Cytoplasmic male sterility DNA factor and utilization thereof

Details Cytoplasmic male sterility DNA factor and utilization thereof Cytoplasmic male sterility DNA factor and utilization thereof

1999-04-14

2001-10-02

Whisenant, Ethan (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S023100, C536S024300

Reexamination Certificate

active

06297012

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a cytoplasmic male sterility DNA factor and a method of discriminating a plant having the same.
A desirable cytoplasmic male sterility line as a parent line for producing a useful F1 hybrid seed has been obtained by a conventional process of repeated crossing of pollen from a cultivar to which sterilization is desired with a plant providing cytoplasmic male sterility for the purpose of replacing the nucleus of the latter with that of the former cultivar having desired traits to be introduced. Alternatively, asymmetrical cell fusion has been used for the same purpose.
Since, however, it required laborious work and took a long time to confirm the fertility of flowers of respective candidate plant provided by each crossing or cell fusion, there has been a strong demand for checking cytoplasmic male sterility traits in a short time without a time loss of flower breeding.
Under such circumstances, the present inventors have conducted extensive researches and, as a result, discovered a cytoplasmic male sterility DNA factor specifically expressed in a male sterility cytoplasm, successfully isolated the said DNA, and found a method to check the presence of the same gene, whereby completing the present invention.
The present invention provides:
1. A DNA comprising a nucleotide sequence selected from the group consisting of:
(a) a nucleotide of 171 bp characterized by a restriction map shown by FIG.
1
and has restriction sites, HinfI (5 bp), MboI (24 bp), MboI (43 bp) and HapII (122 bp);
(b) a nucleotide sequence shown by SEQ ID NO: 1;
(c) a nucleotide sequence encoding the amino acid sequence depicted in SEQ ID NO:2; and
(d) a nucleotide sequence hybridizing with a nucleotide sequence of (b) or (c);
2. A method of discriminating a plant containing a cytoplasmic male sterility DNA factor as defined above, which comprises:
(a) carrying out a PCR-amplification of a genomic DNA of a plant using, as a primer, an oligonucleotide which can amplify a cytoplasmic male sterility DNA factor as defined above or a part of it or a DNA containing said DNA or an equivalent thereof,
(b) separating the amplified genomic DNA by electrophoresis, and then
(c) visually detecting the amplified genomic DNA (hereinafter, this method is referred to as the method of discrimination of the present invention);
3. A method of discriminating a plant containing a cytoplasmic male sterility DNA factor as defined above, which comprises: conducting Southern or Northern hybridization on a genomic DNA or RNA of a plant as a sample for analysis using, as a probe, a cytoplasmic male sterility DNA factor as defined above or an equivalent thereof (hereinafter, this method is referred to as the method of discrimination through hybridization of the present invention); and
4. A DNA comprising a nucleotide sequence selected from the group consisting of:
(a) a nucleotide sequence of 651 bp and having a restriction map as depicted in FIG.
2
and has restriction sites, Ndel (80 bp), EcoRI (117 bp), Mval(152 bp), AccIII(165 bp), NspV(249 bp), SalI (275 bp), BamHI (300 bp) and HinfI (485 bp);
(b) a nucleotide sequence shown by SEQ ID NO: 3;
(c) a nucleotide sequence encoding the amino acid sequence depicted in SEQ ID NO:4; and
(d) a nucleotide sequence hybridizing with a nucleotide sequence of (b) or (c).
The present invention further provides a plasmid containing the cytoplasmic male sterility DNA factor and a method conferring the cytoplasmic male sterility to a plant, plant cell or microorganisms by transfecting the plant, plant cells or microorganisms with the plasmid, and a plant, plant cells or microorganisms into which said cytoplasmic male sterility is introduced and expressing the same.


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Krishnasamy et al., Plant Molecular Biology, 26:935-946 (1994).
Laver et al., Plant J., 1(2):185-193 (1991).
Walters et al., Plant Cell Report, 10:624-628 (1992).

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