CZ424098A3 - Glyceraldehyde-3-phosphatodehydrogenase and nuclear reconstruction of cytoplasmatic male sterility - Google Patents

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 reconstruction of cytoplasmic male sterility.

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Technical field

The invention relates to an indicator of nuclear reconstruction of cytoplasmic male sterility, particularly when such a marker is complementary DNA of glyceraldehyde-3-phosphate dehydrogenase. The invention also relates to a gene for nuclear reconstruction of cytoplasmic male sterility, in particular to the use of a form of the gene encoding glyceraldehyde-3-phosphate dehydrogenase, which has been created for this purpose. The present invention relates to the production of a restorer which results from the genetic transformation of plants by said gene.

BACKGROUND OF THE INVENTION

Hybrids of different crop varieties can show yields that are significantly higher than those of the parental lines. This phenomenon is known as hybrid vitality. Tiby has achieved hybrid growth, it is necessary to have a method available to prevent self-pollination of one or both parental lines in hybrid crossing. Mechanical, chemical and genetic methods can be used to accomplish this. The established genetic method includes a feature of cytoplasmic male sterility (CMS). The genetic determinants of CMS, which means the inability to produce viable pollen that is transmitted from the mother, are found on the mitochondrial genome. Because in plants with CMS, males are sterile, all the seeds produced on them will necessarily be hybrid. Due to maternal CMS transfer, males will normally also be sterile in F1 hybrids and therefore will not be able to fertilize themselves and produce seeds. In order to solve this problem, specific nuclear genes that suppress the phenotype of mammalian sterility can be introduced into the pollinating parents of the hybrid cross. These genes are called fertility restorers (Rf). Genotypes that cause male cytoplasmic sterility are called 'maintainers', while genotypes that carry Rf genes are called restorers; genes for maintaining and normalizing CMS are considered different alleles (rf and Rf) at the same locus.

Imperfect current solutions.

In order to produce a different set of hybrids using CMS, an adequate number of restorer lines containing the Rf genes as well as a "maintainer line" sterilized by the CMS cytoplasm must be available. The use of such lines in hybrid crop production is illustrated in Figure 1. The development of these lines through classical genetics is a slow process requiring at least several years of effort, while at the same time the main obstacle is the formation of CMS-based hybrids in a number of crops including rape type canola, which is Canada's main crop. In order to create a new restoration line, it is first necessary to generate a hybrid between an existing restorer strain which is a donor of the Rf gene and a recipient strain; then a series of back crosses is made on the recipient strain to incorporate the Rf gene without altering other desired strain characteristics. This process is called introgression. Even for many generations, some donor DNA that is linked to the Rf gene on donor DNA is retained, a phenomenon called "linkage drag; this donor DNA may carry harmful events 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 accelerated through the general process of indirect selection in progeny plants first looking for genetic markers associated with the restorer gene rather than the restorer gene itself. These markers were selected so that their presence can be tested at a very early stage of plant development. This bypasses the costly process of generating a number of adult offspring of plants and can noticeably speed up the process.

introgrese. The restriction fragment length polymorphism (RFLP) represents the type of marker DNA that is suitable for this purpose. RFLPs (between two phenotypes) differ in restriction fragment patterns that are detected using specific DNA probes. Probes that detect fragment pattern differences between normalizer and maintainer lines and that cosegregate with the Rf gene can be used in indirect selection of the restorer gene in a plant breeding program. Several probes have been obtained that associate with Rfpl, the Polima restorer gene or pol CMS, one of the two forms of CMS in canola (B. napus) rape, which is currently used in the production of hibernal seeds. None of these markers are fully linked to the gene. This brings some confusion into their use in indirect selection; the presence of any one marker in a plant does not mean that a restorer gene is also present in the same plant. It is therefore necessary to use a number of markers in the indirect selection of plants containing the restorer gene.

It is necessary to create a marker that is perfectly associated with nuclear normalization of cytoplasmic mammalian sterility.

This process can be further accelerated by the introduction of a cloned restorer gene. It is believed that an identified probe that shows a perfect fit with Rfpl is able to detect the restorer gene itself.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a marker for nuclear reconstruction associated with cytoplasmic mammalian sterility.

Furthermore, the invention describes the use of complementary DNA glyceraldehyde-3-phosphate dehydrogenase as said restorer marker.

Further, the invention describes the ability to use this gene in the production of restorer lines directly through genetic transformation.

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The invention further provides a probe specific for nuclear reconstruction of cytoplasmic mammalian sterility of plants comprising a glyceraldehyde-3-phosphate dehydrogenase cDNA, or a genomic DNA sequence, a hybridizing fragment thereof, or any DNA sequence derived therefrom, which can be used as primers in glyceraldehyde-3 amplification. -phosphate dehydrogenase, wherein said DNA sequence or hybridizing fragment thereof hybridizes under limited conditions to specific DNA fragments that are characteristic of plants having a nuclear restorer gene.

The invention also provides a gene for nuclear reconstruction of cytoplasmic male sterility in plants comprising a DNA sequence encoding glyceraldehyde-3-phosphate dehydrogenase and surrounding sequences.

Surrounding sequences may be located at the 3 'end and / or at the 5' end of the glyceraldehyde-3-phosphate dehydrogenase sequence and may contain about 50 kb.

In accordance with the invention, there is provided a method for producing a line of restorers comprising genetically transformed plants with nuclear normalization of the cytoplasmic male sterility gene of the invention.

Any plant species can be used to match the specific form of GAPC in the plant species to the restorer gene of the invention.

Such species include, but are not limited to, Brassica napus, other Brassica species, maize (Zea mays), rice (Oryza sativum), sunflower (Helianthus annuum) and millet (Sorghum bicolor).

The analysis of genetic cross-breeding continued, resulting in plant populations in which the restorer gene was segregated (shown in Figure 2). In any case, the nature of the cross was that in the case of pooled markers, most sterile offspring individuals would exhibit RFLPs characteristic of a sterile male parent, while most mammalian fertile offspring of plants would exhibit RFLPs

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0 0 0 · · characteristic of a fertile parent. It has been found that a new marker, designated cRF1, is perfectly linked to this gene. Especially out of 175 subjects in two crosses, all fertile offspring carry the allele (or form) of the fertile parent, while all sterile plants have the allele of the sterile parent (Table 1). The cRF1 marker therefore represents a particularly potent instrument for indirect restorer gene selection.

Table 1

Co-segregation of the Rfpl-specific RFLP allele by probe detection cRF1 (GAPC) with male fertility reconstruction at 2 backwards crossed Brassica napus populations. Cross Fertile offspring Sterile descendants With Rfplspecific cRF1 allele Without the Rfplspecific cRF1 allele With Rfplspecific cRF1 allele Without the Rfplspecific cRF1 allele Westar x Westar-Rf 30 0 0 34 Karat x Westar-Rf 56 0 0 55 Total 86 0 0 89

Different points compared to previous solutions

Due to the perfect linkage between cRF1 and Rpf1, confusion in using this probe in indirect selection of the restorer gene was eliminated.

Moreover, no restorer gene for Polima or pol CMS has been isolated and therefore restorer lines cannot be produced directly through genetic transformation. This leads to a substantial reduction in the costs of using indirect selection in the development of new restorer lines (Fig. 4).

The DNA probe that detected this polymorphism is complementary B. napus DNA (cDNA), i.e., DNA complementary to a messenger RNA (mRNA) molecule. Determined * r 99

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9-9 the DNA sequence of this cDNA. Analysis of the nucleotide sequence database indicates that the cDNA sequence is 99% similar to the cytoplasmic form of the glycolytic enzyme from Arabidopsis thaliana, glyceraldehyde-3-phosphate dehydrogenase (Figures 3A and 3B), which is encoded by the GAPC gene (Shih, M.C. et al., 1991, Gene, 104: 133-138). The perfect link between the restorer gene and the GAPC polymorphism supports the idea that the restorer gene is probably a specific form of GAPC.

A similar type of analysis was performed on the BC1 population where the restorer gene system for the various B. napus CMS, nap, was segregated and it was found that the nap restorer was simply a different allele of the same genetic locus. Then the different forms of GAPC correspond to two different genes of nuclear normalization of fertility in B. napus. This leads to a further suggestion 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. Previously, there was no indication of a relationship between GAPC and restorer genes.

Therefore, these genes can be used in the construction of restorer lines in one step using genetic transformation to introduce the restorer-specific GAPC gene into the "maintainer" genotype (a genotype that does not naturally involve the restorer). Since this eliminates many steps in the plant breeding process necessary to develop such lines, it extremely reduces costs. If the association between GAPC and restorer genes is extended to other crop types, it will represent a general method for isolating restorer genes and developing restorer lines for many crops.

Overview of the drawings

Figure 1 is a schematic representation of the use of cytoplasmic male sterility (CMS) in hybrid seed production;

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Figure 2 shows the crosses used to identify a marker fully associated with the restorer of the Rpf1 fertility gene;

Figures 3A to 3E show a comparison of cDNA of Brassica napus clone cRF1 (SEQ ID NO: 1) with cytoplasmic glyceraldehyde-3-phosphate dehydrogenase (GAPC) cDNA from Sinapis alba (SEQ ID NO: 2) and Arabidopsis thaliana (SEQ ID NO: 3); and

Figure 4 shows a polymorphism gel detected by the cRF1 probe in Brassica napus in a genetic population segregating the Rfpl1 gene.

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Use of GAPC as an indirect selection marker in the production of a new restorer cell line.

Three plant genotypes are considered:

A line CMS;

B male fertile line that lacks the restorer gene and which includes mammalian fertile cytoplasm; and

R a mammalian fertile line that includes a restorer gene and a male sterile cytoplasm.

Hybrids between lines A and B, which arise from manual genetic crossing, are expected to exhibit substantial hybrid growth; and hybrids between A and R do not show this growth. Since line B does not carry the restorer gene, it is not possible to produce male fertile hybrids of the two lines using CMS. However, if the restorer gene can be transferred from line R to line B without otherwise altering the characteristics of line B, mammalian fertile hybrids A and B can be obtained between the lines using CMS. Traditionally, this is accomplished by a procedure called introgression. Line R crosses as a male with line B to form the male fertile hybrid of line F1.

A- and Β, which contains the male sterile cytoplasm (the hybrid cytoplasm is entirely derived from the female parent), but the male sex is fertile because the hybrid obtained one copy of the restorer gene from the parent line R. The second cross (which is called backcross) is then between a hybrid (female sex) and line B. A large number of growing offspring are in the field. An equal number of fertile and infertile individuals are expected, with fertile individuals carrying the restorer gene. One or more fertile individuals are then used as the female sex in a second backcross with line B; fertile plants are isolated and crossed for the third time as a female with line B. This process is repeated over many generations. In each new generation, the offspring is expected to become increasingly similar to Line B (except that it will include the restorer gene). Different characteristics associated with line stanoví are determined in each generation. Essentially, a new restoration line is produced with all or most of the desired characteristics of Line B. This line is then used to produce hybrids between lines A and B on a large scale.

GAPC facilitates this process by allowing the presence of the restorer gene in the seed progeny as early as the seed stage. DNA was extracted from a small amount of leaf-derived material, digested with a restriction endonuclease such as Hind III (used in Figure 4), and analyzed using a GAPC probe. The presence of the restriction fragment characteristic of the restorer gene indicates that the seedlings carry the restorer gene. A large number of plants were tested at the seedling stage, which is less expensive than the production of adult plants in the field. In addition, the male fertile phenotype is influenced by a number of conditions, and testing for the presence of the gene by an assay for ideally associated polymorphism more reliably detects the presence of the gene during the introgression process.

Example 2: Production of new restorer cell lines by introducing the form of restorer gene by GAPC transformation.

In accordance with this procedure, three plant genotypes of Example 1 are contemplated.

In this example, the problem is exactly the same as in the example

1. The problem is the transfer of the restorer gene from line R to line B without altering the characteristic of line jinak. In this case, however, it is believed that the form of the GAPC gene that represents the restorer gene has been isolated and is available as a cloned DNA segment in suitable Agrobacterium tumefaciens in a transformation vector such as pRD400 (Datla RSS, Hammerlindl JK, Panchuk, B, Pelcher) LE and Keller W. (1992) Gene 211: 383384). Instead of the widespread backcross program described in Example 1, the GAPC gene is transformed into line B by transformation with Agrobacterium.

For this example, it is also assumed that lines A, B, and R are Brassica napus lines and that the cloned restorer gene is the same as that in line R. The procedure described in Moloney, M., Walker, J. and Sharma (1989) Plant Cell Rep. 8: 238-242), an Agrobacterium strain carrying a gene in the prRD400 vector is used to inoculate uterine uterine strains of strain B. Agrobacterium bacteria are eliminated by application of antibiotics and the resulting plant tissue is placed in a medium containing the antibiotic kanamycin. The pRD400 vector contains a gene that causes kanamycin resistance, and therefore cells that grow in the presence of said antibiotic are likely to carry the kanamycin gene along with a restorer gene that is cloned in pRD400. The presence of the restorer gene in these plants is then estimated directly by testing for the presence of restriction fragments in plant forms that are characteristic of the restorer using a GAPC probe. These plants are expected to be fertile if they contain male sterile cytoplasm and that F1 progeny derived from

Z ········ From the crossing of line A (acting as female) and the new transgenic line will also act as fertile female.

This method has two advantages: it is much faster and cheaper than conventional plant breeding approaches. Years, but only a few months, are not required to develop this line. Moreover, the presence of the restorer gene is the only difference between the line B genome and the new restorer line genome. Then, the integrity of Line B characteristics is less likely to be compromised.

Although the above description applies to specific plant species of Brassica napus, the invention can also be applied to other species, wherein the species restorer gene corresponds to a specific form of GAPC. In such cases, the method of transformation may differ from that described above.

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SEQUENCE PROTOCOL (1)

Data for SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1207 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) Sequence description: SEQ ID NO: 1:

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 TAGAGTACCC GTGTGGTCGA 1020 CTTGATCATT CACATGTCCA AGGCCTAAGT CGATGAAGAT CTCGAGTGAT GTAATGGTGT 1080 TTTTAAATTG TTGTTTTTAT CGAATAAATT TTCTTGGGTT TTGAAACCTT TATGGTTTTG 1140 GCGAATTCTC TACTTTCACG TGACGTGATA AGAAGTTTGT AGACCGGTTG TTTTTTATTT 1200 1207 TTACTGA

(2) Data to SEQ ID NO: 1: (iii) SEQUENCE CHARACTERISTICS: (E) LENGTH: 1091 pairs of cows (F) TYPE: nucleic acid (G) CHAIN TYPE: single chain (H) TOPOLOGY: linear (iv) SPECIES MOLECULES: cDNA (xi) Description SEQ ID NO: 2:

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TTTCGAAATG GCTGACAAGA TTTGGTGGCT AGAGTTATCC CTTCATCACC ACCGAGTACA GAAGCACAAT GAGCTCAAGG CACTGTTTTC GGCATCAGGA TGTTGTTGAG TCTACTGGTG TGGTGCCAAG AAAGTTGTCA TGTCAATGAG CATGAGTACA TAACTGCCTT GCTCCACTTG CATGACTACT GTCCACTCTA GGACTGGAGA GGTGGAAGAG CAAGGCTGTC GGAAAGGTGC TGTTCCCACC GTTGATGTTT CTACGATGAA ATCAAGAAGG TGGTTACACA GAGGATGATG CTTTGACGCC AAGGCTGGAA TGACAACGAA TGGGGTTACA CTAAAACGCT GAAGATCTAC ATTTCTTTGG G

AGATTAAGAT CGGAATCAAC TTCAGAGGAA CGATGTTGAG TGACGTACAT GTTTAAGTAT TGAAGGATGA GAAAACACTT ACCCTGAGGA TATCCCATGG TCTTCACTGA CAAGGACAAG TCTCTGCACC AAGCAAAGAT AGTCTGATCT CAACATTGTT CCAAGGTTAT CAACGACAGG TCACTGCTAC TCAGAAGACA CCGCTTCCTT CAACATCATT TTCCACAGCT CAATGGAAAA CAGTTGTCGA CCTCACGGTT CTATCAAGGA GGAGTCTCAG TTGTCTCAAC TGACTTCGTT TCGCATTGAG TGACAACTTC GTACCCGTGT GGTCGACTTG AATGATGTAA TGGTGTCTTA

GGTTTCGGAA GAATCGGTCG CTCGTCGCTG TTAACGATCC GACAGTGTTC ATGGTCAGTG CTCTTCGGAG AGAAGCCTGT GGTGAGGCCG GAGCTGACTT GCTGCTGCTC ACTTGAAGGG GCTCCTATGT TCGTTGTTGG TCCAACGCTA GTTGCACCAC TTTGGAATTG TCGAGGGACT GTTGATGGTC CATCAATGAA CCCAGCAGCA CCGGAGCTGC TTGACCGGAA TGTCCTTCCG AGACTCGAGA AAGCTGCAAC GGCAAGCTAA AGGGAATCCT GGTGACAACA GGTCGAGCAT GTGAAGCTGG TGTCGTGGTA ATCATTCATA TGTCCAAGGC ATTTGTGGTT TTCGAATAAG

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(3) Data to SEQ ID NO: 2: (in) SEQUENCE CHARACTERISTICS: . (AND) LENGTH: 1295 pairs of cows (J) TYPE: nucleic acid (TO) CHAIN TYPE: single chain (L) TOPOLOGY: linear (vi) SPECIES MOLECULES: cDNA (xi) Description SEQ ID NO: 3:

CTCATCTTCA ACCTCTCTCT AGGATCGGAA TCAACGGATT AGGGACGATG TTGAGCTCGT TACATGTTCA AGTACGACAG GATGAGAAGA CCCTTCTCTT GAGGATATCC CATGGGCCGA ACTGACAAAG ACAAGGCTGC GAACCCAGGA AAGACGCTCC GACCTTGACA TTGTCTCCAA GTTATCAATG ACAGATTTGG GCTACTCAGA AGACTGTTGA TCATTCAACA TTATTCCCAG GCTCTTAACG GAAAGTTGAC GTTGACCTTA CTGTCAGACT AAGGAGGAAT CCGAAGGCAA TCAACTGACT TCGTTGGCGA TTGAGCGACA AGTTTGTGAA CGTGTGGTCG ACTTGATCGT GATAGGGAGT GGAAAGTCAT ATAAAATTTC TTTGAACTTG TGAGGTGATG GGAGTTTGTA ATATATTGAG TTAACGTTAT

AACTCTCGTT TTCGATTCTA CGGAAGAATT GGTCGTTTGG CGCTGTCAAC GACCCCTTCA TGTTCACGGT CAATGGAAAC CGGTGAGAAG CCAGTCACTG GGCTGGAGCT GACTACGTTG AGCTCACTTG AAGGGTGGTG AATGTTTGTT GTTGGTGTCA CGCTAGCTGC ACCACTAACT AATTGTTGAG GGTCTTATGA TGGGCCTTCA ATGAAGGACT CAGCACTGGA GCTGCCAAGG TGGAATGTCT TTCCGTGTCC CGAGAAAGCT GCTACCTACG ACTCAAGGGA ATCCTTGGAT CAACAGGTCG AGCATTTTTG ATTGGTGTCA TGGTACGACA CCACATGTCA AAGGCCTAAG CTGTTCATCC CCTTTTATGG GAACTTTTTT TTTTTTTGGT GACCGATGTT TTACTGGAAG GGTTTTAAAA AAAAA

CAATGGCTGA CAAGAAGATT TTGCTAGAGT TGTTCTCCAG TCACTACTGA GTACATGACC ACAATGAACT GAAGATCAAG TTTTCGGCAT CAGGAACCCT TTGAGTCTAC TGGTGTCTTC CCAAGAAGGT TGTTATCTCT ACGAGCACGA ATACAAGTCC GCCTTGCTCC CCTTGCCAAG CTACAGTCCA CTCAATCACT GGAGAGGTGG AAGAGCTGCT CTGTCGGAAA GGTGCTTCCA CAACCGTTGA TGTCTCAGTT AAGAAATCAA AAAGGCTATC ACACCGAGGA TGATGTTGTC ACGCCAAGGC TGGAATTGCA ACGAATGGGG TTACAGTTCC CTAAGAAGCA GATCTCGAAT TCTGAATTTG TCGTTTTCGA TTTCTTAATT CTCATTCATG CCCTTTGTTT TTGGCTTTTG

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Claims (14)

A probe specific for nuclear reconstruction of cytoplasmic male sterility of plants, characterized in that it comprises a nucleic acid sequence encoding glyceraldehyde-3-phosphate dehydrogenase or a hybridization fragment thereof, wherein said DNA sequence or hybridization fragment thereof hybridizes under limited conditions to specific DNA fragments typical of plants that have a nuclear restorer gene. A probe specific for nuclear reconstruction of cytoplasmic male sterility of plants, characterized in that it comprises the nucleic acid shown in SEQ ID NO: 1. The probe-derived DNA primer sequence of claim 1 or 2. 4. A method for detecting the presence of a restorer gene in nuclear genomic DNA comprising i) hybridizing said nuclear genomic DNA under restricted conditions with a probe specific for nuclear reconstruction of cytoplasmic male sterility of plants, said probe comprising a nucleic acid sequence encoding glyceraldehyde-3-phosphate dehydrogenase and said nucleic acid sequence hybridizing under limited conditions to specific DNA fragments typical of plants have a nuclear restorer gene; and ii) detecting hybridization of said probe to said nuclear genomic DNA, wherein hybridization of said probe to said nuclear genomic DNA indicates said nuclear genomic DNA comprising a restorer gene. Fcfcfc fcfc fcfc fcfc fc 5. A method for detecting the presence of a restorer gene in nuclear genomic DNA, comprising: i) amplifying said nuclear genomic DNA under appropriate conditions with the DNA primer sequence of claim 3; and ii) detecting amplification of said nuclear genomic DNA with said DNA primer sequence, wherein amplification of said nuclear genomic DNA indicates said nuclear genomic DNA comprising a restorer gene. The method of claim 4, wherein the probe comprises the nucleic acid set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. The method of claim 5, wherein the probe comprises the nucleic acid set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. A nuclear reconstruction cytoplasmic male sterility plant gene comprising a DNA sequence encoding glyceraldehyde-3-phosphate dehydrogenase and surrounding sequences and which is detected by the method of claim 4, 5, 6, or 7. The gene of claim 8, wherein the surrounding sequences are located at the 3 'and / or 5' end of the glyceraldehyde-3-phosphate dehydrogenase sequence. 10. A method of producing a reconstruction line comprising plants genetically transforming a nuclear reconstitution gene of cytoplasmic male sterility according to claim 8. Use of a probe according to claim 1 in the nuclear reconstruction of cytoplasmic male sterility of plants. • 9 Use of a probe according to claim 2 in the nuclear reconstruction of cytoplasmic male sterility of plants. The use of claim 11, wherein the probe comprises the nucleic acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3. Use of the DNA primer sequence of claim 3 in nuclear reconstruction of cytoplasmic male sterility of plants.