58. Chromosomal localization of rice rbcS genes

2) Section of Biochemistry, Molecular and Cell Biology, Cornell University, Wing Hall, Ithaca, New York, 14853, USA

In assigning locations of genes to chromosomes, in situ hybridization would be one of the best ways, as was shown in cases of human (Harper and Saunders 1981) and Drosophila melanogaster (Spierer et al. 1983). However, this method has not been used for higher plants. Rice genes have been traditionally assigned to their linkage groups based on genetic recombination results. The relationships between rice linkage groups and their corresponding chromosomes have not yet been unambiguously established for all chromosomes. We believe that the use of in situ hybridization would be faster and more exact. This communication reports our preliminary results in testing the feasibility of this method for the chromosomal localization of rice genes.

Two genes coding for the rice ribulose bisphosphate carboxylase small subunit (rbcS) have been cloned and sequenced. The coding sequence shared over 95% DNA sequence homology in a 230 base pair segment which has been sequenced. One of the cloned genes, pOSrbcS-1, was labelled with all four tritiated deoxynucleotides by nick translation. The pOSrbcS-1 probe, with a specific activity of around 2.5X10⁸ cpm/ug, was then used to hybridize with the prometaphase chromosomes of an Indica rice cultivar IR 26 by in situ hybridization (Harper and Saunders 1981). After hybridization, the chromosome preparation was coated with Kodak NTB-2 emulsion and exposed for 10 days. The autoradiographs showed that two to three chromosomes out of the twelve were labelled with silver grain. Mostly, the nucleolar organizer of chromosome 8 (Fig. 1) was involved. Other chromosomes involved might be 2 and 10. Genomic blot experiments using the coding sequence of pOSrbcS-1 as probe gave two or three hybridizing bands. Thus, this result and that of the chromosomal in situ hybridization are consistent. More cells, however, should be examined before a definite conclusion can be made. The preliminary results described here convinced us that chromosomes prepared by the flame dried method (Kurata 1978) can be successfully labeled by in situ hybrization and that genes can be assigned to specific chromosomes. In the future, we plan to use the 3'- noncoding regions of pOSrbcS-1 and pOSrbcS-2, which have different sequences, as probes to localize each rbcS gene to a specific rice chromosome.

Fig. 1. Autoradiogragh of IR 36 chromosomes showing the nucleolar organizer of chromosome 8 has been labelled with a silver grain (arrow).

Harper, M.E. and G.F. Saunders, 1981. Localization of single copy DNA sequences on G-banded human chromosomes by in situ hybridization. Chromosoma (Berl.) 83: 431-439.

Kurata, N. and T. Omura, 1978. Karyotype analysis in rice. I. A new method for identifying all chromosome pairs. Jpn. J. Genet. 53: 251-255.

Spierer, P., A. Spierer, W. Bender and D.S. Hogness, 1983. Molecular mapping of genetic and chromomeric units in Drosophila melanogaster. J. Mol. Biol. 168: 35-50.