U.S. patent application number 13/708459 was filed with the patent office on 2013-07-04 for fertility restorer gene and fertility restoration method for cw-type male sterile cytoplasm of rice.
The applicant listed for this patent is Sota Fuji, Kinya TORIYAMA. Invention is credited to Sota Fuji, Kinya TORIYAMA.
Application Number | 20130174296 13/708459 |
Document ID | / |
Family ID | 41064922 |
Filed Date | 2013-07-04 |
United States Patent
Application |
20130174296 |
Kind Code |
A1 |
TORIYAMA; Kinya ; et
al. |
July 4, 2013 |
FERTILITY RESTORER GENE AND FERTILITY RESTORATION METHOD FOR
CW-TYPE MALE STERILE CYTOPLASM OF RICE
Abstract
Mainly provided is a technique for directly identifying the
genotype at locus Rf17 based on the specific base sequence data
thereof. Also provided is a technique for artificially constructing
a fertility-restored line. A method of restoring the fertility of
CW-type cytoplasmic male sterile rice by inhibiting or reducing the
expression of a gene comprising the base sequence represented by
SEQ ID NO:2 in the above-described rice, and a method for
determining the presence or absence of gene Rf17, which is a
fertility restorer gene for CW-type cytoplasmic male sterility,
comprising identifying a single nucleotide polymorphism (SNP) in
the base at the 1812 position of the base sequence represented by
SEQ ID NO:1 in the rice to be examined.
Inventors: |
TORIYAMA; Kinya; (Miyagi,
JP) ; Fuji; Sota; (Miyagi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TORIYAMA; Kinya
Fuji; Sota |
Miyagi
Miyagi |
|
JP
JP |
|
|
Family ID: |
41064922 |
Appl. No.: |
13/708459 |
Filed: |
December 7, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12922071 |
Nov 18, 2010 |
8344122 |
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PCT/JP2009/000753 |
Feb 21, 2009 |
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13708459 |
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Current U.S.
Class: |
800/274 ;
435/6.11; 800/278; 800/320.2 |
Current CPC
Class: |
A01H 1/02 20130101; A61P
15/08 20180101; C07K 14/415 20130101; C12Q 2600/156 20130101; C12Q
1/6895 20130101; C12N 15/8289 20130101 |
Class at
Publication: |
800/274 ;
800/278; 800/320.2; 435/6.11 |
International
Class: |
A01H 1/02 20060101
A01H001/02 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 12, 2008 |
JP |
2008-062879 |
Claims
1-14. (canceled)
15. A polynucleotide consisting of a base sequence selected from
the group consisting of: (a) a base sequence at least 1611-4835
positions of the base sequence represented by SEQ ID NO:1; (b) a
base sequence that hybridizes with the base sequence (a) or its
complementary sequence under stringent conditions, and (c) a base
sequence having homology of about 95% or more with the base
sequence (a).
16. A vector comprising the polynucleotide according to claim
15
17. A plant that is transformed with the vector according to claim
16.
18. The plant according to claim 17, said plant is transformed by
means of Agrobacterium method.
19. A plant of CW-type cytoplasmic male sterile rice, said plant is
transformed with the vector according to claim 16, so that its
fertility has been restored.
20. The plant according to claim 19, said plant is transformed by
means of Agrobacterium method.
21. A polynucleotide consisting of contiguous 10-30 bp comprising a
base "T" at position 1812 of the base sequence represented by SEQ
ID NO:1.
22. A primer, marker or probe comprising the polynucleotide
according to claim 21 for use in the identification of a single
nucleotide polymorphism in a base at 1812 position of the base
sequence represented by SEQ NO:1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S. Ser.
No. 12/922,071 filed Nov. 18, 2010, now allowed, which is a 371
U.S. National Stage application of PCT/JP2009/000753, filed Feb.
21, 2009, which claims priority from Japanese Application Serial
No. 2008-062879, filed Mar. 12, 2008. The entirety of all the
aforementioned applications are incorporated herein by
reference.
[0002] The present invention relates to a method for restoring the
fertility of CW-type cytoplasmic male sterile rice, and to a method
for determining the presence or absence of a fertility restorer
gene and the like. Cytoplasmic Male Sterility (referred to
hereinafter as "CMS") means that the formation of pollen having the
ability to germinate is inhibited due to mutation in a
mitochondrial gene so that a seed will develop into seeds. It is
known that fertility can be restored by the function of a pollen
fertility restorer gene (Rf) encoded in a nuclei in some cases of
CMS.
BACKGROUND
[0003] A method for breeding a first filial generation is also
called a method for breeding of a hybrid-variety. This method is
utilized in variety-breeding since a variety having excellent
traits inherited from its parents and showing heterosis hybrid
vigour can be bred by means of this method. In order to
economically harvest F1 hybrid seeds in large quantity, a
three-line method utilizing cytoplasmic male sterility is adopted
in the case of seed-harvesting of a hybrid variety of rice. The
three-line method refers to a method that utilizes a sterile line
having male sterility, a fertility restorer line, and a maintainer
line having the same nuclear genes as the sterile line but not
having sterile cytoplasm. According to this three-line method, (1)
a hybrid seed can be harvested by crossing the sterile line with
pollen of the restorer line, while (2) the sterile line can be
maintained by crossing the sterile line with the pollen of the
maintainer line.
[0004] BT-type male sterile cytoplasm and WA-type male sterile
cytoplasm have been used worldwide in order to breed the hybrid
variety with the three-line method. On the other hand, CW-type male
sterile cytoplasm has been hardly_utilized since the structure and
function of a fertility restorer gene Rf17 (Non Patent Document 1)
have not been clarified. However, since there may be a risk of
variety collapse due to limit in the genetic resources provided by
the male sterile cytoplasm that have been used until now,
utilization and development of novel male sterile cytoplasm are now
desired.
[0005] In the prior arts, it has been necessary to breed plant body
(F1) from a hybrid seed obtained by test cross, and then to make
the F1 self-cross and to investigate frequency of appearance of a
individual plant having seed-development rate ovevarietyrtain level
(for example, 90% or more) for the estimation of a genotype of Rf17
locus in the plant. It has not been possible to determine said
genotype by means of DNA markers. [0006] Non Patent Document 1:
Sota Fujii and Kinya Toriyama (2005) Molecular mapping of the
fertility restorer gene for ms-CW-type cytoplasmic male sterility
of rice. Theor. Appl. Genet. 111:696-701.
SUMMARY
Problem to be Solved by the Invention
[0007] If the CW-type male sterile cytoplasm is utilized in the
three-line method, it will be necessary to confirm that rice is
holding Rf17 gene in each step of breeding the rice of the
restorer-line, and that it holds Rf17 gene in both alleles at a
final stage. It is therefore a main purpose of this invention to
provide a technique to directly identify the genotype of Rf17 locus
on the basis of its specific base sequence, and to provide a
technique to artificially produce a fertility restorer line and the
like.
Means for Solving the Problem
[0008] The present inventors have determined a base sequence
comprising the fertility restorer gene Rf17 for the CW-type male
sterile cytoplasm, which is represented by SEQ ID NO:1. We also
have succeeded in the restoration of fertility of CW-type
cytoplasmic male sterile rice by inhibiting the expression of a
gene comprising the base sequence represented by SEQ ID NO:2,
leading to the completion of the present invention.
[0009] Thus, the present invention relates to the following
aspects:
[0010] 1. A method for restoring the fertility of CW-type
cytoplasmic male sterile rice, comprising inhibiting or reducing
the expression of a gene comprising the base sequence represented
by SEQ ID NO:2 in said rice.
[0011] 2. The method according to claim 1, wherein the fertility is
restored by introducing a genomic fragment into the CW-type
cytoplasmic male sterile rice, wherein the fragment is derived from
the chromosome No. 4 of a fertility restorer line for the CW-type
cytoplasmic male sterility and comprises nucleic acids consisting
of the base sequence of at least at the 1611-4835 positions of the
base sequence represented by SEQ ID NO:1.
[0012] 3. The method according to claim 2, wherein the genomic
fragment consists of the whole base sequence represented by SEQ ID
NO:1.
[0013] 4. The method according to claim 1, wherein the fertility is
restored by inhibiting the expression of the gene represented by
SEQ ID NO:2 by means of RNA interference method.
[0014] 5. The method according to claim 4, wherein the RNA
interference is induced by introducing a vector comprising a gene
consisting of the base sequence represented by SEQ ID NO:2 or a
continuous 100-500-base sequence in its 3' non-translation region
and their complementary sequence and expressing a double-stranded
RNA that is capable of inducing the RNA interference in a cell into
the CW-type cytoplasmic male sterile rice.
[0015] 6. The method according to claim 5, which uses a vector
comprising a continuous base sequence at the 638-815 positions of
the base sequence represented by SEQ ID NO:2 and its complementary
sequence and expressing a double-stranded RNA that is capable of
inducing the RNA interference in a cell.
[0016] 7. Rice whose fertility has been restored by the method
according to any one of claims 1-5.
[0017] 8. A method for breeding a first filial generation variety
using the rice according to claim 7.
[0018] 9. F1 hybrid seed harvested from the first filial generation
variety bred by the method according to claim 8.
[0019] 10. A method for determining the presence or absence of gene
Rf17, which is a fertility restorer gene for the CW-type
cytoplasmic male sterility, comprising identifying a single
nucleotide polymorphism (SNP) in a base at the 1812 position of the
base sequence represented by SEQ ID NO:1 in rice to be
examined.
[0020] 11. The method according to claim 10, wherein the SNP is
identified by means of CAPS method.
[0021] 12. The method according to claim 11, which uses a
restriction enzyme recognizing the cleavage site GT(A)AAC.
[0022] 13. The method according to Claim 12, wherein the
restriction enzyme is MaeIII.
[0023] 14. A kit used for the method according to any one of claims
10-13.
Advantages of the Invention
[0024] The present invention has revealed that the CW-type male
sterility is restored by the reduction of the expression of ORF11.
And, the present inventors have succeeded in the identification of
a base sequence that is effective in the restoration of the CW-type
male sterility and in the restoration of said fertility.
BRIEF DESCRIPTION OF DRAWINGS
[0025] FIG. 1 A pattern diagrams showing a gene present in the gene
fragments that restored fertility in a complementarity test.
[0026] FIG. 2 Photos showing the presence or absence of
decomposition of the starch of pollen in a transformed plant into
which a vector inducing RNA interference has been introduced. The
decomposition of starch was observed in the fertility restorer line
(CWR) and an individual plant (RNAi ORF11.sub.--3) into which an
RNA interference vector for ORF11 has been introduced, but it was
not observed in the male sterile line (CWA) and an individual plant
(RNAi PPR2.sub.--1) into which an RNA interference vector for PPR2
has been introduced.
[0027] FIG. 3 Photos of electrophoresis showing the determination
of the Rf17 genotype by means of PCR with a CAPS marker. While the
fertility restorer line (CWR) generated a band of 370 bp, a variety
having no fertility restorer capability (Taichung 65: T65)
generated bands with 276 bp and 84 bp.
BEST MODE FOR CARRYING OUT THE INVENTION
[0028] The present inventors have succeeded in the determination of
a genomic region comprising the fertility restorer gene Rf17 for
the CW-type male sterile cytoplasm in the chromosome No. 4 of rice,
which is represented by SEQ ID NO:1, by means of a conventional
map-base cloning method. We also have further succeeded in the
restoration of fertility of the CW-type cytoplasmic male sterile
rice by inhibiting or reducing the expression of a gene (ORF11)
represented by SEQ ID NO:2. The function of the gene represented by
SEQ ID NO:2 has not yet known.
[0029] The map-base cloning method is also called a "chromosome
walking", which focuses a genomic region in which a gene to be
isolated exists by means of a DNA marker that is present in the
vicinity of said gene. This method is used as one of isolation
methods of a gene when the function or translation product of the
gene can not estimated. Although this method has been utilized for
the gene-isolation in some kinds of animals and plants, it is more
effectively used in the plants from which a segregating population
for laboratory use may be produced according to a plan. The genomic
sequence of nipponbare-type rice
(http://rgp.dna.affrc.go.jp/J/index.html) was utilized in the
map-base cloning according to the present invention.
[0030] The present inventors have found a way of introducing a
genomic fragment into the CW-type cytoplasmic male sterile rice,
wherein the fragment is derived from the chromosome No. 4 of a
fertility restorer line for the CW-type cytoplasmic male sterility
and comprises nucleic acids consisting of the base sequence of at
least at the 1611-4835 positions of the base sequence represented
by SEQ ID NO:1 in order to inhibit or reduce the expression of the
gene comprising the base sequence represented by SEQ ID NO:2. The
whole base sequence represented by SEQ ID NO:1 is listed as one of
the examples of the above genomic fragments.
[0031] The examples of the above genomic fragment further include a
nucleic acid which may hybridize with a nucleic acid consisting of
the base sequence complementary with the base sequence represented
by SEQ ID NO:1 or its part under stringent conditions, and a
nucleic acid consisting of the base sequence having homology of
about 80% or more, preferably about 95% or more with the above
nucleic acids. Those nucleic acids can restore the fertility of
CW-type cytoplasmic male sterile rice.
[0032] The hybridization may be carried out by or according to a
method known in the art such as, for example, Molecular cloning
third. ed. (Cold Spring Harbor Lab. Press, 2001). Commercially
available libraries may be used in accordance with the description
of an attached instruction for use.
[0033] The "stringent conditions" in the present specification
means, for example, the temperature of 60-68.degree. C., sodium
concentration of 150-900 mM, preferably 600-900 mM and pH6-8.
[0034] Accordingly, the above nucleic acid which may hybridize with
a nucleic acid consisting of the base sequence complementary with
the base sequence represented by EQ ID NO:1 or its part may be a
nucleic acid comprising the base sequence having homology of about
80% or more, preferably about 95% or more, more preferably 99% or
more with the whole base sequence of said nucleic acids.
[0035] The sequences may be pre-treated into a suitable state for
comparison before homology (identity) is determined between two
base- or amino acid-sequences. For example, a gap may be introduced
into the sequence of one of them so as to optimize alignment with
the other sequence. The amino acid or base sequences will be
compared in each part thereof. When a part of the first sequence
has the same amino acid or base sequence as a corresponding part of
the second sequence, their sequences are deemed to be identical
with each other in those parts. Homology between the two sequences
is shown as a percentage of the number of the same amino acids or
bases for the total number of the amino acids or bases in that
part.
[0036] According to the above principle, homology (sequence
homology) between two base sequences may be determined by means of
algorism of Karlin and Altschul (Proc. Natl. Acad. Sci. USA
87:2264-2268, 1990 and Proc. Natl. Acad. Sci. USA 90:5873-5877,
1993). BLAST and FAST programs based on the above algorism are used
to investigate a sequence having a high homology with a given
sequence from data base. These programs are available in the
website of the National Center for Biotechnology Information on the
Internet.
[0037] The nucleic acid having the above homology in the base
sequence may be obtained using hybridization as an index, or
obtained from a group of unidentified DNAs that have been obtained
by the analysis of genomic base sequences or from public database
by using any method that those skilled in the art usually uses,
such as BLAST software. The gene according to the present invention
may also be obtained by a known mutation-introducing method.
[0038] The expression of the gene comprising the base sequence
represented by SEQ ID NO:2 in the CW-type cytoplasmic male sterile
rice may be inhibited or reduced by any other methods known for
those skilled in the art. For example, the fertility of the CW-type
cytoplasmic male sterile rice may be restored with anti-sense RNA
method or by the inhibition of the expression of the gene
represented by SEQ ID NO:2 with RNA interference method in
accordance with the Example of the present specification.
[0039] Thus, the RNA interference may be induced by introducing a
vector into the CW-type cytoplasmic male sterile rice, wherein the
vector is designed to express a double-stranded RNA capable of
inducing the RNA interference so as to inhibit or reduce the
expression of the gene comprising the base sequence represented by
SEQ ID NO:2. The vector includes one comprising a gene consisting
of the base sequence represented by SEQ II) NO:2 or a continuous
100-500-base sequence, preferably a continuous 150-200-base
sequence in its 3'' non-translation region and their complementary
sequence and expressing a double-stranded RNA that is capable of
inducing the RNA interference in the cell. An example of said
vector is one comprising a continuous 178-base sequence at the
638-815 positions of the base sequence represented by SEQ ID NO:2
(its first base is numbered as "1 position") and its complementary
sequence and expressing a double-stranded RNA that is capable of
inducing the RNA interference.
[0040] The above vector may be prepared by combing the above DNA
within a vector according to any suitable gene-engineering
technique known for those skilled in the art. The above vector may
comprise a suitable promoter and any other controlling sequences,
for example, enhancer sequence, terminator sequence,
polyadenylation sequence and the like in order to express the gene
according to the present invention in a host cell.
[0041] The genomic region or the vector to be used for the
inhibition or reduction of the expression of the gene comprising
the base sequence represented by SEQ ID NO:2 in the CW-type
cytoplasmic male sterile rice may be introduced into a subject rice
by any method known for those skilled in the art such as
Agrobacterium method, free-thaw method and electroporation
method.
[0042] As a result, the fertility is restored in the CW-type
cytoplasmic male sterile rice. Accordingly, a first filial
generation variety is bred using the rice whose fertility has thus
been restored so that an F1 hybrid seed can be harvested from the
variety.
[0043] The present invention is further relates to a method for
determining the presence or absence of a fertility restorer gene
for the CW-type cytoplasmic male sterility, Rf17 or a genomic
region comprising the gene, comprising identifying a single
nucleotide polymorphism (SNP:A/T) in a base at the 1812 position of
the base sequence represented by SEQ ID NO:1 in the rice to be
examined.
[0044] The determination of SNP may be carried out by any method
known for those skilled in the art, such as base
sequence-determination method, SSCP (single strand conformation
polymorphism) method, ASA (Allele specific amplification), primer
extension method, Taqman method, invasion method, dot-blot-SNP
Method, FRIP (Fluorogenic Ribonuclease Protection) method, and
TILLING (Targeting Induced Local Lesion in Genome) method.
[0045] There is also CAPS (Cleaved Amplified Polymorphic Sequence)
method for the above determination, as described in the Example of
the present specification. In this method, a primer is so designed
that a restriction enzyme-recognition site will be formed at a
location where the SNP exists, followed by PCR-RFLP analysis on
polyacrylamide gel. This method has an advantage that its results
will hardly be affected by the conditions of an experimenter or a
sample, or the kind of a DNA extraction method so as to easily
obtain constant results. Furthermore, the method can be performed
in a relatively simple way and does not need an expensive apparatus
such as a DNA sequencer or advanced technique.
[0046] Primers, markers or probes that are used in the above method
for the identification of SNP may be easily designed and prepared
by those skilled in the art in accordance with the principles of
each method on the basis of the database described above and the
information about the DNA sequence of SEQ ID:NO1 or NO:2 disclosed
in the present specification. For example, a primer used in PCR of
the CAPS method usually has the length of several tens of by such
as 10-30 bp.
[0047] Various kinds of oligonucleotides that will be used as the
above primers, markers or probes may be synthesized in vitro by any
method known for those skilled in the art, such as chemical
synthesis methods that are described, for example, in Carruthers
(1982) Cold Spring Harbor Symp. Quant. Biol. 47:411-418; Adams
(1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acid Res.
25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380;
Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth.
Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981)
Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066. Any known labeling
substance may be attached to them for detection.
[0048] As a sample of rice to be examined, any part of the rice may
be used such as its seed, leaf and stem. DNA may be extracted and
prepared by any method known for those skilled in the art from the
above sample. The kind of DNA to be prepared from the sample has no
limitation, a genomic DNA (gDNA) and cDNA being listed for example.
They may be extracted and purified by any method known for those
skilled in the art depending on their properties, and kinds and
properties of the sample. For example, gDNA may be obtained by CTAB
method, boiling method and enzyme method using amylase or protease,
if necessary.
[0049] If a sufficient amount the DNA is extracted from the sample
rice for detection, it will be subjected to the following
procedures without amplification. However, the DNA will be usually
amplified to an amount suitable for the identification of SNP by
means of any gene-amplification method known for those skilled in
the art, such as PCR (Polymerase Chain Reaction) or RT-PCR method,
ICAN (Isothermal and chimeric primer-initiated amplification of
nucleic acids) method, NASBA (Nucleic acid sequence based
amplification) method, TMA (Transcription-mediated amplification)
method and SDA (Strand Displacement Amplification) method.
[0050] The present invention also relates to a kit used for the
method for determining the presence or absence of gene Rf17.
Depending on the kinds of SNP analysis and the like, the kit
comprises primers, markers or probes for the identification of SNP.
It may further optionally comprise various primer set and/or marker
for the amplification of DNA, a restriction enzyme and other
elements or components known for those skilled in the art such as,
for example, various agents, enzymes, and buffer, a reaction plate
(vessel) depending on its structure and purpose.
[0051] Although the present invention will be explained in more
detail with reference to the following Examples, the technical
scope of the present invention shall not be construed to be limited
by them. The terms described in the present specification are used
in such meaning as is usually used in the art unless particularly
noted otherwise.
[0052] Unless otherwise described, each procedure may be carried
out in accordance with standard techniques in gene engineering and
molecular biology known for those skilled in the art, such as those
described in Sambrook and Maniatis, in Molecular Cloning-A
Laboratory Manual, Cold Spring Harbor Laboratory Press, New York,
1989; Molecular cloning third.ed. (Cold Spring Harbor Lab. Press,
2001); Ausubel, F. M. et al., Current Protocols in Molecular
Biology, John Wiley & Sons, New York, N.Y., 199 and the like.
The content of the publications cited in the present specification
will constitute a part of the disclosure of the present
specification.
Example 1
Restoration of the Fertility by Introduction of a Genomic Fragment
of the Base Sequence Represented by SEQ ID NO:1
[0053] Mapping of Rfl1 using the fertility restorer line (CWR line)
for the CW-type CMS line revealed that Rf17 exists in a region of
77 kb of the chromosome No. 4, so that the base sequence of that
region was determined. The genomic region was divided into seven
fragments and subjected to sub-cloning. Each genomic fragment was
then introduced into the CW-type CMS line by means of Agrobacterium
method and the seed fertility of the transformed plant was
investigated. A plant individual whose fertility had restored was
obtained from the plant transformed with the genomic fragment No. 5
(Table.1). Fertility was restored in four (4) individuals among 44
re-differentiation lines. The four individuals showed the seed
fertility of 79.3, 23.1, 25.3 and 68.1%, respectively. The base
sequence of the fragment No. 5 is shown as SEQ ID NO:1.
TABLE-US-00001 TABLE 1 No. of Individuals of No. of Individuals of
No. of Gene Introduction Re-differentiation Fertility Restoration 1
17 0 2 9 0 3 8 0 4 10 0 5 44 4 6 7 0 7 6 0
[0054] Two genes were predicted in the above fragment, which were
PPR gene and a functionally unidentified gene (named as "ORF11")
(FIG. 1). The comparison in expression between the CMS line and the
fertility restored line revealed that the expression of ORF 11 was
high in the CMS line but low in the fertility-restored line, while
there is no difference in the expression of PPR gene between them.
The reduction of the expression of ORF 11 was also observed in the
next generation of the transformation line whose fertility had been
restored. The comparison of base sequence between the CMS line and
the fertility-restored line showed that there was a single
base-substitution within the PPR gene, which generated a stop codon
in the fertility-restored line (FIG. 1). As the single
base-substitution was located 5'-upstream of ORF11, it was
considered that the expression of ORF11 was reduced in the
fertility-restored line due to the above mutation. It was therefore
concluded that the fertility was restored due to the reduction of
the expression of ORF 11. Accordingly, it has been revealed that
the fertility is restored by introducing a genomic fragment
comprising the base sequence represented by SEQ ID NO:1, into the
CW-type cytoplasmic male sterile line.
[0055] The "PPR" is an abbreviation for "pentatricopeptide repeat"
that is protein having a repeated conserved sequence consisting of
35 amino acids. The protein is considered to bond to the RNA of
arganelle and be involved in RNA processing and regulation of
translation.
Example 2
Restoration of the Fertility by of the Gene of SEQ ID NO:2
[0056] Mapping of the fertility restorer gene, Rf17, for the
CW-type CMS line revealed 14 candidate genes within the candidate
region of 77 kb. According to polymorphism analysis of the CMS line
and the fertility restorer (CWR) line, an amino acid mutation had
occurred only in the PPR gene ("PPR2 gene" in FIG. 1) and the stop
codon was generated in an allele of the CWR line. According to
expression analysis done for each of the 14 candidate genes, only
ORF 11 gene showed the difference between the CMS line and the CWR
line. The base sequence comprising a coding region, and its 5'- and
3'-non translation regions are shown as SEQ ID NO:2. A base
sequence corresponding to that at the 638-815 positions of the base
sequence represented by SEQ ID NO:2 (its first base is numbered as
"1 position") and a base sequence corresponding to that at the
772-1505 positions of the base sequence represented by SEQ ID NO:1
(its first base is numbered as "1 position"), which corresponds to
PPR2 gene, were amplified by means of PCR using the primers
represented by SEQ ID NO:3 and NO:4, and the primers represented by
SEQ ID NO:5 and NO:6, respectively, and linked down stream of a
ubiquinone promoter of pANDA vector to give a vector that induces
the RNA interference. The resulting vectors were introduced into
the CW-type CMS line by means of Agrobacterium method, and shape of
pollen and seed fertility were investigated. As a result, the
expression of ORF11 was reduced down to 30-77% in seven (7) lines
into which the RNA interference-inducing vector for ORF11 had been
introduced. And, four (4) lines out of said seven lines showed
decomposition of the starch of pollen, which is characteristic to
the fertility restorer line (FIG. 2), and their seed fertility was
partially restored (2-3%). On the other hand, although the
expression of ORF11 was reduced down to 27-75% in six (6) lines
into which the RNA interference-inducing vector for PPR2 had been
introduced, no decomposition of the starch of pollen was observed
like the CMS line (FIG. 2) showing the seed fertility of 0%. The
above results demonstrated that the fertility of the CW-type CMS
line can be restored by inhibiting or reducing the expression of
the gene ORF 11.
Example 3
Determination of Rf17 Genotype
[0057] Comparison of the base sequences represented by SEQ ID NO:1
comprising the fertility restorer gene Rf17 with that of
Nipponbare-type rice revealed that a base at the 1812 position of
the base sequence represented by SEQ ID NO:1 is "T" in the
fertility restorer (CWR) line, while "A" in the nipponbare-type
rice that has no fertility-restoring capacity. A CAPS marker was
prepared for a simple identification of this mutation. PCR was
carried out using two primers represented by SEQ ID NO:7 and NO:8.
Treatment with a restriction enzyme MaeIII and electrophoresis gave
a band with 370 bp in the fertility restorer line (CWR), and bands
with 276 bp and 84 bp in the nipponbare-type rice (FIG. 3). The
above results showed that the present or absence of Rf17 could be
simply determined. By the way, the restriction enzyme that can be
used in the above determination is not limited to MaeIII, but any
restriction enzyme may be used as long as it recognizes the
cleavage site GT(A)AAC.
INDUSTRIAL APPLICABILITY
[0058] According to the present invention, a hybrid variety can be
bred by the three-line method using the CW-type male sterile
cytoplasm.
Sequence CWU 1
1
814871DNARiceGenome fragment No.5 derived from chromosome No.4
1ggatccggca gctcttcacc attctcttca accttcaagc cttcatgaaa tgctccacaa
60tttgttgact gttgattaga aataggaaat ttagaagaag aaaaaaaaat actccacatg
120agcttgaaaa atgtaacatg cagttttgaa atcaacctca tggcccacag
tacttgagct 180aaagagatga cggtgcagtt caacaacaaa gaatacacat
caggggggaa tggtggtgca 240cctaaaattt taggtgtttg ggaattgaaa
tatcaccact caatgactat ggttcaatga 300gataattact gcacaaatta
ttggtcatta ttggacgatg taccaaaaca gtcaagagca 360tatccactgg
ttgcagaata gtaccacaaa tgaagtataa atctccgtgt atatgcaatc
420ttaatcttcc caatcatatg atcagtacaa tcaacaggta taaaatttag
aaattttatt 480tggattgggt gtggaaaagg tcaaccaggt aactgcatga
ccatagatgt aactgtaatc 540tgaagttctg aacagtacct taatcatcaa
attattgacc atggaattac tgcaagcgca 600acagaccttg acatccaact
gagcccaata atctgacttc ctttaaggcc aaaccaaaga 660atggatgcta
caatcctaca agcttgctta gcccagcaac aagagaagac caaccataaa
720atttgctgat gtcctcaagt accatgcaca aacagtaggc tgccctgaat
tcgcagactc 780tcaacaaggt taacactttc agcaaacttc ccacatttgt
gaaagtaccg gagtaagcat 840gtcatactga tggcatcaag gttgattcct
tccacatgca tacgatagag aaccatacgg 900ataactgaag cattctgctg
cctgcaggct gcagagagga tagtgttgaa ggagactgaa 960tcacagggaa
caccagtaga gctaaaatgg tccaacatct gcaatgcccc ctgaagatcc
1020agctccatac agaagccttt cagtatagtg ttatatgttg tagtagatgg
tacaagtcct 1080ctctccatca ttccactaac caaagctaat gctttcttaa
cctgcccatc acagcataag 1140ctatggaggt atatattcca agtgataata
tccacatgat tgctcctgtt acataccatt 1200cgtataaaat tctgacaggt
catgacatcc ccagttttca gaaagccagc agccagagaa 1260gaaatggtga
aatagtcagg gaccaatcct gcctcctcca tcaaatcaac aatcacccga
1320gcttctggta ttctatcttc gcagcagagt gcatggagta gcacattgca
tatggtgcta 1380tcaagacaac agccattttt gcatgccatc ctgaaaacat
gcatcgcagc atcaatctta 1440ccatctctac agaggaaatt cacaattacg
gtatatgtta caacagaggg tttgcactct 1500tcatcgcaca tcttctccaa
tgccctgtat gcgtcggccc acatcttctc cttgcataat 1560cctaagacca
atgctgtata cgaataaacg tttagagaga gtcctctctt cttcaagtta
1620cagaacagag tcaatgcctt attgctgcaa cctgacttgc aaagaccaaa
cagaatttgg 1680ttgtacatat acacatctgg cacaaacttc ctatttacaa
gcaccatgaa aacctcatat 1740gcaatctcag cgctgccact tttcaataga
atcgagacaa tggcattggc gatagggaga 1800gtcggaaagt atcacagact
gagcattagg tcgaacacct tcagtgcaag ttcaaagtta 1860gagccactaa
cagctttctt gatgagtcta tctaccgtgg tgaacgaagg cacgaatccg
1920cagcgagacc acatctcttc gagtaccgcg tgcgaggcgt cgaggtggcc
tagccggagc 1980agcccgttga aggcggtatt gaaggtgagg gcgttgggcg
ccgggccgac ggagcgcatg 2040tgacacagca cggcggctac atgctccgcg
gcggcgacgt gttgcgcggc ggcttgcccg 2100gaccggaggt aggcgtggag
gagggcgttg tagtcgtggg cgcctgcggg cgcgggccgg 2160aggagggcga
gcgcgtccag cgccgcggcc aggttcccct cggcggccga cgcccgcatg
2220cggcggcgga ggagcgtggc atccgcgggg ccgtgggatt cggggtccac
gtcggagtcg 2280gaggtggaaa tggaagcttc gacgggggag gcggcggcgg
tggcgagagc ggagcggaag 2340cgcaggcgga cgcggcggca tgaggcgagc
ataggatggg gggcagttcc taccagctgc 2400gatgtgatgt tcgattcatg
aggaggacgg tgatgccgga ggcggaggtg gtggtggtgg 2460ttgggcggcg
ccgccgacgc gcgggctgtg gaggagcggt ggcggcgccg acggtggcgg
2520agggaggcag gaagcggcgt gtggaggagg aggcggcggc gccgcgtcgc
gcgtcggcgt 2580gtgggcgtgg aggaggcgga ctggcggttg ctgggctgga
tggttaggtt tttcggttaa 2640ttgtctaatt gatgggctga atgggccagt
atagtgtgtc ttatggccga ataaaatgga 2700ataagttcat cataggtccc
ttaaccgaat ccgattttcg tccttcaact ggaaaaccag 2760atcgggtccc
tcaactatca cacccggtgc agatgaggtc cctcagcgtt ttagatggcg
2820gttttggcag acgtgacgct tagtggctag tttgtctctg tcttcatctg
acgtgacgct 2880tacgtggcaa ttcgaaccgg aaaagtaata aaactcgtgg
gacccacgga tcagtttcac 2940acataaacta ataaaaaata gtgagcccca
catgtcatcc tcactccctt cttccctcta 3000tcccctctct ctcttcttcc
ctacaatgtg gactggggcg gcgggcggac gagcgcaacc 3060gcggccgaag
cagcagcgca cgaagccgac ggggcggtgg caatcctccg ccgacgccga
3120tatccacagc agaggtgcgc gtcctcctcc tccttaaacc cgtcaccatc
ggtggccgtc 3180ccccctgggc ctccgcccgc ggctcctctc cttccgtcgg
ccggcaagca agatacacaa 3240acaattagca gaaaggccga ccctatcggc
atcggcaagc attccccgat ccaagctagc 3300ttctaccaaa catcgactca
gtggatctca aggatgtgtc cgaccagcca gattttactt 3360aggcctccat
ccaaacagtc tctaaaacca ccttctacct atggcctgtt tggcacagct
3420ccagctccac ccctcctgga gctagagctc agccaaacag ttttagctcc
accaaaactg 3480agagtggagc taggtggagc tctctcacaa aatgaactag
agttgttgag ctgggtttag 3540gcagctccac aactttactc cagacccaac
ttctagagct aaatttagga gttggagcta 3600aattgagggc ttgttaaatt
tgtttggttt aaataattaa aaatgtcaaa tgtctggtcg 3660tgtaattcgg
atcaggagcg aatccagaaa atgaatggta ggagggcttg tctcattcct
3720cctctaaggg catctccaac agcttcccca aatcgaactc tccaaacact
catatagcca 3780actctccatc tgatttagct agtcaaatta gatactcact
ccaacagact ctctattaat 3840cctctccaaa ataaaaatag accctcagcc
tctaccatct tctttctctc tctcccccca 3900ccttccttct ctttccttct
ttttcccctt ttccttcctt cccgtcgacc acgttgatgc 3960cgacggtggc
tgacacatga cgcgcggtgg tgcagcaacg cggggaggcg gaaggcggtg
4020cggggaggcg gcgctcgctg aggtgctctc cctctccctt tgcggcgacg
aggcgacggc 4080ggcggagctc ggagcgggat ggcgatggcg gcggcggagc
tcggggtggg gatggcaaag 4140gcggcggctc tccacaacga cgacttggcg
aggcgacgac gacggcgagg aagatgcagc 4200cacgacagca catgggcagg
gatggcactg gaggcacatg gcggccacgg cgacggagtc 4260gagggagagg
cgcgggaagc cgcaaggggt ggccctctgc tcctcgccgg ccttgcctgg
4320tcctccgccg ccttgtcgcc gtggctccgc tggttccggg aggcaggggc
gagagagagg 4380agggagggag tgaggtggag cgcggggact cggttccagg
cgatggtgga agggggacgg 4440caggggaggc agggcaaaag aggcgctcgc
gcctccggtc catttcggcg gagggcggcg 4500atggaagcag ccgaggcagc
gttgaagagt ttggggacag agagaatgcg agatgtggga 4560agaaaaatat
ggttgtgggt cccacaatcc cacagttgga gagccagttt tggctggcca
4620agtttagcca cctagggagc ccctttggcc aaccaaatag cttcgagagt
aggatagcca 4680gtatgttgga gctcgttttt tttttcaaaa tttctaaatt
ttagtttggg gagtgaaata 4740gctatgctgt tagagatgct ctaagcccct
actctttttc tccctcttct tcctcctccc 4800atatctccct ttctcttaat
ggtcattcag cggggtaggg ggcttgagct cctagcacct 4860acgctggatc c
48712854DNARiceFragment comprising ORF11 gene 2tctttctctc
tctcccccca ccttccttct ctttccttct ttttcccctt ttccttcctt 60cccgtcgacc
acgttgatgc cgacggtggc tgacacatga cgcgcggtgg tgcagcaacg
120cggggaggcg gaaggcggtg cggggaggcg gcgctcgctg aggtgctctc
cctctccctt 180tgcggcgacg aggcgacggc ggcggagctc ggagcgggat
ggcgatggcg gcggcggagc 240tcggggtggg gatggcaaag gcggcggctc
tccacaacga cgacttggcg aggcgacgac 300gacggcgagg aagatgcagc
cacgacagca catgggcagg gatggcactg gaggcacatg 360gcggccacgg
cgacggagtc gagggagagg cgcgggaagc cgcaaggggt ggccctctgc
420tcctcgccgg ccttgcctgg tcctccgccg ccttgtcgcc gtggctccgc
tggttccggg 480aggcaggggc gagagagagg agggagggag tgaggtggag
cgcggggact cggttccagg 540cgatggtgga agggggacgg caggggaggc
agggcaaaag aggcgctcgc gcctccggtc 600catttcggcg gagggcggcg
atggaagcag ccgaggcagc gttgaagagt ttggggacag 660agagaatgcg
agatgtggga agaaaaatat ggttgtgggt cccacaatcc cacagttgga
720gagccagttt tggctggcca agtttagcca cctagggagc ccctttggcc
aaccaaatag 780cttcgagagt aggatagcca gtatgttgga gctcgttttt
tttttcaaaa tttctaaatt 840ttagtttggg gagt 854324DNAArtificial
SequencePrimer for the construction of RNA interference vector of
ORF11 3caccagcgtt gaagagtttg ggga 24420DNAArtificial SequencePrimer
for the construction of RNA interference vector of ORF11
4cgagctccaa catactggct 20524DNAArtificial SequencePrimer for the
constrcution of RNA interference vector of PPR2 5cacctgaaga
gtgcaaaccc tctg 24620DNAArtificial SequencePrimer for the
constrcution of RNA interference vector of PPR2 6ttcgcagact
ctcaacaagg 20726DNAArtificial SequencePrimer for determination of
Rf17 gene type 7tcgttcacca cggtagatag actcat 26825DNAArtificial
SequencePrimer for determination of Rf17 gene type 8cccacatctt
ctccttgcat aatcc 25
* * * * *
References