Chromosome Observation Methods in the Genus Fragaria

Chromosome Observation Methods in the Genus Fragaria P. Nathewee Faculty of Agricultural Production Maejo University Chiangmai Thailand T. Yanagib Faculty of Agriculture Kagawa University Kagawa Japan Keywords: strawberry, staining method, karyotype analysis, chromosome number, DAPI Abstract Distinguishing strawberry chromosomes using light microscopy is extremely difficult, not only because of their small size and large number, but also because the current observation methods warrant improvement. To date, karyotype analysis in strawberry plants has not been completely examined. The objective of the present research was to improve the chromosome staining methods, to obtain clear images for counting, and to conduct karyotype analysis. For improved staining methods, the root tips of diploid, F. vesca L. and octoploid F. xananassa Duchesne ex Rozier and F. chiloensis (L.) Miller were collected, pretreated in a 2 mm 8-hydroxyquinoline solution for 1 h at room temperatures (around 23 C), and stored at 4 C for 16 h. Subsequently, they were fixed in a fresh solution of pure alcohol:glacial acetic acid (3:1) for 1 h, hydrolyzed in 1 mol/l HCI solution at 23 C for 1 h, then at 60 C for 11.5 min and stained separately with 1.5% facto-propionic orcein (LPO), 60% acetic acid (AA) solution, and the two-step staining method (60% AA and 1.5% LPO). For 4',6-diamidino-2-phenylindole (DAPI) staining, the root tips were macerated using an enzyme solution for 25 min at 42 C, and soaked in 60% AA for 5 min before staining. Clear digital images of the F. vesca, F. xananassa and F. cltiloensis were obtained using light and fluorescent microscopy. Fluorescent microscopy displayed clear chromosome images at the pro-metaphase in F. vesca, F. xananassa and F. cltiloensis samples. The clearest chromosome images were obtained with the two-step staining method in both F. xananassa and F. chi/oensis. However, clear chromosome images can also be obtained by staining with 1.5% LPO alone. Results of genomic in situ hybridization (GISH) analysis of F. xananassa with F. iinumae, R nipponica and R vesca strongly support a proposed allo-polyploid origin of F. xananassa (2n = 8x = 56). The GISH results also confirmed that both F. iinumae and R vesca have similar genomes to the diploid progenitors of F. xananassa 'Umenoka'. INTRODUCTION To understand the function for each chromosome of strawberry plants, each chromosome. Many attempts have been made to improve the methodology to observe chromosomes of strawberry plants, but in many cases poor quality results were obtained, especially for octoploids (Kafkas et al., 2002; Owen and Miller, 1993). There are many difficulties to clearly observe strawberry chromosomes. The major ones are: 1) strawberry chromosomes at the metaphase stage are small (Iwatsubo and Naruhashi, 1989, 1991; Yamaguchi, 1980), 2) chromosomes occupy a small volume of mitotic cells, which clumpe together (Ichijima, I 926; Kafkas et al., 2002; Owen and Miller, 1993; Yarnell, 1929), and 3) metaphase-stage chromosomes may not easily separate (Yarnell, 1931 a). These characteristics rendered it difficult to obtain well spread, clear images of somatic cells and chromosomes. Therefore, the objective of this paper was to present a method to obtain clear images of well-spread somatic chromosomes in Fragaria. Our second objective was to determine if the genomes off vesca and F iinumae could be diploid contributors to that of the octoploid cultivated strawberries. preeda_n@mju.ac.th byanagi@ag.kagawa-u.ac.jp Proc. 7th Inti. Strawberry Symp. Eds.: Yuntao Zhang and J. Maas Acta Hort. 1049, ISHS 2014 201

MATERIALS AND METHODS Plant Materials Chromosome observation was performed in meiotic cells of young flowers and meristematic cells of the root tips for Fragaria plants. These plants were grown in a greenhouse at the Faculty of Agriculture, Kagawa University, Japan and the US Department of Agriculture, ARS National Clonal Germplasm Repository, Oregon, USA. Pre-Treatment and Fixation For study of mitotic chromosome, both pre-treatment and fixation were carried out using the respective methods reported by lwatsubo and Naruhashi (1991). For the meiotic chromosome behavior study, young flower buds were fixed in Newcomer's fluid at l7 C for 3 h and macerated with the same procedure as for the root tips. Staining Method For separate staining by 60% AA (acetic acid) and 1.5% LPO (!acto-propionic orcein), the single pretreated root tip was expelled onto a glass slide with drops of the respective staining solutions and allowed to stand for several minutes. The root tip was covered with a cover slip. Then the cover slip was gently tapped with a pair of fine forceps to break the root tip into particles. The slide was warmed for a few seconds using a spirit lamp and slightly pressed with the thumb. For the two-step staining method, the slide in which the sample was stained with 60% AA was frozen at -80 C for at least 5 min. Then the cover slip was removed using a razor blade. The specimens were air-dried, stained with the 1.5% LPO for more than 3 min, warmed using a spirit lamp, and pressed with the thumb. For pollen mother cells (PMCs), the anthers were stained and squashed in 1.5% LPO. Chromosome pairing at metaphase I was examined in PMCs. For DAPI staining, the fixed root tips (2-3 mm) of the diploid and octoploid plants were hydrolyzed in the 1 mol/l HCl solution at room temperature for 2 h, and then digested using the enzyme mixture of 4% cellulose Onozuka RS (Yakult Co. Ltd., Tokyo), 0.3% pectolyase Y-23 (Seishin Pharmaceutical Co. Ltd., Tokyo), 2.1% macerozyme Rl 0, and 1 mm ethylene diamine tetra-acetic acid (EDTA) ph 4.2 at 42 C for 25 min. The sample of chromosome preparations and staining method were conducted by the method described by Preeda et al. (2007). Genomic In Situ Hybridization The chromosome preparations were conducted following the DAPI staining. For DNA extraction and probe labeling, total genomic DNA off iinumae, F nipponica and F vesca were extracted from younger leaves using a commercial DNA extraction kit (DNeasy Plant kits; Qiagen Inc.). The DNA of each species was labeled with biotin-16-utp using nick translation according to the manufacturer's recommendation (Invitrogen Corp, Carlsbad, CA). In situ hybridization was performed as described by Ma et al. (1997). Chromosome Observation Chromosomes were observed using a light microscope (BX51, Olympus Corp., Japan) for 60% AA, 1.5% LPO and two-step staining method, and an epifluorescence microscope (BX50; Olympus Optical Co. Ltd.) for DAPI staining method at the 100 x magnifications. Well spread chromosomes at the metaphase stage were photographed and stored using a 3CCD camera (XD500; Olympus Corp., Japan) connected to a personal computer equipped with image filing software (FLVFS-LS; Flovel Co. Ltd., Japan). The images were treated for color contrast and brightness uniformity. 202

RESULTS 60% Acetic Acid Staining The results showed that the cytoplasm of samples stained with 60% AA was clearer, but the chromosomes were pale (Fig. 1). However, the results of this staining method provided a possible observation method. 1.5% Lacto-Propionic Orcein Staining The somatic chromosomes of strawberry plants stained with 1.5% LPO exhibited higher color intensity than that of chromosomes with 60% AA. Clear images of samples at all ploidy levels were obtained in this staining method (Fig. 3). The meiotic chromosome of octoploid F. xananassa 'Umenoka' were very clear. Many good images of the chromosome pairing at metaphase I were also obtained by staining with 1.5% LPO (Fig. 3). Two-Step Staining Method The samples of two-step staining method exhibited clearer chromosome images than those obtained using any other method (Fig. 2). The chromosomes treated with the two-step staining method were rendered in the clean cytoplasm in the cell. They were stained evenly with higher color intensity. DAPI Staining Method and GISH Many good images of the well-spread somatic chromosomes at the pro-metaphase and metaphase stage were obtained from diploid and octoploid plants (Fig. 4a). The GISH signals of genomic DNA of F. iinumae were observed clearly on 35 chromosomes of F. xananassa (Fig. 4d). The GISH results revealed that 12-14 chromosomes of F. xananassa were labeled with corresponding genomic DNA of F. vesca at telomeric regions of short arm chromosomes (Fig. 4b). The genomic DNA of F. nipponica hybridized with the proximal segment at telomeric regions for both sites of meiotic chromosome of F. xananassa (Fig. 4c). DISCUSSION In this study, the method for chromosome observation developed by Iwatsubo and Naruhashi (1991) was adopted to obtain clear images of the somatic cells of strawberry plants for use in counting chromosomes and conducting genome analyses. The 60% AA, 1.5% LPO, two-step-staining and DAPI staining solutions were used to stain chromosome at metaphase and pro-metaphase stages. Any of the methods in this study could be used for counting chromosomes. Particularly, the samples stained with 1.5% LPO and two-step-staining gave good images of chromosomes with clear cytoplasm and high chromosome staining intensity. In addition, although 60% AA could stain the somatic chromosomes of strawberry plants, but the shape of chromosomes were not distinguished properly and chromosomes were pale. However, the results from this study revealed that the samples stained with 60% AA before DAPI or 1.5% LPO staining was effective to clean cytoplasm in the somatic cell even the chromosomes were stained slightly. In addition, high concentration acetic acid was sufficient to destroy chromosomal protein; it made the DNA in the chromosome accessible to staining (Schwarzacher and Heslop-Harrison, 2000; Sugiyama et al., 2004). The results obtained from GISH experiments in which DNA of diploid species of F. iinumae, F nipponica and F vesca were hybridized to the chromosomes of cultivated strawberry, F. xananassa, are in agreement with the result of molecular marker analysis (Potter et al., 2000; Rousseau-Gueutin et al., 2009). Bringhurst (1990) reported the genome structure of the octoploid strawberries was AAA' A'BBB 'B'. Current molecular evidence is consistent with classical predictions that three or more distinct genomes are represented in octoploid strawberries, but it remains a possibility that genome composition may vary within and between octoploid species (Davis et al., 2009; 203

Rousseau-Gueutin et al., 2009). In our study, F. iinumae hybridized onto a maximum of 35 chromosomes in the whole set of 56 chromosomes. To get such a large amount of chromosomes hybridized with genomic DNA of F. iinumae, this may indicate as many as five copies of a genome similar to F. iinumae. Rousseau-Gueutin et al. (2009) indicated that F. iinumae may share a common genome donor as paternal parent with the octoploid strawberries. In addition, the hypothesis for genome donor of the diploid F. vesca to octoploid strawberry proposed by Senanayake and Bringhurst (1967) was markedly supported by the results of GISH. The GISH analysis in this study strongly support the proposed allopolyploid origin of F. xananassa (2n = 8x = 56). The GISH results also confirmed that the genomes of F. iinumae, F. nipponica and F. vesca are likely to be similar to the diploid progenitors of F. xananassa 'Umenoka'. Results of GISH analysis of F. xananassa with the genomic DNA F. iinumae, F. nipponica and F vesca strongly support the proposed allopolyploid origin of F. xananassa (2n = 8x = 56). The GISH results also confirmed that those F. iinumae, F nipponica and F. vesca are diploid progenitors of F. xananassa. In addition, this is the first time to report using GISH technique for clarifying chromosomes constitution of F. xananassa. Literature Cited Darrow, G. 1966. The Strawberry. Holt, Rinehart and Winston, New York, New York, USA. Ichijima, K. 1926. Cytological and genetic studies on Fragaria. Genetics 11 :590-604. Iwatsubo, Y. and Naruhashi, N. 1989. Karyotypes of three species Fragaria (Rosaceae). Cytologia 54:493-497. lwastubo, Y. and Naruhashi, N. 1991. Karyotypes of Fragaria nublicola and F. daltoliana (Rosaceae). Cytologia 56:453-457. Kafkas, E., Paydas, S. and Celiktas, N. 2002. Studies on chromosome counting of strawberry root cells. Acta Hort. 567:235-237. Owen, H.R. and Miller, A.R. 1993. A comparison of staining techniques for somatic chromosome of strawberry. Hort. Sci. 28:115-116. Preeda, N., Yanagi, T., Sone, K., Taketa, S. and Okuda, N. 2007. Chromosome observation method at metaphase and pro-metaphase stages in diploid and octoploid strawberries. Sci. Hort. 114:133-137. Ma, Y., Islam-Faridi, M.N., Crane, C.F., Ji. Y., Stelly, H.D.M., Price, J. and Byrne, D.H. 1997. In situ hybridization of ribosomal DNA to rose chromosomes. J. Hered. 88:158-161 Nathewet, P., Yanagi, T., Iwatsubo, Y., Sone, K., Tamura, T. and Okuda, N. 2009a. Improvement of staining method for observation of mitotic chromosome in octoploid strawberry plants. Sci. Hort. 120:431-435. Potter, D., Luby, J.J. and Harrison, R.E. 2000. Phylogenetic relationships among species of Fragaria (Rosaceae) inferred from non-coding nuclear and chloroplast DNA sequences. Syst. Bot. 25:337-348. Rousseau-Gueutin, M., Gaton, A., Alouche, M.L., Noucl1e, A., Olbricht, K., Staudt, G., Richard, L. and Denoyes-Rothan, B. 2009. Tracking the evolutionary history of polyploid in Fragaria L. (Strawberry): new insights from phylogenetic analyses of low-copy nuclear genes. Mol. Phylogenet. Evol. 51:515-530. Senanayake, Y.D.A. and Bringhurst, R.S. 1967. Origin of Fragaria polyploids. I. cytological analysis. J. Am. Bot. 54:221-228. 204

Figures b Fig. 1. Somatic chromosome of (a) F. vesca and (b) F. xananassa 'Umenoka' stained with 60% acetic acid. a b Fig. 2. Somatic chromosome of (a) F. chiloensis CHI-24-1 and (b) F. xananassa 'Nyoho' stained with two step staining (60% acetic acid + 1.5%!acto-propionic orcein staining). 205

d g Fig. 3. Somatic chromosome at metaphase stage of (a) wild diploid F viridis, (b) tetraploid F orientali, (c) hexaploid F moschata, (d) aneuploid (2n=56-2) F chiloensis subsp. Pacifica, (e) F virginiana subsp. Glauca, (f) decaploid F iturupensis, and (g) meiotic chromosome at metaphase I of cultivated strawberry F. xananassa 'Hokowase'. Fig. 4. (a) Somatic metaphase of cultivated strawberry F. xananassa 'Hokowase' with DAPI. (b) In situ hybridization of total genomic F vesca, (c) F nipponica and (d) F iinumae to somatic and meiotic chromosome at methaphase stage of F. xananassa 'Hokowase'. 206