Abstract:
The instant invention provides a method for detecting the presence of a restorer gene in the nuclear genomic DNA of a Brassica plant comprising the use of a probe/primer comprising the sequence of the Brassica glyceraldehyde-3-phosphate dehydrogenase (SEQ ID NO: 1) or a sufficient hybridizing fragment thereof.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of PCT/CA97/00424 filed Jun. 16, 1997 which claimed benefit under 35 U.S.C. §119 of U.S. provisional application No. 60/020,533, filed Jun. 26, 1996. 
    
    
     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 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. 
     (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 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 F1 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 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.  1 . 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&#39;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&#39;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. 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 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 Rfp1, 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 number of the markers for indirect selection of plant containing the restorer gene. 
     It would be highly desirable to be provided with a marker that is perfectly associated with nuclear restoration 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 Rfp1 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 DNA sequence, a hybridizing fragment thereof or any DNA sequence derived therefrom for use as primers for amplification of glyceraldehyde-3-phosphate dehydrogenase, 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 dehydrogenase 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 50 kb. 
     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 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 limitation,  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 production; 
     FIG. 2 shows the crosses used to identify a marker completely linked to the Rfp1 restorer of fertility gene; 
     FIGS. 3A to  3 E 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 by cRF1 probe in  Brassica napus  in a genetic population segregating for the Rfp1 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 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 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). cRF1 therefore represents a particularly powerful tool for indirect selection of the restorer gene. 
     
       
         
               
             
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Co-segregation of an Rfp1-specific RFLP allele detected by the 
               
               
                 probe cRF1 (GAPC) with male fertility restoration in 2 
               
               
                   Brassica napus  backcross populations 
               
             
          
           
               
                   
                 Fertile progeny plants 
                 Sterile progeny plants 
               
             
          
           
               
                   
                   
                 without 
                   
                 without 
               
               
                   
                 with Rfp1- 
                 Rfp1- 
                 with Rfp1- 
                 Rfp1- 
               
               
                   
                 specific 
                 specific 
                 specific 
                 specific 
               
               
                   
                 cRF1 
                 cRF1 
                 cRF1 
                 cRF1 
               
               
                 Cross 
                 allele 
                 allele 
                 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 cRF1 and Rfp1, 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), i.e., 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&#39;s sequence is 99% similar to that of a cytoplasmic form of a glycolytic enzyme from  Arabidopsis thaliana,  glyceraldehyde-3-phosphate dehydrogenase (FIGS.  3 A and  3 B), 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 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 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. 
     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 F1 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 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 is available as a cloned DNA segment in a suitable plant  Agrobacterium tumefaciens  transformation vector such as pRD400 (Datla RSS, Hammerlindl J K, Panchuk B, Pelcher L E &amp; 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, M., Walker, J. &amp; 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 F1 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. 
     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 variations, 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.