AU732094B2

AU732094B2 - Glyceraldehyde-3-phosphate dehydrogenase and nuclear restoration of cytoplasmic male sterility

Info

Publication number: AU732094B2
Application number: AU30857/97A
Authority: AU
Inventors: Gregory G. Brown; Martine Jean; Benoit Landry
Original assignee: Universite de Montreal; McGill University
Current assignee: Universite de Montreal; McGill University
Priority date: 1996-06-26
Filing date: 1997-06-16
Publication date: 2001-04-12
Anticipated expiration: 2017-06-16
Also published as: CZ424098A3; CA2258561A1; CA2258561C; EP0954604A1; PL330793A1; JP2000512153A; HUP9904008A3; CN1228126A; AU3085797A; WO1997049831A1; HUP9904008A2

Description

-WO 97/49831 PCT/CA97/00424 1 GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE AND NUCLEAR RESTORATION OF CYTOPLASMIC MALE STERILITY BACKGROUND OF THE INVENTION Field of the Invention The invention relates to a marker for nuclear restoration of cytoplasmic male sterility, and more particularly to the use of glyceraldehyde-3-phosphate dehydrogenase complementary DNA as such a marker. The invention also relates to a gene for nuclear restoration of cytoplasmic male sterility, and more particularly to the use of a form of the gene encoding glyceraldehyde-3-phosphate dehydrogenase for this purpose.

Finally, the invention relates to the production of restorer lines directly through genetic transformation of plants with such a gene.

Description of Prior Art Hybrids of different crop varieties may show yields that are considerably greater than those of the parental lines. This phenomenon is known as hybrid vigor. To implement the use of hybrid vigor it is necessary to have a method available for preventing self-pollination of one or both of the parent lines in the hybrid cross. Mechanical, chemical and genetic methods are available for accomplishing this. One established genetic method involves the trait of cytoplasmic male sterility (CMS). The genetic determinants for CMS, the maternally transmitted inability to produce viable pollen, reside on the mitochondrial genome.

Because CMS plants are male sterile, all of the seed that forms on them will necessarily be hybrid. Due to the maternal transmission of CMS, however, such Fl hybrids will also normally be male-sterile and hence be unable to self-fertilize and produce seed. To address this problem, specific nuclear genes that suppress the male sterile phenotype, termed restorers of fertility -WO 97/49831 PCT/CA97/fllM24 -WO 97/49831 PCTICA97/004l24 -2 can be incorporated into the pollinating parent of the hybrid cross. Genotypes on which the male sterile cytoplasm confers sterility are termed maintainers whereas those carrying Rf genes are termed restorers; the genes for the maintenance and restoration of CMS can be considered as different alleles (rf and Rf, respectively) at the same locus.

Shortcomings of present solutions To produce a diverse set of hybrids using CMS, adequate numbers of restorer lines, that contain Rf genes, as well as "maintainer" lines, that are sterilized by the CMS cytoplasm, must be available. The use of such lines in hybrid crop production is outlined in Fig. i. The development of these lines through conventional genetics is a slow process that minimally requires several years of effort and currently poses a major bottleneck in the generation of CMS-based hybrids in a number of crops, including canola, Canada's major cash crop. For example, to create a new restorer line it is necessary to first generate a hybrid between an existing restorer strain, which donates the Rf gene, and a recipient strain; a series of backcrosses to the recipient strain are then performed to incorporate the Rf gene without altering the strain's other desirable characteristics, a process termed introgression. Even after many generations some donor DNA that is linked to the Rf gene on the donor DNA will remain, a phenomenon termed linkage drag; this donor DNA may carry deleterious traits and compromise the quality of the recipient strain (Jean, M. et al., 1993, Current Topics in Molecular Genetics, 1:195-201).

This process can be expedited through the general process of indirect selection: progeny plants are first screened for genetic markers linked to the restorer gene rather than the restorer gene itself.

3 These markers are chosen such that they can be screened for at a very early stage in plant development. This circumvents the costly procedure of raising many progeny.

.plants to maturity and can considerably accelerate the introgression process. Restriction fragment length polymorphisms (RFLPs) represent a type of DNA marker that is ideally suited for this purpose. RFLPs are differences (between two genotypes) in restriction fragment patterns detected by specific DNA probes. Probes that detect fragment pattern differences between restorer and maintainer lines and that co-segregate with the Rfgene can be used to indirectly select for the restorer gene in a plant breeding program. We have obtained several probes that are linked to Rfpl, a restorer of the Polima or pol CMS, one of the two forms of CMS in canola napus)that is currently being used.in hybrid seed production. None of these markers is completely linked to the gene. This introduces an element of uncertainty into their use for indirect selection the presence of any one marker in a 20 plant does not guarantee the presence of the restorer gene in that plant. It therefore is necessary to employ a number of the markers for indirect selection of plant containing the restorer gene.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common 30 general knowledge in the field relevant to the present invention as it existed in Australia before the priority .date of each claim of this application.

It would be highly desirable to be provided with a marker that is perfectly associated with nuclear restoration of cytoplasmic male sterility.

4 This process can be further expedited through direct introduction of a cloned restorer gene. We believe that the probe we have identified, which show perfect linkage to Rfpl is detecting the restorer gene itself.

SUMMARY OF THE INVENTION One aim of the present invention is to provide a marker for nuclear restoration associated with cytoplasmic male sterility.

Another aim of the present invention is to provide the use of glyceraldehyde-3-phosphate dehydrogenase complementary DNA as such a restorer marker.

Another aim of the present invention is to be able to use this gene to produce restorer lines directly through genetic transformation.

In accordance with the present invention there is provided a probe specific for nuclear restoration of cytoplasmic male sterility of plants, which comprises a glyceraldehyde-3-phosphate dehydrogenase cDNA or genomic 20 DNA sequence, derived therefrom for use as primers for amplification of glyceraldehyde-3-phosphate dehydrogenase, wherein said DNA sequence or hybridising fragment thereof hybridizes to specific DNA fragments characteristic of plants possessing a nuclear restorer gene under stringent conditions.

Accordingly, in one aspect the invention provides a specific marker for detecting nuclear restoration of cytoplasmic male sterility of plants, which comprises a nucleic acid sequence encoding glyceraldehyde-3-phosphate 30 dehydrogenase or a hybridizing fragment thereof, wherein said nucleic acid sequence or hybridizing fragment thereof hybridizes to specific DNA fragments characteristic of plants possessing a nuclear restorer gene under stringent conditions, said nucleic acid sequence being as set forth in SEQ ID NO:1.

In accordance with the present invention there is also provided a gene for nuclear restoration of cytoplasmic male sterility in plants which comprises a DNA sequence encoding glyceraldehyde-3-phosphate dehydrogenase and surrounding sequences.

The surrounding sequences may be located 3' and/or relative to the glyceraldehyde-3-phosphate dehydrogenase sequence and may be of about In accordance with the present invention there is also provided a method of production of restorer lines, which comprises genetically transforming plants with the nuclear restoration of cytoplasmic male sterility gene of the present invention.

In another aspect the invention provides a method for detecting the presence of a restorer gene in nuclear genomic DNA, said method comprising the steps of: i) hybridizing said nuclear genomic DNA under stringent conditions with a probe specific for nuclear restoration of cytoplasmic male sterility of plants, said 20 probe comprises a nucleic acid sequence encoding for glyceraldehyde-3-phosphate dehydrogenase, wherein said nucleic acid sequence hybridizes to specific DNA fragments characteristic of plants possessing a nuclear restorer gene under stringent conditions; and ii) detecting hybridization of said probe with said nuclear genomic DNA, wherein hybridization of said probe with said nuclear genomic DNA is indicative of said nuclear genomic DNA containing a restorer gene.

In accordance with the present invention, any plant 30 species may be used provided that the restorer gene in the plant species corresponds to a specific form of GAPC.

Such species include, without limitation, Brassica napus, other Brassica species, maize (Zea mays), rice (Oryza sativum), sunflower (Helianthus annuum) and sorghum (Sorghum bicolor).

In another aspect the invention provides a primer DNA Sequence derived from a novel marker as previously described.

In another aspect the invention provides a gene for nuclear restoration of cytoplasmic male sterility in plants which comprises a DNA sequence encoding glyceraldehyde-3-phosphate dehydrogenase and surrounding sequences and which is detected by the novel methods previously described.

In another aspect the invention provides a method of production of restorer lines, which comprises genetically transforming plants with the nuclear restoration of cytoplasmic male sterility gene as previously described.

In another aspect the invention provides the use of a novel marker as previously described for detecting nuclear restoration of cytoplasmic male sterility of plants.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic representation of the use of 20 cytoplasmic male sterility (CMS) in hybrid seed production; Fig. 2 shows the crosses used to identify a marker completely linked to the Rfpl restorer of fertility gene; Figs. 3A to 3E show the comparison of Brassica napus cDNA clone cRF1 (SEQ ID NO:1) with cytoplasmic glyceraldehyde-3-phosphate dehydrogenase (GAPC) cDNAs from Sinapis alba (SEQ ID NO:2) and Arabidopsis thaliana (SEQ ID NO:3); and Fig. 4 illustrates a gel of the polymorphism detected 30 by cRFl probe in Brassica napus in a genetic population segregating for the Rfpl gene.

DETAILED DESCRIPTION OF THE INVENTION We continued an analysis of two genetic crosses which gave rise to plant populations in which the restorer gene was segregating (outlined in Fig. In each case, the nature of the cross was such that for linked markers, most sterile progeny individuals would show the RFLP characteristic of the male sterile parent of the cross, while most male fertile progeny plants would show the RFLP characteristic of the fertile parent. A new marker, designated cRF1, was found that is perfectly linked to this gene. Specifically, of the 175 individuals tested in the two crosses, all fertile o* **oe* e* *,o -WO 97/49831 PCT/CA97/f0424 6 progeny were found to possess the allele (or form) of the fertile parent while all sterile plants were found to possess the allele of the sterile parent (Table 1).

cRFl therefore represents a particularly powerful tool for indirect selection of the restorer gene.

Table 1 Co-segregation of an Rfp 1-specific RFLP allele detected by the probe cRF1 (GAPC)with male fertility restoration in 2 Brassica napus backcross populations Cross Fertile progeny plants Sterile progeny plants with Rfpl- without Rfpl- with Rfpl- without Rfplspecific cRF1 specific cRF1 specific cRF1 specific cRF1 allele allele allele allele Westar x Westar-Rf 30 0 0 34 Karat x Westar-Rf 56 0 0 Total 86 0 0 89 Points of difference with previous solutions Because of the perfect linkage between cRF1 and Rfpl, the uncertainty in the use of this probe for indirect selection of the restorer gene is virtually eliminated.

In addition, no restorer gene for the Polima or pol CMS system has been isolated and hence production of restorer lines directly through genetic transformation is not possible. This should result in a significant reduction of the cost of the use of indirect selection in the development of new restorer (Fig. 4) lines.

The DNA probe that detected this polymorphism is a B. napus complementary DNA (cDNA), a DNA complementary to a messenger RNA molecule (mRNA). The DNA sequence of this cDNA was determined. Analysis of a nucleotide sequence database indicated that the cDNA's sequence is 99% similar to that of a cytoplasmic form of a glycolytic enzyme from Arabidopsis thaliana, mmm VO 97/49831 PCT/CA97/00424 7 glyceraldehyde-3-phosphate dehydrogenase (Figs. 3A and 3B), which is encoded by the GAPC gene (Shih, et al., 1991, Gene, 104:133-138). The perfect linkage between the restorer gene and the GAPC polymorphism leads us to believe that the restorer gene is likely to be specific form of GAPC.

We have conducted a similar type of analysis on a BCl population in which the restorer gene for a different B. napus CMS, the nap, system was segregating and found that the nap restorer was simply a different allele of the same genetic locus. Thus different forms of GAPC correspond to two different nuclear fertility restorer genes in B. napus. This result further suggests that other restorer genes may correspond to GAPC isoforms and that the relationship between GAPC and restorer genes may extend to other CMS systems in other plant species. No relationship between GAPC and restorer genes for any plant species has been suggested previously.

With this gene it may therefore be possible to construct restorer .lines in a single step by using genetic transformation to introduce the restorer-specific GAPC gene into maintainer genotypes (genotypes that do not naturally contain the restorer).

This would be extremely cost effective as it would eliminate many steps in the plant breeding process necessary for the development of such lines. If the association between GAPC and restorer genes is extended to other crop species, this would represent a general method for the isolation of restorer genes and the development of restorer lines in many crops.

The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

-WO 97/49831 PCT/CA97/00424 8 EXAMPLE I Use of a GAPC probe as an indirect selection marker in the production of a new restorer cell line Three plant genotypes will be considered: A a CMS line; B a male fertile line that lacks the restorer gene and contains a male fertile cytoplasm; and R a male fertile line that contains the restorer gene and a male sterile cytoplasm.

It will be assumed that hybrids between lines A and B that are produced by manual genetic crosses show considerable hybrid vigour; hybrids between A and R do not. As line B lacks a restorer gene, it is not possible to produce male fertile hybrids of these two lines using CMS. If, however, the restorer gene could be transferred from line R to line B without otherwise altering the characteristics of line B, it would be possible to obtain male fertile hybrids between lines A and B using CMS. Traditionally, this would be done through a process termed introgression. Line R is crossed as a female with line B to produce a male fertile Fl hybrid of A and B that contains the male sterile cytoplasm (the cytoplasm of a hybrid is derived exclusively from the female parent) but is also male fertile because it has received a single copy of the restorer gene from the line R parent. A second cross (termed a backcross) is then performed between the hybrid (as female) and the line B. Large numbers of progeny grown are in the field, and equal numbers of steriles and fertiles are expected, fertiles possessing the restorer gene. One or more fertiles are then used as females in a second backcross to line B; fertile plants are recovered and crossed as females to line B for a third time. This process is repeated for many generations; with each new generation the progeny are -WO 97/49831 PCT/CA97/00424 9 expected to become more similar to line B (except they will possess the restorer gene). At each generation various characteristics associated with line B will be assessed. Eventually, new restorer line, with all or most of the desirable characteristics of line B will be produced. This line could then be used for the large scale production of hybrids between lines A and B.

The GAPC probe facilitates this process because it allows for the assessment of the presence of the restorer gene in progeny plants at the seedling stage.

DNA is extracted from a small amount of leaf material, digested with a restriction endonuclease, such as HindIII (used in Fig. 4) and analyzed using the GAPC probe. The presence of the restriction fragment characteristic of the restorer gene indicates that the seedling has the restorer gene. Very large numbers of plants at the seedling stage are screened at much lower cost that the cost of raising the same plants to maturity in the field. In addition, the male fertile phenotype is affected by many different conditions and screening for the presence of the gene by screening for a perfectly linked polymorphism more reliably detect the presence of the gene during this introgression procedure.

EXAMPLE II Production of new restorer cell lines through the introduction of the restorer gene form of GAPC via transformation The three plant genotypes of Example I will be considered in accordance with this procedure.

In this example, the problem is precisely the same as that of Example I, namely the transfer of the restorer gene from line R into line B without otherwise altering the characteristics of line B. In this case, however, we will assume that the form of the GAPC gene that represents the restorer gene has been isolated and WO 97/49831 PCT/CA97/n1424 10 is available as a cloned DNA segment in a suitable plant Agrobacterium tumefaciens transformation vector such as pRD400 (Datla RSS, Hammerlindl JK, Panchuk B, Pelcher LE Keller W. (1992) Gene 211:383-384).

Instead of the lengthy backcrossing program described in Example I, the GAPC gene is transferred to line B through Agrobacterium-mediated transformation.

For the sake of this example, we will also assume that lines A, B and R are Brassica napus lines, and that the cloned restorer gene is identical to that of line R. Using the procedure described by Moloney et al. (Moloney, Walker, J. Sharma, K. (1989) Plant Cell Rep. 8:238-242) an Agrobacterium strain harboring the gene in the prRD400 vector is used to inoculate cotyledons from strain B seedlings. The Agrobacterium is eliminated by antibiotic treatment and the resulting plant tissue is placed on media containing the antibiotic kanamycin. pRD400 contains a gene that confers resistance to kanamycin, and hence cells that grow on this antibiotic are likely have acquired the kanamycin gene, along with the restorer gene which is cloned into pRD400. The presence of the restorer gene in these plants is then assessed directly by testing the plants form the presence of restriction fragments characteristic of the restorer using a GAPC probe. It is expected that these plants will be made fertile if they contain the male sterile cytoplasm and that Fl progeny from a cross between line A (as female) and the new transgenic line will also be male fertile.

This method has two distinct advantages: it is much faster and cheaper than conventional plant breeding approaches, requiring only a few months as opposed to years to develop this line. In addition, the presence of the restorer gene will be the only difference between the genome of line B and that of the new restorer line. Thus the integrity of the characteristics of line B are less likely to be compromised.

Although the above description relates to a specific plant species, Brassica napus, the invention could be applied to other species provided that the restorer gene in the species corresponds to a specific form of GAPC. In such cases the technique for transformation may differ from that described above.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variation, uses, or adaptions of the invention following, in general, the 20 principals of the invention and including such departures from the present disclosure as come within known or o customary practice within the art to which the invention pertains and as may be applied to the essential features *hereinbefore set forth, and as follows in the scope of the appended claims.

.ooooi oooo oooo oooo ooooS -WO 97/49831 PCT/CA97/00424 12 SEQUENCE LISTING GENERAL INFORMATION APPLICANT: McGILL UNIVERSITY et al.

(ii) TITLE OF THE INVENTION: GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE AND NUCLEAR RESTORATION OF CYTOPLASMIC MALE

STERILITY

(iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: SWABEY OGILVY RENAULT STREET: 1981 McGill College Ave. Suite 1600 CITY: Montreal STATE: QC COUNTRY: Canada ZIP: H3A 2Y3 COMPUTER READABLE FORM: MEDIUM TYPE: Diskette COMPUTER: IBM Compatible OPERATING SYSTEM: DOS SOFTWARE: FastSEQ for Windows Version (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 60/020,553 FILING DATE: 26-JUN-1996 (viii) ATTORNEY/AGENT INFORMATION: NAME: C6te, France REGISTRATION NUMBER: 4166 REFERENCE/DOCKET NUMBER: 1770-152"PCT" FC (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 514 845-7126 TELEFAX: 514-288-8389

TELEX:

INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1207 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA -WO 97/4983 13 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: PCT/CA97/00424

TCTCGATCTC

TCAACGGTTT

TTGAGCTCGT

AGTATGACAG

CACTTCTCTT

CCATGGGGTG

GACAAGGCTG

AAAGATGCTC

ATTGTTTCCA

ACAGGTTTGG

AGACAGTTGA

TCATTCCCAG

GAAAGCTGAC

GGTTAGACTC

TGAGGGCAAG

CGTTGGTGAC

CTTTGTGAAG

CTTGATCATT

TTTTAAATTG

GCGAATTCTC

TTACTGA

ATCGACACCC

CGGAAGALTC

CGCTGTTAAC

TGTTCACGGT

CGGTGAGAAG

AGGCTGGAGC

CTGCTCACTT

CCATGTTCGT

ACGCTAGTGC

AATTGTCGAG

TGGTCCATCA

CAGCACCGGA

CGGTATGTCC

GAGAAAGCTG

CTAAAGGGAA

AACAGGTCGA

CTGGTGTCGT

CACATGTCCA

TTGTTTTTAT

TACTTTCACG

TCTGATATCG

GGTCGCTTGG

GACCCCTTCA

CAGTGGAAGC

CCTGTCACTG

TGACTTTGGG

GAAGGGTGGT

TGTTGGTGTC

ACCACTAACT

GGACTCATGA

ATGAAGGACT

GCTGCCAAGG

TTCCGTGTTC

CAACCTACGA

TCCTTGGTTA

GCATTTTTGA

GGTACGACAA

AGGCCTAA.GT

CGAATAAATT

TGACGTGATA

AAATGGCTGA

TGGCTAGAGT

TCACCACCGA

ACAACGAGCT

TTTTCGGCAT

GTTGAGTCTA

GCGAAGAAAG

APLTGAGCATG

GCCTTGCTCC

CCACCGTCCA

GGAGAGGTGG

CTGTCGGAAA

CCACCGTTGA

TGAAATCAAG

CACAGAGGAT

CGCAAAGGCT

CGAATGGGGT

CGATGAAGAT

TTCTTGGGTT

AGAAGTTTGT

CAAGAAGATT

TATCCTTCAG

GTACATGACG

CAAGGTTAAG

CAGGAACCCT

CTGGTGTCTT

TTGTCATCTC

AGTACAAGTC

ACTTGCCAAG

CTCTATCACT

AAGAGCCGCT

GGTTCTTCCA

TGTTTCAGTT

AAGGCTATCA

GATGTTGTCT

GGAATCGCGT

TACAGTACCC

CTCGAGTGAT

TTGAAACCTT

AGACCGGTTG

AAGATCGGAA

AGGAACGATG

TACATGTTTA

GATGAGAAGA

GAGGATATGC

CACCGACAAG

TGCACCAAGC

TGATCTCAAC

GTTATCANCG

GCAACTCAGA

TCCTTCAACA

CAGCTCAACG

GTTGACTCAC

AGGAGGAATC

CAACCGACTT

TGAGTGACAA

GTGTGGTCGA

GTAATGGTGT

TATGGTTTTG

TTTTTTATTT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1207 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 1091 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

TTTCGAAATG

TTTGGTGGCT

CTT CAT CACC

GAAGCACAAT

CACTGTTTTC

TGTTGTTGAG

TGGTGCCAAG

TGTCAATGAG

TAACTGCCTT

CATGACTACT

GGACTGGAGA

CAAGGCTGTC

TGTTCCCACC

CTACGATGAA

TGGTTACACA

CTTTGACGCC

TGACAACGAA

CTAAAACGCT

ATTTCTTTGG

GCTGACAAGA

AGAGTTATCC

ACCGAGTACA

GAGCTCAAGG

GGCATCAGGA

TCTACTGGTG

AAAGTTGTCA

CATGAGTACA

GCTCCACTTG

GTCCACTCTA

GGTGGAAGAG

GGAAAGGTGC

GTTGATGTTT

ATCAAGAAGG

GAGGATGATG

AAGGCTGGAA

TGGGGTTACA

GAAGATCTAC

G

AGATTAAGAT

TTCAGAGGAA

TGACGTACAT

TGAAGGATGA

ACCCTGAGGA

TCTTCACTGA

TCTCTGCACC

AGTCTGATCT

CCAAGGTTAT

TCACTGCTAC

CCGCTTCCTT

TTCCACAGCT

CAGTTGTCGA

CTATCAAGGA

TTGTCTCAAC

TCGCATTGAG

GTACCCGTGT

AATGATGTAA

CGGAATCAAC

CGATGTTGAG

GTTTAAGTAT

GAAAACACTT

TATCCCATGG

CAAGGACAAG

AAGCAAAGAT

CAACATTGTT

CAACGACAGG

TCAGAAGACA

CAACATCATT

CAATGGAAAA

CCTCACGGTT

GGAGTCTCAG

TGACTTCGTT

TGACAACTTC

GGTCGACTTG

TGGTGTCTT.A

GGTTTCGGAA

CTCGTCGCTG

GACAGTGTTC

CTCTTCGGAG

GGTGAGGCCG

GCTGCTGCTC

GCTCCTATGT

TCC.AACGCTA

TTTGGAATTG

GTTGATGGTC

CCCAGCAGCA

TTGACCGGAA

AGACTCGAGA

GGCAAGCTAA

GGTGACAACA

GTGAAGCTGG

ATCATTCATA

ATTTGTGGTT

GAATCGGTCG

TTAACGATCC

ATGGTCAGTG

AGAAGCCTGT

GAGCTGACTT

ACTTGAAGGG

TCGTTGTTGG

GTTGCACCAC

TCGAGGGACT

CATCAATGAA

CCGGAGCTGC

TGTCCTTCCG

AAGCTGCAAC

AGGGAATCCT

GGTCGAGCAT

TGTCGTGGTA

TGTCCAAGGC

TTCGAATAAG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1091 WO 97/49831 -14 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 1295 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: PCT/CA97/00424

CTCATCTTCA

AGGATCGGAA

AGGGxACGtATG

TACATGTTCA

GATGAGAAGA

GAGGATATCC

ACT GACAAAG

GAACCCAGCA

GACCTTGACA

GTTATCAATG

GCTACTCAGA

TCATTCAACA

GCTCTTAACG

GTTGACCTTA

AAGGAGGAAT

TCAACTGACT

TTGAGCGACA

CGTGTGGTCG

GATAGGGAGT

ATAAAATTTC

TGAGGTGATG

ATATATTGAG

ACCTCTCTCT

TCAACGGATT

TTGAGCTCGT

AGTACGACAG

CCCTTCTCTT

CAT GGGCCGA

ACAAGGCTGC

AAGACGCTCC

TTGTCTCCAA.

ACAGATTTGG

AGACTGTTGA

TTATTCCCAG

GAAAGTTGAC

CTGTCAGACT

CCGAAGGCAA

TCGTTGGCGA

AGTTTGTGAA

ACTTGATCGT

GGAAAGTCAT

TTTGAACTTG

GGAGTTTGTA

TTAACGTTAT

AACTCTCGTT

CGGAAGAATT

CGCTGTCAAC

TGTTCACGGT

CGGTGAGAAG

GGCTGGAGCT

AGCTCACTTG

AATGTTTGTT

CGCTAGCTGC

AATTGTTGAG

TGGGCCTTCA

CAGCACTGGA

TGGAATGTCT

CGAGAAAGCT

ACT CAAGGGA

CAACAGGTCG

ATTGGTGTCA

CCACATGTCA

CTGTTCATCC

GAACTTTTTT

GACCGATGTT

GGTTTTAAAA

TTCGATTCTA

GGTCGTTTGG

GACCCCTTCA

CAATGGAA.AC

CCAGTCACTG

GACTACGTTG

AAGGGTGGTG

GTTGGTGTCA

ACCACTAACT

GGTCTTATGA

ATGAAGGACT

GCTGCCAA.GG

TTCCGTGTCC

CAATGGCTGA

TTGCTAGAGT

TCACTACTGA

ACAATGAACT

TTTTCGGCAT

TTGAGTCTAC

CCAAGAAGGT

ACGAGCACGA

GCCTTGCTCC

CTACAGTCCA

GGAGAGGTGG

CTGTCGGAAA

CAACCGTTGA

CAAGAAGATT

TGTTCTCCAG

GTACATGACC

CAAGATCAAG

CAGGAACCCT

TGGTGTCTTC

TGTTATCTCT

ATACAAGTCC

CCTTGCCAAG

CTCAATCACT

AAGAGCTGCT

GGTGCTTCCA

TGTCTCA.GTT

AAAGGCTATC

TGATGTTGTC

TGGAATTGCA

TTACAGTTCC

GATCTCGAAT

TCGTTTTCGA

CTCATTCATG

TTGGCTTTTG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1295 GCTACCTACG AAGAAATCAA ATCCTTGGAT ACACCGAGGA AGCATTTTTG ACGCCAAGGC TGGTACGACA ACGAATGGGG A.AGGCCTAAG CTAAGAAGCA CCTTTTATGG TCTGAATTTG TTTTTTTGGT TTTCTTAATT TTACTGGAAG CCCTTTGTTT

AAAAA

Claims

1. A specific marker for detecting nuclear restoration of cytoplasmic male sterility of plants, which comprises a nucleic acid sequence encoding glyceraldehyde-3-phosphate dehydrogenase or a hybridizing fragment thereof, wherein said nucleic acid sequence or hybridizing fragment thereof hybridizes to specific DNA fragments characteristic of plants possessing a nuclear restorer gene under stringent conditions, said nucleic acid sequence being as set forth in SEQ ID NO:1.

2. A primer DNA sequence derived from a marker as defined in claim 1.

3. A method for detecting the presence of a restorer gene in nuclear genomic DNA, said method comprising the steps of: i) hybridizing said nuclear genomic DNA under stringent conditions with a probe specific for nuclear 20 restoration of cytoplasmic male sterility of plants, said probe comprises a nucleic acid sequence encoding for glyceraldehyde-3-phosphate dehydrogenase, wherein said nucleic acid sequence hybridizes to specific DNA fragments characteristic of plants possessing a nuclear restorer gene under stringent conditions; and ii) detecting hybridization of said probe with said nuclear genomic DNA, wherein hybridization of said probe with said nuclear genomic DNA is indicative of said nuclear genomic DNA containing a restorer gene. S.4. A method for detecting the presence of a restorer gene in nuclear genomic DNA, said method comprising the steps of: i) amplifying said nuclear genomic DNA under suitable conditions with a primer DNA sequence as defined o' in claim 2; and h 16 ii) detecting amplification of said nuclear genomic DNA with said primer DNA sequence, wherein amplification of said nuclear genomic DNA is indicative of said nuclear genomic DNA containing a restorer gene. The method of claim 3, where the probe comprises a nucleic acid as set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

6. The method of claim 4, where the probe comprises a nucleic acid as set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

7. A gene for nuclear restoration of cytoplasmic male sterility in plants which comprises a DNA sequence encoding glyceraldehyde-3-phosphate dehydrogenase and surrounding sequences and which is detected by the method of claim 3, 4, 5 or 6. S 20 8. The gene of claim 7, wherein the surrounding sequences are located 3' and/or 5' relative to the glyceraldehyde-3-phosphate dehydrogenase sequence. S* 9. A method of production of restorer lines, which comprises genetically transforming plants with the nuclear restoration of cytoplasmic male sterility gene of claim 7.

10. Use of a marker as defined in claim 1 for detecting nuclear restoration of cytoplasmic male sterility of 30 plants.

11. A marker according to claim 1 substantially as hereinbefore described with reference to the examples. Qq 17

12. A primer DNA sequence according to claim 2 substantially as hereinbefore described with reference to the examples.

13. A method according to 3 or 9 substantially as hereinbefore described with reference to the examples.

14. A gene according to claim 7 or 8 substantially as hereinbefore described with reference to the examples. 0 Use according to claim 10 substantially as hereinbefore described with reference to the examples. Dated this 1 3 t h day of February 2001 McGill University and Universite De Montreal Patent Attorneys for the Applicant: F B RICE CO S.. S. S. S S 5.55 S S 555555 S S5 S W~