36. Location of two rDNA genes by linkage analysis in rice

S. MATSUURA1, M. YAN02 and A. SAIT03

1) Tohoku Seed Co., Kiyohara Breed. Farm, Utsunomiya, 321-32 Japan

2) Hokuriku Natl. Agric. Exp. Station, Jyoetsu-shi, Niigata-ken, 943-01 Japan

3) Natl. Inst. Agrobiol. Resources, Kannondai, Tsukuba City, 305 Japan

Rice ribosomal DNA genes (RDNA) of the O. sativa-rufipogon complex consist of a conservative coding region and a highly polymorphic spacer, and the poly- morphic region shows Mendelian segregation pattern in segregating populations (Sano and Sano 1990). From somatic observations, the nucleolar chromosomes are found on the short arm of both chromosomes 9 and 10 (Chang and Wu 1987;Khush et al. 1984), or only on chromosome 10 (Kurata et al. 1981).

By analysis of restriction fragments of the whole genome DNA electrophorsis, we identified two loci of the rDNA gene. In this report, we describe the segregation pattern of the RDNA gene in F2 populations and its linkage relationships with several RFLP loci.

Using an appropriate electrophoresis condition for detection of rDNA (for details see Fig. 1), 5.0 and 4.8 kb fragments from variety Kasalath (Indica type) could be separated from each other. To reveal linkage relationships of rDNA and some RFLP loci, we analyzied segregations of these loci within F2 populations from crosses between strains carrying spacer regions of different sizes. In the F2 populations, the 5 and 4.5 kb BamHI fragments segregated alielically, while the 4.8 kb BamHI fragment showing lower copies than 5 and 4.5 kb ones segregated in a 3: 1 ratio. We named these two loci rDNA-1 and rDNA-2, respectively. As shown in Tables 1 and 2, rDNA-1 and rDNA-2 were linked with RFLP loci on chroniosomes 9 and 10, respectively. The order of these loci was determined by MDS method (Ukai et al. 1990). This result shown in Fig. 2 agreed well with both the location of nucleolar chromosomes, as mentioned above, and observation by in situ hybridization (Islam-Faridi et al. 1991).


Fig. 1. Autoradiograph of southern blot showing two parental and some F2 phenotypes for restriction fragment of RDNA. Loading DNA (ca. 500 ng) digested by BamHI and electrophoresed until 3.8 kb coding region comes 14cm from loading well using 0.5% agarose gel. The rDNA probe for southern hybridization is clone which cloned by Dr. F. Takaiwa from Nipponbare. We digested the 7.8 kb EcoRl insert by SacII, and 2.0 kb fragment corresponded to the spacer region was used for this experiment. P\1\: Kasalath, P\2\: FL134. 

Table 1.  F2 segregation data for linkages between rDNA-1 and 7 RFLP loci on
chromosome 9
===============================================================================
rDNA-1/RFLP    P\1\             H            P\2\           X2L      Recombi-
             ____________  ____________  ____________ Total  
RF Lp        P\1\  H P\2\  P\1\  H P\2\  P\1\  H P\2\                nation(%)
===============================================================================
XNpb 095     20    2   0     2  42   0     0   1  35   102  212.29**  2.5+/-1.1
     315     23    0   0     1  45   0     0   1  34   104  225.81**  1.0+/-0.7
     036     23    0   0     1  43   3     0   1  35   106  218.06**  2.4+/-1.1
     103     13   10   0    12  29   6     3  16  17   106   31.51** 26.8+/-3.6
     385     10   10   1    14  23   4    10  15   7    94    2.32n.s. indep.
     123     19    -   4    37   -  10    32   -   4   106    2.32n.s. indep.
     295      9   10   2    13  19  11     9  22   3    98    6.98n.s. indep.
===============================================================================
P\1\-Kasalath(Indica); P\2\-FL134(Japonica).


Table 2.  F2 segregation data for linkages between rDNA-2 and 6 RFLP loci on
chromosome 10
===============================================================================
rDNA-2/RFLP          P\1\         P\2\                    X2L        Recombi-
             _______________  ________________   Total               nation(%)
             P\1\  H  P\2\    P\1\   H  P\2\
===============================================================================
XNpb 032       32  49   0        0   0  25        106    98.62**     0.0+/-12.4
     089-2     77   -   4        5   -  20        106    55.45**     9.2+/-3.0
     333       33  40   8        2   9  14        106    24.66**    22.2+/-4.5
     037       33  34  14        6  12   7        106     3.36n.s.   indep.
     291       29  34  12        5  12   6         98     3.06n.s.   indep.
     323       60   -  15       17   -   5         97     0.09n.s.   indep.
===============================================================================
P\1\-Kasalath; P\2\-FL134.

Fig. 2. Linkage maps of rDNA-1, rDNA-2 and some RFLP loci on chromosome 9 and 10. (8; located on each chromosome by trisomics analysis. Yano et al. 1990).

References

Chung, M. C. and H. K. Wu, 1987. Karyotype analysis of IR36 and two trisomic lines of rice. Bot. Bull. Acad. Sinica 28: 289-304. (Chinese/English)

Islam-Faridi, M. N., T. Ishii, V. Kumar, L. A. Sitch and D. S. Brar, 1991. Chromosomal location of ribosomal RNA genes by in situ hybridization. RGN 7: 143-144.

Khush, G. S., R. J. Singh, S. C. Sur and A. L. Librojo, 1984. Primary trisomics of rice: Origin, morphology, cytology and use in linkage mapping. Genetics 107: 141-163.

Kurata, N., T. Omura and N. Iwata, 1981. Studies on centromere, chromomere and nucleolus in pachytene nuclei of rice, Oryza sativa, microsporocytes. Cytologia 46: 791-800.

Sano, Y. and R. Sano, 1990. Variation of the intergenic spacer region of ribosomal DNA in cultivated and wild rice species. Genome 33: 209-218.

Ukai, Y., R. Osawa and A. Saito 1990. Automatic determination of the order of RFLPs in linkage group by metric multi-dimensional scaling method. Jpn. J. Breed. 40 (Sup. 2): 302-303.

Yano, M., A. Saito, A. Yoshimura, S. Kuhara, S. Yoshimura, 0. Ideta, N. Kishimoto, K. Saito, R. Osawa, M. Kawase, T. Nagamine, T. Ogawa, M. Nakagahra, and N. Iwata, 1990. Chromosome identification of rice RFLP linkage groups. Jpn. J. Breed. 40 (Suppl. 1): 468- 469. (in Japanese)