20. A new source of cytoplasmic male sterility found in boro varieties of Indica rice

Toshiro KINOSHITA and Koh-ichi MORI

Plant Breeding institute, Faculty of Agriculture, Hokkaido University, Sapporo, 060 Japan

For producing hybrids in Japonica rice, only the male-sterile cytoplasm [cms- bo] derived from the boro variety, Chinsurah boro II, is widely used. There is a possibility of genetic vulnerability caused by the use of single source of cytoplasmic male sterility.

New isonuclear male sterile lines of the Hokkaido variety, Norin 9 (A-133) were established by using three sources of cytoplasm of boro varieties, namely Bhutmuri 36(I-44), Assam III(I-88) and Chinsurah boro II by ten successive backcrosses. According to the previous experiment (Kinoshita et al. 1980), it was concluded that the three lines indicated an identical pattern of pollen restoration when they were crossed with two strong and one weak restorer lines. Thus, it 

Table 1. Male sterile and fertile lines
==============================================================================
                            Constitution
Line or strain       ===========================              Pollen
                        Nucleus      Cytoplasm               fertility
==============================================================================
A-133 Norin 9           A-133        A-133                    Fert
K-11 CMS                A-133        I-44                     CMS*
I-44 Bhutmuri 36        I-44         I-44                     Fert
K-13 CMS                A-133        I-88                     CMS
I-88 Assam III          I-88         I-88                     Fert
A-133 CMS               A-133        [cms-bo]                 CMS
A-58 CMS                A-58         [cms-bo]                 CMS
I-127 Taichung 65 CMS  Taichung 65   [cms-bo]                 CMS
I-129 Taichung 65 Rf    I-129        [cms-bo]                 Fert
=============================================================================
*CMS means cytoplasmic male sterility.

Fig. 1. BamHI restriction profiles of mtDNA prepared from six rice strains. The strains are A-133(A), I-44 having the cytoplasm equivalent with K-11(B), K-13(C), A-133CMS(D),I-129[cms-bo]Rf\1\Rf\1\(E) and A-58CMS[cms-bo](F). The DNA fragments were separated by electrophoresis in 0.8% agarose slab gel.

was assumed that the three lines possess the same cytoplasm, [cms-bo]

In further experiments, the molecular nature of mitochondrial genomes was examined by restriction endonuclease analysis. First, seed calli were induced from the lines or strains shown in Table 1. Since the callus induction was unsuccessful in K-11, the original line, Bhutmuri (I-44) having the identical cytoplasm with K-11 was used for comparision with the other two isolines. Mitochonodrial DNAs isolated from cultured cells in suspension were surveyed following the procedures used by Yamato et al. (1992). Restriction endonuclease digestion patterns of mtDNAs are shown in Fig. 1 and Table 2. Three [cms-bo] lines indicated a common pattern in all endonucleases used. Therefore it is proved that there is no recogniz- 

Table 2. Restriction endonuclease analysis of mitochondrial DNAs in CMS and
fertile lines
===============================================================================
Line                            EcoR  I
            ===================================================================
            1   2   3   4   5   6   7   8   9   10   11   12   13
===============================================================================
A-133           +   +   +       +                +    +         +
I-44            +   +   +   +       +       +    +         +
K-13            +   +                   +   +              +
A-133 CMS*  +                               +              +
I-129*      +                               +              +
A-58 CMS*   +                               +              +
===============================================================================

================================================
Line            Hind     III
            ====================================  
            1   2   3   4   5   6   7
================================================
A-133               +   +           +
I-44        +       +       +   +
K-13        +       +       +   +
A-133 CMS*  +   +           +   +
I-129*      +   +           +   +
A-58 CMS*   +   +           +   +
================================================

===============================================================================
Line                                        Pst   I
            ===================================================================
            1   2   3   4   5   6   7   8   9   10   11   12   13   14   15
===============================================================================
A-133       +                       +   +        +    +    +    +
I-44            +       +       +           +                   +    +    +
K-13            +   +       +   +           +                        +    +
A-133 CMS*          +   +       +   +       +                        +    +
I-129*              +   +       +   +       +                        +    +
A-58 CMS*           +   +       +   +       +                        +    +
===============================================================================
* [cms-bo] cytoplasm.
Numerals show cytoplasm-specific bands.
able change during repeated backcrosses to introduce the cytoplasm. It is also noted that the new cytoplasmic sources involved in the isonuclear lines are clearly distinguished from the pattern of [cms-bo] cytoplasm. In addition, a Japonica cytoplasm represented by A-133 showed another distinctive pattern. Thus, the molecular nature of the new cytoplasms differed from the [cms-bo] cytoplasm used for the commercial CMS strains.

Southern hybridization analysis of mtDNAs was also done with the gene probes, rrn18 and coxII. Polymorphisms of mtDNA were detected as shown in Fig. 2 and 3. In both cases, there were three groups, namely two new cytoplasm lines (K-11, I-88), three [cms-bo] lines and A-133 Norin 9. Therefore it is estimated that the male sterile cytoplasms from I-44 and I-88 constitute a new subgroup of [cms-bo] cytoplasm, as shown in the case of the cms S group of maize (Sisco et al. 1985).


Fig. 2. Southern hybridization analysis of rrn]8 gene. BamHI DNA fragments shown in Fig. 1 were transferred to a nylon membrane and hybridized with a 32P labelled probe containing a portion of pea mitochondrial rrn18 sequence. The lanes are identical to those of Fig. 1.

Fig. 3. Southern hybridization analysis of coxII gene. BamHI DNA fragments were used as explained in Fig. 2. The lanes are identical to those of Fig. 1.

References

Kinoshita, T., K. Mori and M. Takahashi, 1980. Inheritance studies on cytoplasmic male sterility induced by nuclear substitution -Genetical studies on rice plant LXX-. J. Fac. Agr. Hokkaido Univ. 60: 23-41.

Sisco, P. H., V. E. Gracen, H. I. Everett, E. D. Earle, D. R. Pring, J. W. McNay and C. S. Levings, III. 1985. Fertility restoration and mitochondrial nucleic acids distinguish at least five subgroups among cms-S cytoplasm of maize (Zea mays L.). Theor Appl Genet 71: 5-15.

Yamato, K., Y. Ogura, T. Kanegae, Y. Yamada and K. Ohyama, 1992. Mitochondrial genome structure of rice suspension culture from cytoplasmic male sterile line (A-58 CMS): reappraisal of the master circle. Theor Appl Genet 83: 279-288.