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1)National Institute of Agrobiological Resources, Kannondai, Tsukuba, Ibaraki- ken, 305 Japan
2)Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606 Japan
3)Faculty of Science, Toho University, Funabashi, Chiba-ken, 274 Japan
4)Tropical Agriculture Research Center, Ohwashi, Tsukuba, Ibaraki-ken, 305 Japan
All 12 chromosomes of rice in the somatic prometaphase have been identified by
Kurata and Omura (1978), but it is not always feasible to identify all the
chromosomes objectively in a given nuclear plate. We have been engaged in
characterization of plant chromosomes with the aid of a newly developed
chromosome image analyzing system (CHIAS; Fukui 1985, 1986). In the present
work, the root tips of a rice variety, "Nipponbare" were used. They were
pretreated in 0.1% colchicine dissolved in phosphate buffer (1/15 M, pH6.8),
and after enzymatic maceration (Fukui et al. 1988), were fixed in 1:3 acetic
alcohol for 30 min., smashed, air-dried, and stained in 4% Giemsa solution.
The slides were examined under microscope to search for prometaphase nuclei in which all chromosomes were well scattered without overlapping (Fig. 1). Such nuclear plates obtained were each photographed three time with different exposures. The photographs were printed with a certain magnification, and were put in the digital image-frame memory of the system with a 512 x 512 "pixel matrix".
Fig. 1. A prometaphasic plate of rice chromosomes. Bar shows
5 micra.
Fig. 2. Sequential steps of image analysis for rice chromosome.
Explanations in text.
The over-exposed picture was processed by the CHIAS to divide the image into
white and black portions or to make a binary image, and the border line between
the two portions was drawn so as to show the outline of each chromosome (Fig.
2a). The under-exposed picture was used to adequately represent the pattern of
desity distribution within the border line. The border lines were superimposed
on it to make an overlapped figure (Fig. 2b).
The figure was enlarged two folds, and the density distribution in each chromosome was represented by different colors so as to facilitate visual judgement of the actual density distribution by man (Fig. 2c.). Then, in each chromosome, a transverse line showing the position of centromere and a longitudinal line showing the mid-rib of a chromatid were drawn by hand, and were put in the memory of computer.
Curves showing the profile of density distribution along the mid-rib or the condensation pattern (CP) of respective chromosomes were printed out by the CHIAS (Fig. 2d). This curve is characteristic of each chromosome. Together with the lengths and arm ratios of the chromosomes and other information obtained from the normally explosed picture, the 12 chromosomes can be each identified with certainty. Their relative lengths and arm ratios are given by the CHIAS in each nuclear plate.
Each chromosome exists in duplicate in a somatic complement, but the two homologues are not always both suited for taking measurements, as one of them may sometimes be bent strongly or twisted. Therefore, in the present study, each chromosome was represented by one which was relatively straight.
By the method outlined above, we analyzed 10 prometaphase plates. It was confirmed by the t-test of the total length of 12 chromosoems that they were in a certain stage of prometaphase. The mean measurements of relative length and arm ratio with standard deviations are given in Table 1. The 12 chromosomes each identified are numbered in the table in the descending order of mean relative length.
The characteristics of each chromosome thus obtained were compared with those presented by Kurata and Omura (1978) to examine how our characterization corresponds to theirs designated by K numbers. It was found that K2 and K3, as well as K7 and K9, had to be replaced with each other in order to arrange them in the descending order of relative length.
Table 1. Mean relative length and arm ratio with standard deviations of rice
chromsomes obtained in 10 somatic prometaphase nuclei.
===============================================================================
Chromosome Relative length Arm Ratio Correspondence
Number ===================== =============== to K number by
Mean S.D. % Mean S.D. Kurata & Omura
===============================================================================
1 6.32 0.30 12.64 1.71 0.21 K1
2 5.60 0.48 11.20 1.25 0.08 K3
3 5.04 0.42 10.08 1.60 0.21 K2
4 4.49 0.45 8.98 2.91 0.42 K4
5 4.26 0.31 8.52 1.46 0.20 K5
6 4.14 0.41 8.28 1.12 0.06 K6
7 3.89 0.28 7.78 1.59 0.13 K9
8 3.60 0.17 7.20 1.16 0.08 K8
9 3.34 0.12 6.68 1.24 0.13 K7
10 3.19 0.29 6.38 3.85 0.68 K10
11 3.06 0.11 6.12 1.28 0.17 K11
12 3.06 0.27 6.12 1.93 0.34 K12
===============================================================================
Total 49.99 99.98
===============================================================================
Fukui, K., 1985. Identification of plant chromosome by image analysis method. The Cell (Tokyo) 17: 145-149.
Fukui, K., 1986. Standardization of karyotyping plant chromosomes by a newly developed chromosome image analyzing system (CHIAS). Theor. Appl. Genet. 72: 27-32.
Fukui, K., K. Iijima and K. Kakeda, 1988. Analysis and utility of chromosome information, 21. Karyotype of rice chromosomes. Jpn. J. Breed. 38, Suppl. 1: 474-475. (in Japanese)
Kurata, N. and T. Omura, 1978. Karyotype analysis in rice, 1. A new method for identifying all chromosome pairs. Jpn. J. Genet. 53: 251-255.
