Estimation of the Nuclear DNA Content of Strawberries (Fragaria Spp.) Compared with Arabidopsis Thaliana by Using Dual-Step Flow Cytometry
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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 study- ing 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 pro- cedure for DNA content estimation by flow cytometry. It is important to choose appropriate standard material for accurate plant DNA content estima- tion. 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 en- dopolyploidy, 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 nu- clear 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 cy- tometry, 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, fluo- rescent 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.