WO1997049831A1

WO1997049831A1 - Glyceraldehyde-3-phosphate dehydrogenase and nuclear restoration of cytoplasmic male sterility

Info

Publication number: WO1997049831A1
Application number: PCT/CA1997/000424
Authority: WO
Inventors: Gregory G. Brown; Benoit Landry; Martine Jean
Original assignee: Mcgill University
Priority date: 1996-06-26
Filing date: 1997-06-16
Publication date: 1997-12-31
Also published as: CZ424098A3; CA2258561A1; CA2258561C; AU732094B2; EP0954604A1; PL330793A1; JP2000512153A; HUP9904008A3; CN1228126A; AU3085797A; HUP9904008A2

Abstract

The present 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. There is provided a gene for nuclear restoration of cytoplamic male sterility, and more particularly to the use of a form of the gene encoding glyceraldehyde-3-phosphate dehydrogenase for this purpose. Finally, there is provided a method for the production of restorer lines directly through genetic transformation of plants with such a gene.

Description

GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE AND NUCLEAR RESTORATION OF CYTOPLASMIC MALE STERILITY

BACKGROUND OF THE INVENTION (a) 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 restora¬ tion of cytoplasmic male sterility, and more particu¬ larly to the use of a form of the gene encoding glycer- aldehyde-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, (b) 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 nec¬ essary 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 cyto¬ plasmic male sterility (CMS). The genetic determinants for CMS, the maternally transmitted inability to pro¬ duce 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 FI 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 (Rf), can be incorporated into the pollinating parent of the hybrid cross. Genotypes on which the male ster¬ ile 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 steril¬ ized by the CMS cytoplasm, must be available. The use of such lines in hybrid crop production is outlined in Fig. 1. The development of these lines through conven- tional 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 deleteri¬ ous 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 gen¬ eral process of indirect selection: progeny plants are first screened for genetic markers linked to the restorer gene rather than the restorer gene itself. 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 prog¬ eny 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 Rf gene 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 ( B. 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 plant does not guarantee the presence of the restorer gene in that plant. It therefore is necessary to employ a num¬ ber of the markers for indirect selection of plant con¬ taining the restorer gene. It would be highly desirable to be provided with a marker that is perfectly associated with nuclear res¬ toration of cytoplasmic male sterility.

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 cyto¬ plasmic male sterility. Another aim of the present invention is to pro¬ vide the use of glyceraldehyde-3-phosphate dehydro¬ genase 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 DNA sequence, a hybridizing fragment thereof or any DNA sequence derived therefrom for use as primers for amplification of glyceraldehyde-3-phosphate dehy¬ drogenase, wherein said DNA sequence or hybridizing fragment thereof hybridizes to specific DNA fragments characteristic of plants possessing a nuclear restorer gene under stringent conditions.

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 dehy¬ drogenase and surrounding sequences.

The surrounding sequences may be located 3 ' and/or 5 ' relative to the glyceraldehyde-3-phosphate dehydrogenase sequence and may be of about 50kb. 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 ste¬ rility gene of the present invention. In accordance with the present invention, any plant species may be used provided that the restorer gene in the plant species corresponds to a specific form of GAPC. Such species include, without limita- tion, Brassica napus, other Brassica species, maize ( Zea mays ) , rice ( Oryza sativum) , sunflower ( Helianthus annuum) and sorghum ( Sorghum bicolor) .

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic representation of the use of cytoplasmic male sterility (CMS) in hybrid seed pro¬ duction;

Fig. 2 shows the crosses used to identify a marker completely linked to the Rfpl restorer of fer- tility gene;

Figs. 3A to 3E show the comparison of Brassica napus cDNA clone cRFl (SEQ ID NO:l) with cytoplasmic glyceraldehyde-3-phosphate dehydrogenase (GAPC) cDNAs from Sinapis alba (SEQ ID NO:2) and Arabidopsis tha - liana (SEQ ID NO:3); and

Fig. 4 illustrates a gel of the polymorphism detected 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. 2). 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 par¬ ent. A new marker, designated cRFl, was found that is perfectly linked to this gene. Specifically, of the 175 individuals tested in the two crosses, all fertile 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 Rfpl -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 Rfpl- specific cRF1 specific cRF1 specific cRF1 specific cRF1 allele ^a"^ele allele allele

Westar x Westar-Rf 30 0 0 34

Karat x Westar-Rf 56 0 0 55

Total 86 0 0 89

Points of difference with previous solutions Because of the perfect linkage between cRFl 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 transforma¬ tion is not possible. This should result in a signifi¬ cant 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), i.e., a DNA com¬ plementary 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 , - 7 -

glyceraldehyde-3-phosphate dehydrogenase (Figs. 3A and 3B), which is encoded by the GAPC gene (Shih, M.-C. et al., 1991, Gene, 104:133-138). The perfect linkage between the restorer gene and the GAPC polymorphism 5 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 BC1 population in which the restorer gene for a dif¬ ferent B. napus CMS, the nap, system was segregating 0 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 sug¬ gests that other restorer genes may correspond to GAPC 5 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. 0 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). 5 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 0 method for the isolation of restorer genes and the development of restorer lines in many crops.

The present invention will be more readily un¬ derstood by referring to the following examples which are given to illustrate the invention rather than to 5 limit its scope. 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 possi¬ ble 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 fer¬ tile FI hybrid of A and B that contains the male ster¬ ile 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 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 char- acteristic 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 matur¬ ity in the field. In addition, the male fertile pheno- type 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 pro¬ cedure. 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 - 10 -

is available as a cloned DNA segment in a suitable plant Agrobacteri um tumefaciens transformation vector such as pRD400 (Datla RSS, Hammerlindl JK, Panchuk B, Pelcher LE & Keller W. (1992) Gene 211:383-384). 5 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, 0 and that the cloned restorer gene is identical to that of line R. Using the procedure described by Moloney et al. (Moloney, M., Walker, J. & Sharma, K. (1989) Plant Cell Rep. 8:238-242) an Agrobacteri um strain harboring the gene in the prRD400 vector is used to inoculate 5 cotyledons from strain B seedlings. The Agrobacterium is eliminated by antibiotic treatment and the resulting plant tissue is placed on media containing the antibi¬ otic kanamycin. pRD400 contains a gene that confers resistance to kanamycin, and hence cells that grow on 0 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 character- 5 istic 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 FI progeny from a cross between line A (as female) and the new transgenic line will also be male fertile. 0 This method has two distinct advantages: it is much faster and cheaper than conventional plant breed¬ ing approaches, requiring only a few months as opposed to years to develop this line. In addition, the pres¬ ence of the restorer gene will be the only difference 5 between the genome of line B and that of the new restorer line. Thus the integrity of the characteris¬ tics 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 trans¬ formation may differ from that described above.

While the invention has been described in con- nection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any varia¬ tions, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or 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.

- 12 -

SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) 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:

(A) ADDRESSEE: SWABEY OGILVY RENAULT

(B) STREET: 1981 McGill College Ave. - Suite 1600

(C) CITY: Montreal

(D) STATE: QC

(E) COUNTRY: Canada

(F) ZIP: H3A 2Y3

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: DOS

(D) SOFTWARE: FastSEQ for Windows Version 2.0

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 60/020,553

(B) FILING DATE: 26-JUN-1996

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Cόte, France

(B) REGISTRATION NUMBER: 4166

(C) REFERENCE/DOCKET NUMBER: 1770-152"PCT" FC

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 514 845-7126

(B) TELEFAX: 514-288-8389

(C) TELEX:

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1207 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

TCTCGATCTC ATCGACACCC TCTGATATCG AAATGGCTGA CAAGAAGATT AAGATCGGAA 60

TCAACGGTTT CGGAAGAATC GGTCGCTTGG TGGCTAGAGT TATCCTTCAG AGGAACGATG 120

TTGAGCTCGT CGCTGTTAAC GACCCCTTCA TCACCACCGA GTACATGACG TACATGTTTA 180

AGTATGACAG TGTTCACGGT CAGTGGAAGC ACAACGAGCT CAAGGTTAAG GATGAGAAGA 240

CACTTCTCTT CGGTGAGAAG CCTGTCACTG TTTTCGGCAT CAGGAACCCT GAGGATATGC 300

CCATGGGGTG AGGCTGGAGC TGACTTTGGG GTTGAGTCTA CTGGTGTCTT CACCGACAAG 360

GACAAGGCTG CTGCTCACTT GAAGGGTGGT GCGAAGAAAG TTGTCATCTC TGCACCAAGC 420

AAAGATGCTC CCATGTTCGT TGTTGGTGTC AATGAGCATG AGTACAAGTC TGATCTCAAC 480

ATTGTTTCCA ACGCTAGTGC ACCACTAACT GCCTTGCTCC ACTTGCCAAG GTTATCANCG 540

ACAGGTTTGG AATTGTCGAG GGACTCATGA CCACCGTCCA CTCTATCACT GCAACTCAGA 600

AGACAGTTGA TGGTCCATCA ATGAAGGACT GGAGAGGTGG AAGAGCCGCT TCCTTCAACA 660

TCATTCCCAG CAGCACCGGA GCTGCCAAGG CTGTCGGAAA GGTTCTTCCA CAGCTCAACG 720

GAAAGCTGAC CGGTATGTCC TTCCGTGTTC CCACCGTTGA TGTTTCAGTT GTTGACTCAC 780

GGTTAGACTC GAGAAAGCTG CAACCTACGA TGAAATCAAG AAGGCTATCA AGGAGGAATC 840

TGAGGGCAAG CTAAAGGGAA TCCTTGGTTA CACAGAGGAT GATGTTGTCT CAACCGACTT 900

CGTTGGTGAC AACAGGTCGA GCATTTTTGA CGCAAAGGCT GGAATCGCGT TGAGTGACAA 960

CTTTGTGAAG CTGGTGTCGT GGTACGACAA CGAATGGGGT TACAGTACCC GTGTGGTCGA 1020

CTTGATCATT CACATGTCCA AGGCCTAAGT CGATGAAGAT CTCGAGTGAT GTAATGGTGT 1080

TTTTAAATTG TTGTTTTTAT CGAATAAATT TTCTTGGGTT TTGAAACCTT TATGGTTTTG 1140

GCGAATTCTC TACTTTCACG TGACGTGATA AGAAGTTTGT AGACCGGTTG TTTTTTATTT 1200

TTACTGA 1207

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1091 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

TTTCGAAATG GCTGACAAGA AGATTAAGAT CGGAATCAAC GGTTTCGGAA GAATCGGTCG 60

TTTGGTGGCT AGAGTTATCC TTCAGAGGAA CGATGTTGAG CTCGTCGCTG TTAACGATCC 120

CTTCATCACC ACCGAGTACA TGACGTACAT GTTTAAGTAT GACAGTGTTC ATGGTCAGTG 180

GAAGCACAAT GAGCTCAAGG TGAAGGATGA GAAAACACTT CTCTTCGGAG AGAAGCCTGT 240

CACTGTTTTC GGCATCAGGA ACCCTGAGGA TATCCCATGG GGTGAGGCCG GAGCTGACTT 300

TGTTGTTGAG TCTACTGGTG TCTTCACTGA CAAGGACAAG GCTGCTGCTC ACTTGAAGGG 360

TGGTGCCAAG AAAGTTGTCA TCTCTGCACC AAGCAAAGAT GCTCCTATGT TCGTTGTTGG 420

TGTCAATGAG CATGAGTACA AGTCTGATCT CAACATTGTT TCCAACGCTA GTTGCACCAC 480

TAACTGCCTT GCTCCACTTG CCAAGGTTAT CAACGACAGG TTTGGAATTG TCGAGGGACT 540

CATGACTACT GTCCACTCTA TCACTGCTAC TCAGAAGACA GTTGATGGTC CATCAATGAA 600

GGACTGGAGA GGTGGAAGAG CCGCTTCCTT CAACATCATT CCCAGCAGCA CCGGAGCTGC 660

CAAGGCTGTC GGAAAGGTGC TTCCACAGCT CAATGGAAAA TTGACCGGAA TGTCCTTCCG 720

TGTTCCCACC GTTGATGTTT CAGTTGTCGA CCTCACGGTT AGACTCGAGA AAGCTGCAAC 780

CTACGATGAA ATCAAGAAGG CTATCAAGGA GGAGTCTCAG GGCAAGCTAA AGGGAATCCT 840

TGGTTACACA GAGGATGATG TTGTCTCAAC TGACTTCGTT GGTGACAACA GGTCGAGCAT 900

CTTTGACGCC AAGGCTGGAA TCGCATTGAG TGACAACTTC GTGAAGCTGG TGTCGTGGTA 960

TGACAACGAA TGGGGTTACA GTACCCGTGT GGTCGACTTG ATCATTCATA TGTCCAAGGC 1020

CTAAAACGCT GAAGATCTAC AATGATGTAA TGGTGTCTTA ATTTGTGGTT TTCGAATAAG 1080

ATTTCTTTGG G 1091 (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1295 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

CTCATCTTCA ACCTCTCTCT AACTCTCGTT TTCGATTCTA CAATGGCTGA CAAGAAGATT 60

AGGATCGGAA TCAACGGATT CGGAAGAATT GGTCGTTTGG TTGCTAGAGT TGTTCTCCAG 120

AGGGACGATG TTGAGCTCGT CGCTGTCAAC GACCCCTTCA TCACTACTGA GTACATGACC 180

TACATGTTCA AGTACGACAG TGTTCACGGT CAATGGAAAC ACAATGAACT CAAGATCAAG 240

GATGAGAAGA CCCTTCTCTT CGGTGAGAAG CCAGTCACTG TTTTCGGCAT CAGGAACCCT 300

GAGGATATCC CATGGGCCGA GGCTGGAGCT GACTACGTTG TTGAGTCTAC TGGTGTCTTC 360

ACTGACAAAG ACAAGGCTGC AGCTCACTTG AAGGGTGGTG CCAAGAAGGT TGTTATCTCT 420

GAACCCAGCA AAGACGCTCC AATGTTTGTT GTTGGTGTCA ACGAGCACGA ATACAAGTCC 480

GACCTTGACA TTGTCTCCAA CGCTAGCTGC ACCACTAACT GCCTTGCTCC CCTTGCCAAG 540

GTTATCAATG ACAGATTTGG AATTGTTGAG GGTCTTATGA CTACAGTCCA CTCAATCACT 600

GCTACTCAGA AGACTGTTGA TGGGCCTTCA ATGAAGGACT GGAGAGGTGG AAGAGCTGCT 660

TCATTCAACA TTATTCCCAG CAGCACTGGA GCTGCCAAGG CTGTCGGAAA GGTGCTTCCA 720

GCTCTTAACG GAAAGTTGAC TGGAATGTCT TTCCGTGTCC CAACCGTTGA TGTCTCAGTT 780

GTTGACCTTA CTGTCAGACT CGAGAAAGCT GCTACCTACG AAGAAATCAA AAAGGCTATC 840

AAGGAGGAAT CCGAAGGCAA ACTCAAGGGA ATCCTTGGAT ACACCGAGGA TGATGTTGTC 900

TCAACTGACT TCGTTGGCGA CAACAGGTCG AGCATTTTTG ACGCCAAGGC TGGAATTGCA 960

TTGAGCGACA AGTTTGTGAA ATTGGTGTCA TGGTACGACA ACGAATGGGG TTACAGTTCC 1020

CGTGTGGTCG ACTTGATCGT CCACATGTCA AAGGCCTAAG CTAAGAAGCA GATCTCGAAT 1080

GATAGGGAGT GGAAAGTCAT CTGTTCATCC CCTTTTATGG TCTGAATTTG TCGTTTTCGA 1140

ATAAAATTTC TTTGAACTTG GAACTTTTTT TTTTTTTGGT TTTCTTAATT CTCATTCATG 1200

TGAGGTGATG GGAGTTTGTA GACCGATGTT TTACTGGAAG CCCTTTGTTT TTGGCTTTTG 1260

ATATATTGAG TTAACGTTAT GGTTTTAAAA AAAAA 1295

Claims

- 15 -WHAT IS CLAIMED IS:

1. A probe specific for nuclear restoration of cytoplasmic male sterility of plants, which comprises a glyceraldehyde-3-phosphate dehydrogenase cDNA or genomic DNA sequence, a hybridizing fragment thereof or any DNA sequence derived therefrom for use as primers for amplification of glyceraldehyde-3-phosphate dehy¬ drogenase, wherein said DNA sequence or hybridizing fragment thereof hybridizes to specific DNA fragments characteristic of plants possessing a nuclear restorer gene under stringent conditions.

2. A gene for nuclear restoration of cytoplasmic male sterility in plants which comprises a DNA sequence encoding glyceraldehyde-3-phosphate dehydrogenase and surrounding sequences.

3. The gene of claim 2, wherein the surrounding sequences are located 3 ' and/or 5 ' relative to the glyceraldehyde-3-phosphate dehydrogenase sequence.

4. The gene of claim 3, wherein the surrounding sequences are of about 50kb.

5. A method of production of restorer lines, which comprises genetically transforming plants with the nuclear restoration of cytoplasmic male sterility gene of claim 2.