Plantech Research Institute, 1000 Kamoshida-cho, Midori-ku, Yokohama, 227 Japan
The development of transformation systems in rice has enabled us to study the expression and regulation of foreign genes in transformed plants as one model of monocot plants (Toriyama et al. 1988; Shimamoto et al. 1989). It is important to determine how many copies of a single gene were incorporated into one transformed rice plant. However, it appears that information on the size of the rice genome would enable us to estimate the copy number of a single foreign gene in transformed plants, using a Southern hybridization technique. Thus, I estimated the relative DNA content of the metaphase chromosomes of a cultivated rice variety Nipponbare (japonica) and of Arabidopsis thaliana L. Heyhn ecotype Colombia as a control plant using an epifluorescent microfluorometer, and calculated the size of the rice genome based on the relative ratio in the two plants (Table 1).
Table 1 - Relative DNA content, size of haploid genome, and size of metaphase chromosome measured in Oryza sativa and Arabidopsis thaliana =============================================================================== Item of observation Oryza sativa A. thaliana 2n=24 2n=10 =============================================================================== No. of metaphase plates observed 38 21 Relative DNA content 246+/-42 105+/-18 Estimated size (bps) 1.6~2.3 x 108 0.7~1.0 X 108 of haploid genome (1C) (1.8~2.5 x 10-13g) (0.8~1.1 X 10-13g) Chromosome length (um), Average+/- S.D. 1.6+/-0.3 1.6+/-0.2 Range 1.2~2.2 1.3~1.9 Total chromosome length(um) 38.9 16.2 Chromosome area (um2), Average 1.6+/-0.3 1.4+/-0.2 Range 1.2~2.2 1.1~1.9 Tatal chromosome area(um2) 37.6 14.1 ===============================================================================Root-tips of 2-7 day-old seedlings of the rice cultivar Nipponbare and of Arabidopsis thaliana grown in darkness at 25 deg C were used for the observation of chromosomes and estimation of the DNA content. The root-tips were immediately fixed in a solution of ethanol-acetic acid (3 : 1) for more than one h at 4-5 deg C, washed with distilled water 2-3 times and treated with a RNase solution (100 ug/ml) in 0.02 X SSC for one h at 27 deg C. Chromosome preparations were made using an enzyme maceration method (Nishibayashi and Kaeriyama 1986). The chromosomes were stained with ethidium bromide (50 ug/ml) in buffer-N (Terada et al. 1987) overnight at room temperature. The relative DNA content of metaphase chromosomes stained with ethidium bromide were measured with an epifluorescent microfluorometer (Olympus) for the estimation of the size of the rice genome. Karyotype analyses of both plants were made after standard Giemsa staining, using an Image Analyzing System (Mitsubishi Kasei).
The size of the haploid genome of Arabidopsis thaliana was estimated at 7 X 107 bps by reassociation kinetics analysis (Leutwiler et al. 1984). However, recently, the size of the haploid genome of Arabidopsis thaliana was reported to be 1 X 108 bps in the International Conference on Arabidopsis Research (Vienna 1990). On the basis of these data and the present results shown in Table 1, it may be considered that the size of the haploid genome of rice is 1.6-2.3 X 108 bps. This size is somewhat smaller than the 0.78 X 10-12 g/C value (7-.1 X 108 bps) obtained by Feulgen microspectrophotometrical measurement (Iyengar and Sen 1978) and the 1.1- 1.3 X10-12 g/C value (0.9-1.2 X 109 bps) obtained by chemical measurement of cellular DNA content (Nagato et al. 1981) which were previously reported. However, the average value of the chromosome size and chromosome area of rice were similar to those reported for Arabidopsis thaliana, and the ratios of chromosome number, total chromosome length and total chromosome area in rice and Arabidopsis thaliana were 2.4:1, 2.4:1 and 2.7:1, respectively. These ob- servations indicate that the size of the haploid genome of rice is 1-iot three times larger than that of Arabidopsis thaliana.
I thank Dr. K. Okada (National Institute for Basic Biology, Okazaki) for providing seeds of Arabidopsis thaliana.
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