49. Chromosome 4 specific DNA library of rice by microdissection technique

1) Faculty of Agriculture, Kyushu University, Fukuoka, 812 Japan

2) Dept. of Human Genetics, School of Medicine, Nagasaki University, Nagasaki, 852 Japan

Studies to decide the regional relationship between chromosomes and the linkage groups are limited. Gustafson and Dille(1992) established the orientation of 8 RFLP linkage groups on each mitotic and meiotic chromosome using the in situ hybridization technique, and we revealed the orientation of 6 linkage groups on each pachytene chromosome and the inferred ranges of centromeres of RFLP linkage groups by the use of the cytogenetical materials. Hence, the new technique is necessary to locate RFLP marker genes on the proper chromosome arms. We report here the construction of chromosome 4 specific DNA library by the microdissection technique.

Procedure for chromosome preparation was the flame dry method described by Kurata and Omura (1978), in which the root-tip cells of Japonica cultivar "Taichung 65" were spread. The microdissection procedure followed Ohta et al. (1993). The entire chromosome 4 was microdissected with a fine glass needle under the inverted microscope, and transferred into a collection chamber. The dewdrop containing the collected chromosome 4 was covered with paraffin oil, digested with proteinase K. DNA extraction and Sau 3AI digestion followed. DNA ligation to a linker/primer set and PCR were carried out, in which the sequence of the linker was 5'-GATCCATGTC-3', and that of the primer was 5'-CGGGAATTCTGGCTCTGCGACATG-3'. Fluorescence in situ hybridization (FISH) method, described by Hori et al. (1990) with a slight modification, was used to examine whether the PCR product belonged to chromosome 4. To separate the RFLP clones of chromosome 4 from the other chromosome clones, PCR product was dropped onto the nylon membrane and hybridized with RFLP clones, Npb 114, 299 and 313 (chromosome 4), and Npb 164, 297 and 315 (chromosomes 3, 5 and 9, respectively).

The size of the PCR products ranged from about 200 to 500 bp (Fig. 1). Chromosome 4 of rice is easy to identify, as it is submetacentric and is the 4th longest chromosome. The short arm is constituted of heterochromatin only. When probed with the PCR product in FISH analysis, only a pair of chromosome 4, particularly its heterochromatin, was detectable with an intense signal (Fig. 2). Moerover, RFLP clones belonging to chromosome 4 hybridized to the PCR product in Southern analysis (Fig. 3). The results suggest that if the arm specific DNA libraries can be made, the RFLP clones would be quickly sorted into each chromosome arm. Thus, the construction of arm specific DNA library is now underway.

This work has been supported by Fellowships of the Japan Society for the Promotion of Science for Japanese Junior Scientists.

References

Gustafson, J. P. and J. E. Dille, 1992. Chromosome location of Oryza sativa recombination linkage groups. Proc. Natl. Acad. Sci. USA 89: 8646-8650.

Hori, T., E. Takahashi, D. Ayusawa, K. Takeishi, S. Kaneda and T. Seno, 1990. Regional assignment of the human thymidylate synthase (TS) gene to chromosome band 18p11.32 by nonisotopic in situ hybridization. Hum. Genet. 85: 576-580.

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.

Ohta, T., T. Tohman, H. Soejima, Y. Fukushima, T. Nagai, K. Yoshiura, Y. Jinno and N. Niikawa, 1993. The origin of cytologically unidentifiable chromosome abnormalities: six cases ascertained by targeted chromosome-band painting. Human. Genet. 92: 1-5.