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CaM, the major calcium target protein, is a heat-stable, acid, ubiquitous, eukaryotic protein and has been proposed to play a central role in calcium-mediated processes in plants (Poovaiah and Reddy 1993). The involvement of CaM genes in plant signal transduction has been established from experiments, which showed that the expression of CaM genes is regulated by environmental stimuli, including light and auxin, wind and touch, and heat shock (Lu et al. 1996). Ca2+-ATPase is an important CaM binding protein, and is also responsible for the synthesis of important element of signal

transduction chain. There are close relationships between CaM and Ca-2+-ATPase in their structures and functions. The positions of CaM and Ca-2+-ATPase genes have been identified in the high density molecular genetic maps (Causse et al. 1994; Kurata et al. 1994), but the physical locations of these two genes on rice chromosomes remain unknown. Considerable variation can exist between genetic and physical maps (Gustafson and Dill e 1992). Nonradioactive in situ hybridization (ISH), a powerful tool for the molecular cytogenetics, can be used to physically map repetitive, low-copy, and unique DNA sequences in plant chromosomes. This study was designed to physically map these two genes on the chromosomes and compare the relationship between their genetic and physical locations.

The test cultivar, Oryza sativa L. cv. Guang-lu-ai 4, was obtained from the Department of Genetics and Breeding, China National Rice Research Institute. The cDNA C419 (D15295) which was used as probe of CaM gene, was isolated by Sasaki et al. (1994) and supplied by Dr. Nagamura (Rice Genome Research Program, NIAR/STAFF Institute, Tsukuba, Japan), cloned in plasmid pBluescript II SK+, The cDNA SSU304 (D21274) which was used as probe of Ca-2+-ATPase gene, was isolated by Umeda et al. (1994) and supplied by Dr. Nakamura (Iwate Biotechnology Research Center, Kitakami, Japan), and cloned in plasmid pBluescript II SK. These two cDNA clones were 0.8 and 0.3 kb in size respectively. The plasmids containing these cDNA sequences were amplified in E. Coli. DH5 alpha. Chromosome preparations were made with the protoplast technique developed by Dill 6 et al. (1990) and modified by Song and

A total of 550 late-prophase or early-metaphase spreads was analyzed and 34 labeled mitotic cells were detected (6.18%) for two test probes. This ratio of detection was close to previous reports (Gustafson and Dill e 1992; Song and Gustafson 1995). The average arm ratios and standard deviations of detected chromosome for probes C419 and SSU304 were 1.79 ± 0.06 and 1.91 ± 0.08, respectively. All of the hybridization signals were found on only one chromatid of each detected chromosome. CaM gene was located at the end of long arm on chromosome 5, and Ca-2+-ATPase gene located at the part near the centromere of short arm of chromosome 5 (Fig. la and b).

Causse et al. (1994) mapped two RFLP clones CALa and CALb of CaM gene separately to linkage group 5 and 7. Kurata et al. (1994) constructed the high resolution genetic map including the cDNA clone (C419) of CaM gene tested in this study, and located it near the end of chromosome 5 at a distance of 102.2 cM (total distance 123.7 cM). In the molecular maps developed by Tanksley et al. (1992) and Causse et al. (1994), the genetic distance between CaM and RG344 was about 22.5 cM. RG344 was physically mapped near the end of long arm of chromosome 5 by Gustafson and Dill e (1992) using in situ hybridization technique. Thus, our results are consistent with the data reported before to a certain extent.

ATPase gene was closely linked to marker RG344 and RG119 near the end of linkage group 5 in the genetic map (Ahn and Tanksley 1993; Causse et al. 1994; Tanksley et al. 1992). Our results showed that Ca-2+-ATPase gene was physically mapped on the short arm close to the centromere of chromosome 5. These results indicate that there are large differences between the genetic map and physical map (Gustafson and Dill e 1992; Song and Gustafson 1995).

Both CaM and Ca^-ATPase genes are unique sequences as small as 0.8 and 0.3 Kb, respectively. Their localization on rice chromosomes by in situ hybridization is the first report in the world. Although the frequency of signal detection was very low for the ISH of single or low copy DNA probe, the HCI treated root-tip protoplast preparation technique which we adopted compensated for the deficiencies of plant chromosome ISH, such as low mitotic indices, hiding of cytoplasmic debris. Using this technique, large quantities of high quality metaphase cells with well dispersed chromosomes could be obtained and gave us more opportunities to find the detection sites.

It has been reported that there are considerable variations between genetic and physical maps (Linde-Laursen 1979; Baum and Appels 1991; Heslop-Hamson 1991: Gustafson and Dill e 1992). Therefore, the large discrepancy between the results of genetic and physical mapping of Ca^-ATPase gene is not unusual. One of the factors

which may contribute to the variations or discrepancies probably are the differences in the experimental materials adopted for genetic and physical mapping because it is inevitable that the structural variations of chromosomes, such as inversion and translocation, within species, especially between species took place during evolution. The rice genetic map reported by Causse et al. (1994) was constructed based on the population derived from cross 0. saliva (BS125, indica) x 0. longistaminata (WL02), and our results were obtained from 0. sativa L. cv. Guang-lu-ai 4 (indica). Gustafson and Dill 6 (1992) also observed the large discrepancy between the results of genetic and physical mapping of RFLP marker RG207 on chromosome 5 with 0. sativa L. cv. IR36. They physically mapped RG207 near one end of chromosome 5 in the genetic map onto the midway across the centromere. However, the causes for the discrepancies are rather complicated. Thus, more detail and confirmed explanations remain to be further exploited.

Both CaM and Ca-2+-ATPase genes are important components of signal transduction chain which affects the regulation of gene expressions and developments in plants. They are closely linked in the high density genetic map, but they were localized at different arms on the same chromosome in the physical map. It is worth studying what relationships there are between the distributions and functions of the functionally related genes.

We are grateful to Dr. Y. Nagamura for sending the rice CaM cDNA C419, and to Dr. 1. Nakamura for providing the Ca-2+-ATPase gene cDNA clone SSU304. This work was supported by the grants from China National Natural Science Foundation.

Ahn, S. and S.D. Tanksley, 1993. Comparative linkage maps of the rice and maize genomes. Proc. Nat.