way to draw a standard curve between the fluorescent intensity and the DNA content. A. thaliana has also endopolyploidy. Be- cause of these reasons, we chose to use A. thaliana as a standard for estimating the DNA content of Fragaria species. The standard curve, regression line, from A. thaliana shown in Fig. 4. The formula for regression line is DNA amount=1.54 X fluorescent intensity+4.74, correlation coefficient is just 1. This result shows that the relation between DNA content and fluorescent intensity is a direct proportion, but not the proportion of one to one. This result also implies that various kinds of nuclei are necessary to make a standard curve for estimation of DNA content. Furthermore, even if same flow cy- tometer was used, the formula of regression line would easily change by condition of flow cytome- ter. For this reason, internal standard is recommended. Internal standard means measuring standard and sample material simultaneously. This study used external standard due to difficulty in separat- ing sample peak from standard peaks. Therefore, we measured sample immediately after measuring of standard, then we measured standard again to confirm that there is no difference between first and second. As a result, the formula of regression line was almost stable. Table 1 shows the DNA content of the F. vesca strain U5, F. ananassa cv. Hokowase and cv. Toyonoka. They were estimated from fluorescent intensity measured on linear scale and were determined as 164, 562 and 571 Mbp/C, respectively. F. vesca with an AA genome, a diploid species, is considered as one of the ancestors of F. ananassa (Senanayake and Bringhurst 1967). The sex expression of F. vesca is bisexual and hermaphroditic. F. ananassa is octoploid, with an 2001 Nuclear DNA Content of Strawberries 435

AAA'A'BBB'B' genome (Bringhurst 1990). The sex expression of F. ananassa is trioecious. Sex expression seems to correlate well with the ploidy level (Ahmadi and Bringhurst 1991). It is also known that polyploidy plays an important role in evolution (Wendel 2000). There are many polyploid species and most of them have large genome sizes. For example, the nuclear DNA contents of Triticum species range from 4700 to 16700 Mbp/C (Bennett and Smith 1976). The nuclear DNA content of Trillium species ranges from 4300 (Bennett and Smith 1991) to 108000 Mbp/C (Bennett and Smith 1976), whereas that of Gossypium species ranges from 1160 to 3100 Mbp/C (Bennett et al. 1982). A small Fig. 4. The standard curve from A. thaliana. DNA amount =1.536Xfluorescent intensity+4.744, r= 1.0. genome size is one of the essential characteris- tics for a plant model in genome research. For Table 1. Nuclear DNA contents of Fragaria species example A. thaliana and Oryza sativa have determined by flow cytometry 125 Mbp/C and 400 Mbp/C (Ohmido et al. 2000), respectively. Fragaria species also have small genome sizes, as revealed in the present study. Fragaria species may also be considered as appropriate plant candidates for evolution studies, because they show interesting relation- ships between polyploidy and sex differentia- tion.

References

Ahmadi, H. and Bringhurst, R. S. 1991. Genetics of sex expression in Fragaria species. Amer. J. Bot. 78: 504-514. Antonius, K. and Ahokas, H. 1996. Flow cytometric determination of polyploidy level in spontaneous clones of strawberries. Hereditas. 124: 285. Arumuganathan, K. and Earle, E. D. 1991. Nuclear DNA content of some important plant species. Plant Molec. Biol. Rep. 9: 208-218. Bennett, M. D. and Smith, J. B. 1976. Nuclear DNA amounts in angiosperms. Philosophical Transactions Roy. Soc. London B274: 227-274. - and - 1991 . Nuclear DNA amounts in angiosperms. Philosophical Transactions Roy. Soc. London B334: 309-345. -, - and Heslop-Harrison , J. S. 1982. Nuclear DNA amounts in angiosperms. Proc. Roy. Soc. London B216: 179-199. - and Leitch , I. J. 1997. Nuclear DNA amounts in angiosperms-583 new estimates. Ann. Bot. 80: 169-196. - , Bhandol, R. and Leitch, I. J. 2000. Nuclear DNA amounts in angiosperms and modern uses-807 new estimates. Ann. Bot. 86: 859-909. Bringhurst, R. S. 1990. Cytogenetics and evolution in American Fragaria. Hort Science 25: 879-881. Dolezel, J., Lucretti, S. and Schubert, I. 1994. Plant chromosome analysis and sorting by flow cytometry. Crit. Rev. Plant Sci. 13: 275-309. Dynlacht, J. R., Henthorn, J., O'Nan, C., Dunn, S. T. and Story, M. D. 1996. Flow cytometric analysis of nuclear matrix proteins: method and potential application. Cytometry 24: 348-359. Fukui, K. and Niizeki, H. 1983. Chromosome engineering in plants with special reference to somatic reduction with PEP treatment. Bull. Nat. Inst. Agr. Sci. Ser. D 34: 113-185. Genome Sequencing Groups, European Union Chromosome and Sequencing Consortium, European Union Chromosome Sequencing Consortium, The Cold Spring Harbor and Washington University Genome Sequencing Center Consor- tium 2000. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. 408, 6814: 796. Grant, D., Cregan, P. and Shoemaker, R. C. 2000. Genome organization in dicots: Genome duplication in Arabidopsis and 436 Yukio Akiyama et al. Cytologia 66

synteny between soybean and Arabidopsis. Proc. Nat. Acad. Sci. USA 97: 4168-4173. Iwatsubo, Y. and Naruhashi, N. 1989. Karyotypes of Three Species of Fragaria Rosaceae. Cytologia 54: 493-498. Johnston, J. S., Bennett, M. D., Rayburn, A. L., Garbraith, D. W. and Price, H. J. 1999. Reference standards for determination of DNA content of plant nuclei. Amer. J. Bot. 86: 609-613. Lan, T. H., DelMonte, T. A., Reischmann, K. P., Hyman, J., Kowalski, S. P., McFerson, J., Kresovich, S. and Paterson, A. H. 2000. An EST-enriched comparative map of Brassica oleracea and Arabidopsis thaliana. Genome Res. 10: 776-788. Martinez, C. P., Arumuganathan, K., Kikuchi, H. and Earle, E. D. 1994. Nuclear DNA content of ten rice species as determined by flow cytometry. Jap. J. Genet. 69: 513-523. Ohmido, N., Kijima, K., Akiyama, Y., de Jong, J. H. and Fukui, K. 2000. Quantification of total genomic DNA and selected repetitive sequences reveals concurrent changes in different DNA families in indica and japonica rice. Mol. Gen. Genetics 263: 388-394. O'Neill, C. M. and Bancroft, I. 2000. Comparative physical mapping of segments of the genome of Brassica oleracea var. alboglabra that are homoeologous to sequenced regions of chromosomes 4 and 5 of Arabidopsis thaliana. Plant J. 23: 233-243. Sakamoto, K., Akiyama, Y., Fukui, K., Kameda, H. and Satoh, S. 1998. Characterization; Genome sizes and morphology of sex chromosome in Hemp (Cannabis sativa). Cytologia 63: 459-464. Senanayake, Y. D. A. and Bringhurst, R. S. 1967. Origin of Fragaria polyploids. I. Cytological analysis. Amer. J. Bot. 54: 221-228. Wendel, J. F. 2000. Genome evolution in polyploids. Plant Mol. Biol. 42: 225-249.