CA2123751A1 - Self-incompatibility genes associated with the a10 allele - Google Patents

Abstract

Novel DNA molecules have been isolated that encode the S locus glycoprotein (SLG) and the S locus receptor kinase (SRK) of the A10 self-incompatibility allele.
Surprisingly, the gene encoding the SRK-A10 protein was found to contain a one base pair deletion which would result in premature termination of translation and the production of a truncated SRK-A10 protein. The defect in the SRK-A10 gene can be corrected by inserting a nucleotide at position 948. Expression vectors comprising a corrected SRR-A10 gene can be used to produce a self-incompatible plant.

Description

212~751 -Inventors: Steven J. Rothstein Daphne R. Goring Tracy L. Glavin Ulrike Schafer SELF-INCOMPATIBILITY GBNES ASSOCIATED WITH
THE A10 ~T.T.~T.~

CROSS-REFEREN OE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S.
Serial No. 08/208,909, filed on March 11, 1994, which in turn is a continuation of U.S. Serial No. 07/847,564, filed on March 3, 1992, now abandoned.

BACR~uN~ OF T~E lNV~..l-ION

1. Field of the Invention This invention is directed to DNA molecules that encode the S locus glycoprotein and the S locus serine kinase protein associated with the A10 self-incompatibility allele. Moreover, this invention is directed to a method for producing a self-incompatible plant by the use of such DNA molecules.

2. Background Oil seed crops have significant economic importance throughout the world. For example, the oil seed crops Brassica napus ssp. oleifera and Brassica rapa (formerly campestris) are the second most valuable crops in Canada with an annual growth on 7-9 million acres. Since canola seed is required for oil and m~a/l production, yield is extremely important in determining the usefulness of a Brassica line. B. napus F~ hybrid lines have been found to be superior to B. napus established lines. Typically, Fl hybrids produce yields that are greater than 20 to 21237~i1 _ -2-70%, compared with established lines. Thompson, Adv.
Appl. Biol. 7: 1 (1983); Johnston, Euphytica 20: 81 (1971).
The prerequisite for developing hybrid lines is the use of a pollen-control system to prevent self-pollination of the female line. Several approaches have been used for pollen control in the production of hybrid canola: dominant nuclear male-sterility genes, cytoplasmic male-sterility genes, and the self-incompatibility system. The most economic and flexible approach utilizes a self-incompatibility system that is naturally present in B. oleracea and B. campestris.
Gowers, Euphytica 24: 537 (1975).
In the Brassica family, the self-incompatibility system is inherited as a dominant genetic locus called the S locus. Nasrallah et al., Annu. Rev. Plant Physiol.
Plant Mol. Biol. 42: 393 (1991); Dzelzkalns et al., Dev.
Biol. 153: 70 (1992). The presence of a functional S allele results in a barrier to fertilization when the pollen grain originates from a plant carrying the same S allele as the pistil. The pollen phenotype, which is determined by the diploid parental genotype and not by the haploid pollen, is thought to be propagated by putative S allele products deposited in the outside wall of the pollen grain by the surrounding tapetum during pollen development. de Nettancourt, INCOMPATIBILITY IN
ANGIOSPERMS (Springer-Verlag, 1977). Thus, during pollen-pistil interactions, there is a recognition of self versus non-self leading to either a block in pollen germination when both parents carry the same S allele or successful fertilization when different S alleles are present.
The diploid species, Brassica oleracea and B.
campestris are typically self Jncompatible, while B.
napus, an allotetraploid composed of both of these genomes, generally occurs as a self-compatible plant.
Downey et al., "Rapeseed and mustard," in PRINCIPLES OF
CULTIVAR DEVELOPMENT, Fehr (ed.) pages 437-86 (Macmillan Publishing Co. 1987). Although the S-alleles have been 21237~1 _ -3 introgressed into B. napus using traditional genetic approaches, there are two problems that limit this approach for the production of hybrid seed. First, there is some environmental variability in the level of hybridity from year to year when using self-incompatibility as the pollen control system. Second, and most importantly, the level of dominance of the self-compatible alleles is not always sufficient in heterozygotes, with the level of dominance widely variable depending on genetic background.
This second problem is an extremely important one because hybrid breeding schemes using self-incompatibility require the use of heterozygotes for the final production of the hybrid seed. Thus, it would be useful to be able to confer the self-incompatible phenotype by gene transformation since this would be faster than traditional breeding approaches and would allow the co-transfer of herbicide resistance genes which would be useful under some breeding schemes.
Two types of genes have been found to co-segregate with the self-incompatibility phenotype: the S locus glycoprotein (SLG) gene and the S locus receptor kinase (SRR) gene. Transformation experiments have shown that the SLG gene alone is not sufficient to confer self-incompatibility to a self-compatible B. napus. Nishio et al., Sex. Plant Reprod. 5: 101 (1992). Nevertheless, there is evidence that SLG genes are required for self-incompatibility. For example, a naturally occurring variant of B. campestris with normal SRR expression, but low levels of SLG expression has been associated with self-compatibility. Nasrallah et al., Plant J. 2: 497 (1992). Therefore, transformation of a self-compatible plant to a self-incompatible plant requires the introduction of both SRR and SL~ genes.
The expression of any foreign gene in the genome of a plant cell may be repressed due to hypermethylation of the foreign gene, the particular chromosomal context of the foreign gene, or the presence of multiple copies of the foreign gene. Such problems are compounded when a 2~237~i1 _ --4 desired phenotype of a transgenic plant is dependent upon the expression of two foreign genes. This fact may explain the observation that there is no report of a transgenic self-incompatible plant which was produced by transforming a self-compatible plant with both SRK and SLG genes.

SUMMARY OF T~E lNV~.. llON

Accordingly, it is an object of the present invention to provide a method for producing a self-incompatible plant which requires transformation of a single S locus gene.
It is a further object of this invention to provide DNA molecules that encode a functional SRK-A10 protein.
These and other objects are achieved, in accordance with one embodiment of the present invention, by the provision of an isolated DNA molecule, comprised of the nucleotide sequence of SEQ ID NO:5, wherein the DNA
molecule encodes an SRK protein. In particular, these objects are achieved by the provision of a DNA molecule consisting of the nucleotide sequence of SEQ ID NO:6.
In accordance with another embodiment of the present invention, there has been provided an isolated DNA
molecule, comprised of the nucleotide sequence of SEQ ID
NO:4, wherein the DNA molecule encodes an SLG protein.
In accordance with a further embodiment of the present invention, there has been provided an expression vector comprising a regulatory element and a DNA molecule that encodes either an SLG protein or an SRK protein, wherein the regulatory element controls the production of the SLG protein or the SRK protein. A suitable regulatory element is selected from the group consisting of the SLG-910 regulatory element! the SLG-A10 regulatory element, the SRK-A10 regulatory element and the CaMV 35S
35 promoter.
In accordance with another embodiment of the present invention, there has been provided a method of producing a self-incompatible plant, comprising the steps of:

(a) producing a first parent self-compatible plant comprising an expression vector that comprises an SRK-encoding DNA molecule, wherein the first parent plant does not contain the A10 allele;
and (b) cross-fertilizing the first parent plant with a second parent plant having the A10 allele to produce a progeny plant, wherein the progeny plant produces SLG-A10 protein and SRK-A10 protein, resulting in the self-incompatibility phenotype.
In accordance with a further embodiment of the present invention, there has been provided a method of producing hybrid seed, comprising the steps of:
(a) producing a self-incompatible transgenic plant comprising the nucleotide sequence of SEQ ID
NO:6; and (b) cross-fertilizing the transgenic plant with a second plant.
In accordance with another embodiment of the present invention, there has been provided a method for conferring the self-incompatible phenotype on a self-compatible plant, comprising the steps of:
(a) preparing a first expression vector comprising an SLG-encoding DNA molecule consisting of the nucleotide sequence of SEQ ID NO:2;
(b) preparing a second expression vector comprising an SRK-encoding DNA molecule consisting of the nucleotide sequence of SEQ ID NO:6; and (c) transferring the first expression vector and the second expression vector into a self-compatible plant to produce a transformed plant, wherein the transformed p~ant expresses SLG-A10 protein and SRK-A10 protein, resulting in a self-incompatibility phenotype.
In accordance with a further embodiment of the present invention, there has been provided a transformed plant comprising an expression vector, wherein the 21237~i~

expression vector comprises the nucleotide sequence of SEQ ID NO:6.
In accordance with another embodiment of the present invention, there has been provided an isolated DNA
molecule comprising the nucleotide sequence of SEQ ID
NO:8, wherein the DNA molecule is the SLG-A10 promoter.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the predicted amino acid sequence of the SLG-A10 cDNA tSEQ ID NO:1]. The underlined section represents a putative signal peptide. Conserved cysteine residues are marked by stars above the amino acid residues. Double underlining indicates potential N-glycosylation sites.
Figure 2 shows the alignment of the SLG-A10 and SR~-A10 cDNA sequences [SEQ ID NOs: 2 and 3]. Panel A
depicts the coding region of the SLG-A10 cDNA that was aligned with the SRK-A10 cDNA sequence. A region of 100%
homology between the two genes is marked. The 1-bp deletion in the SRR-A10 gene leading to premature termination is indicated by the arrow. Underneath the SRR-A10 cDNA is a representation of the S-receptor kinase domains that would be translated if the deletion was not present. Panel B shows the alignment of the SLG-A10 nucleotide sequence [SEQ ID NO:2] and the SRR-A10 nucleotide sequence [SEQ ID NO:3]. Identical nucleotides are marked by dashes, and gaps are represented by blank spaces. The predicted start and stop codons for both genes are underlined. The 1-bp deletion in the SR~-A10 gene is marked by an arrow.
Figure 3 shows the nucleotide sequence of the corrected SRR-A10 gene [SEQ'~ID NO:6], which is constructed by inserting a nucleotide at position 948 of the SRR-A10 cDNA sequence [SEQ ID N0:3].
Figure 4 shows the nucleotide sequence of the SLG-A10 promoter.

2123~1 DETAIT,~D DESCRIPTION OF PR~FERRED EMBOD

1. Definitions In the description that follows, a number of terms are used extensively. The following definitions are provided to facilitate understanding of the invention.
A structural gene is a DNA sequence that is transcribed into messenger RNA (mRNA) which is then translated into a sequence of amino acids characteristic of a specific polypeptide.
A promoter is a DNA sequence that directs the transcription of a structural gene. Typically, a promoter is located in the 5' region of a gene, proximal to the transcriptional start site of a structural gene.
If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent.
In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter.
An isolated DNA molecule is a fragment of DNA that is not integrated in the genomic DNA of an organism. For example, the SLG-A10 gene is a DNA fragment that has been separated from the genomic DNA of a Brassica napus plant.
Another example of an isolated DNA molecule is a chemically-synthesized DNA molecule that is not integrated in the genomic DNA of an organism.
An enhancer is a DNA regulatory element that can increase the efficiency of transcription, regardless of the distance or orientation of the enhancer relative to the start site of transcription.
Complementary DNA (cDNA) is a single-stranded DNA
molecule that is formed from an mRNA template by the enzyme reverse transcriptase. Typically, a primer complementary to portions of mRNA is employed for the initiation of reverse transcription. Those skilled in the art also use the term "cDNA" to refer to a double-stranded DNA molecule consisting of such a single-stranded DNA molecule and its complementary DNA strand.

- 21237~1 The term expression refers to the biosynthesis of a gene product. For example, in the case of a structural gene, expression involves transcription of the structural gene into mRNA and the translation of mRNA into one or more polypeptides.
A cloninq vector is a DNA molecule, such as a plasmid, cosmid, or bacteriophage, that has the capability of replicating autonomously in a host cell.
Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of an essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance.
An expression vector is a DNA molecule comprising a gene that is expressed in a host cell. Typically, gene expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. Such a gene is said to be "operably linked to" the regulatory elements.
A foreign gene refers in the present description to a DNA sequence that is operably linked to at least one heterologous regulatory element. For example, any gene other than the SRK-A10 structural gene is considered to be a foreign gene if the expression of that gene is controlled by a regulatory element of the SRR-A10 gene.
A recombinant host may be any prokaryotic or eukaryotic cell that contains either a cloning vector or expression vector. This term also includes those prokaryotic or eukaryotic célls that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell.
A transqenic plant is a plant having one or more plant cells that contain an expression vector.

212~7~1 . g Two nucleic acid molecules are considered to have a substantial se~uence similarity if their nucleotide sequences share a similarity of at least 50%. Sequence similarity determinations can be performed, for example, using the FASTA program (Genetics Computer Group;
Madison, WI). Alternatively, sequence similarity determinations can be performed using BLASTP (Basic Local Alignment Search Tool) of the Experimental GENIFO(R) BLAST Network Service. See Altschul et al., J. Mol.
Biol. 215: 403 (1990). Also, see Pasternak et al., "Sequence Similarity Searches, Multiple Sequence Alignments, and Molecular Tree Building," in METHODS IN
PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Glick et al.
(eds.), pages 251-267 (CRC Press, 1993).
As described below, the nucleotide sequence [SEQ ID
NO:3] of the SRK-A10 cDNA contains a one base pair deletion at position 948 which would cause a premature termination of translation of the SRK-A10 protein. It is possible to correct this defect by inserting a nucleotide at position 948 of the SRK-A10 cDNA sequence. In the present context, a DNA molecule containing such a nucleotide sequence is referred to as a corrected SR~-AlO
gene. A corrected SRR-A10 gene is capable of producing SRK-A10 protein.

2. Isolation of cDNA Molecules Encoding Genes Associated with the Al0 Allele cDNA molecules encoding genes associated with the A10 allele can be isolated from a Brassica pistil cDNA
library. Preferably, the RNA used to construct the cDNA
library is obtained from the pistils of the self-incompatible Brassica napus ssp. olifera lines Topas-2, Regent-2, or W1. The generat~on of the Topas-2 and Regent-2 lines has been described in U.S. Serial No.
08/208,909 (filed on March 11, 1994), and in Goring et al., The Plant Journal 2: 983 (1992), the contents of which are hereby incorporated by reference. The generation of the W1 line has been described in U.S.

.

Serial No. 08/208,909, as well as in Goring et al ., Mol .
Gen. Genet. 234: 185 (1992), the contents of which are hereby incorporated by reference. The Wl line can be obtained from the American Type Culture Collection (Rockville, MD) as accession number 75570.
Total RNA can be prepared from pistils using techniques well-known to those in the art. In general, RNA isolation techniques must provide a method for breaking plant cells, a means of inhibiting RNase-directed degradation of RNA, and a method of separating RNA from DNA, protein, and polysaccharide contaminants.
For example, total RNA can be isolated from pistils by freezing plant tissue in liquid nitrogen, grinding the frozen tissue with a mortar and pestle to lyse cells, extracting the ground tissue with a solution of phenol/chloroform to remove proteins, and separating RNA
from the remaining impurities by selective precipitation with lithium chloride. See, for example, Ausubel et al.
(eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, pages 4.3.1-4.3.4 (John Wiley & Sons 1990) [hereinafter "Ausubel"]. Also, see Sharrock et al ., Genes and Development 3:1745 (1989).
Alternatively, total RNA can be isolated from pistils by extracting ground tissue with guanidinium isothiocyanate, extracting with organic solvents, and separating RNA from contaminants using differential centrifugation. See, for example, Strommer et al ., "Isolation and characterization of Plant mRNA," in METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Glick et al . (eds.), pages 49-65 (CRC Press, 1993).
In order to construct a cDNA library, poly(A)+ RNA
must be isolated from a total RNA preparation. Poly(A)+
RNA can be isolated from total RNA by using the standard tec-hnique of olgo(dT)-cellulose chromatography. See, for example, Strommer et al ., supra .
Double-stranded cDNA molecules are synthesized from poly(A)+ RNA using techniques well-known to those in the art. See, for example, Ausubel at pages 5.5.2-5.6.8.
Moreover, commercially available kits can be used to - 21~3751 synthesize double-stranded cDNA molecules. For example, such kits are available from GIBCO/BRL (Gaithersburg, MD), Clontech Laboratories, Inc. (Palo Alto, CA), Promega Corporation (Madison, WI) and Stratagene Cloning Systems (La Jolla, CA).
Various cloning vectors are appropriate for the construction of a pistil cDNA library. For example, a cDNA library can be prepared in a vector derived from bacteriophage, such as a ~gtlO vector. Huynh et al., "Constructing and Screening cDNA Libraries in ~gtlO and Agtll," in DNA Cloning: A Practical Approach, Vol . I, Glover (ed.), pages 49-78 (IRL Press, 1985).
Alternatively, double-stranded cDNA molecules can be inserted into a plasmid vector, such as a pBLUESCRIPT
vector (Stratagene Cloning Systems; La Jolla, CA), or other commercially available vectors. Suitable cloning vectors also can be obtained from the American Type Culture Collection (Rockville, MD).
In order to amplify the cloned cDNA molecules, the cDNA library is inserted into a procaryotic host, using standard techniques. For example, the pistil cDNA
library can be introduced into competent E. coli DH5 cells, which can be obtained from GIBCO/BRL
(Gaithersburg, MD).
cDNA clones encoding SLG-A10 and SRK-A10 proteins can be isolated from a cDNA library using radiolabeled oligonucleotide probes. Suitable nucleotide sequences for such probes are disclosed in Goring et al., The Plant Cell 5: 531 (1993), the contents of which are hereby incorporated by reference. Moreover, oligonucleotide probes can be designed using the unique nucleotide sequences of SLG-A10 and SRR-A10 cDNA molecules, which are provided below. General techniques for screening cDNA libraries with radiolabeIed oligonucleotide probes are described, for example, by Ausubel at pages 2.11.1-2.12.5, 6.1.1-6.1.23, and 6.4.1.-6.4.10.
The above-described methods can be used to obtain SLG-A10 and SRK-A10 cDNA clones. The cDNAs can be analyzed using a variety of techniques such as 21237~1 restriction analysis, Northern analysis, and in situ hybridization. See, for example, Goring et al., The Plant Journal 2: 983 (1992), and Goring et al., The Plant Cell 5: 531 (1993).
In the present invention, a cDNA clone encoding an SLG protein was isolated from a B. napus oleifera cDNA
library. The nucleotide sequence of the SLG-A10 cDNA
clone [SEQ ID N0:2] was found to have a high degree of sequence similarity with the following SLG genes: 56 [Nasrallah et al., Nature 326: 617 (1987)], S8 and S13 [Dwyer et al., Plant Nolec. Biol. 16: 481 tl991)], 529 [Trick et al., Mol. Gen. Genet. 218: 112 (1989)], and 910 [Goring et al., Mol. Gen. Genet. 234: 185 (1992)].
However, the SLG-A10 cDNA also was found to contain the following unique nucleotide sequence [SEQ ID N0:4]:
(395) CACGAATCTT ACTAGACGTA ATGAGAGAAC (424) GTGCTTAGAA TGATCTGCAT TACTCTCTTG.
Thus, the present invention includes an SLG-encoding DNA
molecule comprising the nucleotide sequence of SEQ ID
NO:4.
The isolation and characterization of the SRR-A10 cDNA clone is described in Example 2. The nucleotide sequence of the SRK-A10 cDNA clone [SEQ ID NO:3] was compared with the nucleotide sequences of the SRK-910 gene [Goring et al., Plant Cell 4: 1273 (1992)] and the SRR6 gene [Stein et al., Proc. Natl. Acad. Sci. USA 88:
8816 (1991)]. The results of these analyses revealed that the SRR-A10 cDNA has a high degree of nucleotide sequence similarity with the SRR-910 and SRR6 genes, and that the SRR-A10 cDNA contains the following unique nucleotide sequence [SEQ ID N0:5]:
(1267) TGGAAATCTC GCTGATATGC GGAATTACGT (1296) ACCTTTAGAG CGACTATACG CCTTAATGCA.
Thus, the present invention inclu~es an SRK-encoding DNA
molecule comprising the nucleotide sequence of SEQ ID
NO:5.

3. Use of DNA Nolecules Encoding SLG-A10 and 8RR-A10 Protein~ to Produce Self-Incompatible Plants Surprisingly, the nucleotide sequence of the SRR-A10 cDNA clone contains a one base pair deletion which would lead to premature termination of the translation of SRR-A10 mRNA and the production of a truncated SRK-A10 protein. See Example 2. As described below, plants that contain the A10 allele express the SLG-A10 gene and yet, such plants are self-compatible. This observation suggests that a functional SRR gene is required for self-incompatibility in plants that carry the A10 allele.
A DNA molecule encoding a corrected SRR-A10 gene can be obtained by adding a nucleotide to a DNA molecule containing the naturally occurring SRK-A10 sequence to correct the deletion. Specifically, a "corrected SRK-A10 gene" can be obtained by adding a nucleotide at position 948 ~SEQ ID NO:6]. See Figure 3. Methods that can be used to synthesize such a corrected SRR-A10 gene include oligonucleotide-directed mutagenesis, linker-scanning mutagenesis, mutagenesis using the polymerase chain reaction, and the like. Ausubel at pages 8Ø3-8.5.9.
Also see generally, McPherson (ed.), DIRECTED
MUTAGENESIS: A PRACTICAL APPROACH, IRL Press (1991). A
specific method that has been used to correct the SRR-A10 gene is illustrated in Example 4.
Although Example 4 presents a method to add an adenine residue, a cytosine residue or guanine residue also may be used to correct the SRR-A10 gene. However, the insertion of a thymine residue would result in the presence of a stop codon.
A corrected SRK-A10 gene can be used to confer the self-incompatible phenotype upon a self-compatible plant.
For example, a self-compatible p~ant that lacks the A10 allele can be transformed with a DNA molecule encoding the SLG-A10 protein and a DNA molecule containing a corrected SRR-A10 gene.
A preferred method for producing a self-incompatible plant is to transform a self-compatible plant that ~123751 contains an A10 allele with a DNA molecule that contains a corrected SR~-A10 gene. This approach advantageously allows the use of alternative schemes for breeding hybrids since the required self-incompatibility genes would be located at different chromosomal locations.
Therefore, one could have two self-compatible lines in which one self-compatible line contains the A10 allele and the other self-compatible line would be a transgenic line that contains the corrected SRR-A10 gene, but does not contain an A10 allele. The two self-compatible lines can be propagated and maintained as inbred lines and when crossed would produce self-incompatible progeny. The self-incompatible progeny could be used as the female line and crossed with any pollen donor to give hybrid seed. Self-compatible lines that contain the A10 allele include, for example, Ceres, Regent, Westar, Wl, and certain Topas lines.
In order to express a protein that confers the self-incompatibility phenotype, an expression vector is constructed in which a DNA molecule encoding the SLG-A10 or SRK-A10 protein is operably linked to DNA sequences that regulate gene transcription. The general requirements of an expression vector are described below in the context of a transient expression system. Here, however, the objective is to introduce the expression vector into plant tissue in such a manner that SLG-A10 and/or SRK-A10 proteins are expressed in the tissue of an adult plant. Mitotic stability can be achieved using plant viral vectors that provide epichromosomal replication.
An alternative and preferred method of obtaining mitotic stability is provided by the integration of expression vector sequences into the host chromosome.
Such mitotic stability can be provided by Agrobacterium-mediated transformation, as discussed below.
Transcription of the SLG-A10 and/or SRR-A10 genes may be controlled by a promoter of an S-locus gene, or by a viral promoter, such as a Cauliflower Mosaic Virus (CaMV) promoter, a Figwort Mosaic Virus promoter, and the like.

_ -15-Gruber et al., "Vectors for Plant Transformation," in METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Glick et al . (eds.), pages 89-119 (CRC Press, 1993).
Preferably, the promoter is the SLG-A10 promoter, the SRK-910 promoter, or a CaMV 35S promoter.
As described in Example 1, plant tissue contains high levels of SLG-A10 mRNA, compared with levels of SLG-910 mRNA. This observation indicates that the SLG-A10 promoter is an especially strong promoter. Thus, the o SLG-A10 promoter is a particularly preferred promoter.
In order to select transformed cells, the expression vector contains a selectable marker gene. For example, such genes may confer resistance to kanamycin or hygromycin. Although the expression vector can contain DNA sequences encoding SLG-A10 or SRK-A10 protein under the control of a regulatory element, as well as the selectable marker gene under control of constitutive promoter, the selectable marker gene is preferably delivered to host cells in a separate selection expression vector.

4. I~olation of SLG-A10 and SRR-A10 Genes from a Genomic Library The methodology, described above, can be used to isolate cDNA clones encoding SLG-A10 and SRK-A10 proteins. The SLG-A10 and SRR-A10 genes can be isolated from a genomic library using, for example, the corresponding cDNA clones as probes.
A plant genomic DNA library can be prepared by means well-known in the art. See, for example, Slightom et al.
"Construction of ~ Clone Banks," in METHODS IN PLANT
MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Glick et al . (eds.), pages 121-146 (CRC Press, 1993).J A preferred source of plant genomic DNA is Brassica napus DNA. A more preferred source of plant genomic DNA is the Wl canola line. Genomic DNA can be isolated from Brassica napus tissue, for example, by lysing plant tissue with the detergent Sarkosyl, digesting the lysate with proteinase 21237~1 .

K, clearing insoluble debris from the lysate by centrifugation, precipitating nucleic acid from the lysate using isopropanol, and purifying resuspended DNA
on a cesium chloride density gradient. Ausubel et al., S supra, at pages 2.3.1-2.3.3.
DNA fragments that are suitable for the production of a genomic library can be obtained by the random shearing of genomic DNA or by the partial digestion of genomic DNA with restriction endonucleases. See, for example, Ausubel at pages 5.3.2-5.4.4, and Slightom et al., supra.
Genomic DNA fragments can be inserted into a vector, such as a bacteriophage or cosmid vector, in accordance with conventional techniques, such as the use of restriction enzyme digestion to provide appropriate termini, the use of alkaline phosphatase treatment to avoid undesirable joining of DNA molecules, and ligation with appropriate ligases. Techniques for such manipulation are disclosed by Slightom et al., supra, and are well-known in the art. Also see Ausubel at pages 3Ø5-3.17.5.
Standard techniques can be used to screen a genomic library using a cDNA clone or oligonucleotide probe.
See, for example, Ausubel at pages 6Ø3-6.6.1; Slightom et al., supra; Raleigh et al., Genetics 122:279 (1989).

5. Identification of a Regulatory Element of the SLG-A10 Gene or the SR~-A10 Gene The present invention also contemplates the isolation and use of SLG-A10 and SRR-A10 genomic regulatory elements. In the present context, a "regulatory element"
is a DNA sequence that controls gene expression. A
regulatory element contains at least a promoter, but may include an enhancer, as well as DNA sequences that confer tissue-specific gene expression.
Genomic clones can be analyzed using a variety of techniques such as restriction analysis, Southern analysis, primer extension analysis, and DNA sequence 21~3751 -analysis. Primer extension analysis or S1 nuclease protection analysis, for example, can be used to localize the putative start site of transcription of the cloned gene. Ausubel at pages 4.8.1-4.8.5; Walmsley et al ., "Quantitative and Qualitative Analysis of Exogenous Gene Expression by the S1 Nuclease Protection Assay," in METHODS IN MOLECULAR BIOLOGY, VOL. 7: GENE TRANSFER AND
EXPRESSION PROTOCOLS, Murray (ed.), pages 271-281 (Humana Press Inc. 1991). However, structural analysis per se cannot lead to the identification of a regulatory element associated with either the SLG-A10 gene or the SRR-A10 gene because a model for Brassica S-locus regulatory sequences has not been developed. Thus, the regulatory element of the SLG-A10 gene or the SRK-A10 gene must be identified using functional analysis.
The general approach of such functional analysis involves subcloning fragments of the genomic clone into an expression vector which contains a reporter gene, introducing the expression vector into various plant tissues, and assaying the tissue to detect the transient expression of the reporter gene. The presence of a regulatory element in the genomic subclone is verified by the observation of reporter gene expression in pistils, and the low level of reporter gene expression in anther tissue.
Methods for generating fragments of a genomic clone are well-known. Preferably, enzymatic digestion is used to form nested deletions of genomic DNA fragments. See, for example, Ausubel at pages 7.2.1-7.2.20; An et al., "Techniques for Isolating and Characterizing Transcription Promoters, Enhancers, and Terminators," in METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Glick et al . (eds.), pages 155-16J6 (CRC Press, 1993).
As an example, the possibility that the regulatory element resides "upstream," or 5'-ward, of the transcriptional start site can be tested by subcloning a DNA fragment that contains the upstream region, digesting the DNA fragment in either the 5' to 3' direction or in the 3' to 5' direction to produce nested deletions, and subcloning the small fragments into expression vectors for transient expression.
The selection of an appropriate expression vector will depend upon the method of introducing the expression vector into host cells. Typically, an expression vector contains: (1) prokaryotic DNA elements coding for a bacterial replication origin and an antibiotic resistance marker to provide for the growth and selection of the expression vector in the bacterial host; (2) a reporter gene that is operably linked to the test DNA elements;
and (3) DNA elements that control the processing of reporter gene transcripts, such as a transcription termination/polyadenylation sequence. Useful reporter genes include ~-glucuronidase, B-galactosidase, chloramphenicol acetyl transferase, luciferase, and the like. Preferably, the reporter gene is either the B-glucuronidase (GUS) gene or the luciferase gene. General descriptions of plant expression vectors and reporter genes can be found in Gruber et al., "Vectors for Plant Transformation," in METHODS IN PLANT MOLECULAR BIOLOGY
AND BIOTECHNOLOGY, Glick et al. (eds.), pages 89-119 (CRC
Press, 1993). Moreover, GUS expression vectors and GUS
gene cassettes are available from Clontech Laboratories, Inc. (Palo Alto, CA), while luciferase expression vectors and luciferase gene cassettes are available from Promega Corporation (Madison, WI).
Expression vectors containing test genomic fragments can be introduced into protoplasts, or into intact tissues or isolated cells. Preferably, expression vectors are introduced into intact tissues. General methods of culturing plant tissues are provided, for example, by Miki et al., "Procedures for Introducing Foreign DNA into Plants," in METHODS IN PLANT MOLECULAR
BIOLOGY AND BIOTECHNOLOGY, Glick et al. (eds.), pages 67-88 (CRC Press, 1993).
Methods of introducing expression vectors into plant tissue include the direct infection or co-cultivation of plant tissue with Agrobacterium tumefaciens. Horsch et al., Science 227:1229 (1985). General descriptions of _. --19--Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are provided by Gruber et al., supra, and Miki et al., supra.
Descriptions of techniques for Agrobacterium-mediated transformation of Brassica are provided, for example by Fry et al., Plant Cell Reports 6: 321 (1987), and Toriyama et al., Theor. Appl. Genet. 81: 769 (1991), the contents of which are hereby incorporated by reference.
Alternatively, expression vectors are introduced into lo Brassica tissues using a direct gene transfer method such as microprojectile-mediated delivery, DNA injection, electroporation, and the like. See, for example, Gruber et al., supra; Miki et al., supra; Klein et al., Biotechnology 10:268 (1992).
The above-described methods can be used to identify DNA sequences that regulate expression of the SLG-A10 gene or the SRK-A10 gene. For example, f igure 4 shows the nucleotide sequence of the SLG-A10 promoter tSEQ ID
NO:8]. Variants of such regulatory elements can be produced by deleting, adding and/or substituting nucleotides. Such variants can be obtained, for example, by oligonucleotide-directed mutagenesis, linker-sc~nn;ng mutagenesis, mutagenesis using the polymerase chain reaction, and the like. Ausubel at pages 8Ø3-8.5.9.
Also see generally, McPherson (ed.), DIRECTED
MUTAGENESIS: A PRACTICAL APPROACH, IRL Press (1991).
Thus, the present invention also encompasses DNA
molecules comprising nucleotide sequences that have substantial sequence similarity with naturally occurring SLG-A10 or SRK-A10 gene regulatory elements.

The present invention, thus generally described, will be understood more readily by reference to the following examples, which are provided by-way of illustration and are not intended to be limiting of the present invention.

Example 1 Isolation and Characterization of SLG-AlO cDNA

A cDNA molecule encoding the SLG-A10 protein was isolated from a Regent-2 cDNA library, as described in U.S. Serial No. 08/208,909, and in Goring et al., Plant J. 2: 983 (1992), the contents of which are hereby incorporated by reference.
Genomic blot analyses were used to characterize the SLG-A10 gene. In these studies, genomic DNA was extracted from leaves using methods described by Goring et al., Mol. Gen. Genet. 234: 185 (1992). Approximately 5 to 10 ~g of genomic DNA were digested with HindIII, fractionated through a 0.7~ agarose gel, and transferred to Zetabind membrane (Cuno, Inc. Laboratory Products;
Meriden, CT). The membranes were prehybridized and hybridized as described previously. Id. Radiolabeled probes were obtained by random priming using a full-length SLG-A10 cDNA. Feinberg et al., Anal. Biochem.
132: 6 (1983). Following hybridization, filters were washed using two, 30-minute washes in 0.1 x SSC (lx SSC
is O.lM sodium chloride, 0.015 M sodium citrate), 0.1%
SDS at 50C to 53C for cross-hybridization, and at 65C
to 68C for specific hybridization.
A survey of different canola cultivars revealed that SLG-A10 probes hybridize to an 8.4 kilobase HindIII
fragment and a 9.2 kilobase HindIII fragment. Further analysis revealed that the 8.4 kilobase ~indIII band corresponds to the SLG-A10 gene, while the 9.2 kilobase band results from cross-hybridization of the SLG-A10 probes to the S~R-A10 gene.
In the self-compatible canola cultivars, Ceres, Regent, and Westar, the SLG-A10 gene is present in a homozygous form. The A10 locuS also is occasionally found in the self-compatible Topas line. In the self-incompatible line W1, which was developed from a cross between a self-incompatible B.campestris and the self-compatible canola cultivar Westar, the SLG-A10 gene is present in a homozygous state.

21237~1 _ A cross between Wl and a self-compatible winter canola line, which does not carry the A10 allele, demonstrated the segregation pattern of the A10 allele relative to the self-incompatibility phenotype. Of the five genomic DNA samples from self-incompatible progeny, only two contained the A10 allele, while genomic DNA from four self-compatible progeny carried the A10 allele. The allele responsible for self-incompatibility in the Wl line has been determined to be the 910 allele which was present only in the Wl plant and in the resulting self-incompatible plants from the cross involving Wl. Thus, the A10 allele does not segregate with self-incompatibility in the Wl line.
The sequence of the SLG-A10 cDNA was analyzed to determine if an alteration of the nucleotide sequence was responsible for the inability of the A10 allele to provide a self-incompatibility phenotype. To sequence the full-length SLG-A10 cDNA clone, deletions were made using exonuclease III and mung bean nuclease according to the procedure in the Stratagene kit (Stratagene Cloning Systems; La Jolla, CA). Overlapping deletions were sequenced for both strands. All DNA and protein sequence analyses were performed on the DNASIS and PROSIS software (Hitachi America Ltd.; San Bruno, CA).
The results of these studies demonstrated that the SLG-A10 sequence could be translated into a full-length SLG protein. The predicted amino acid sequence shows that the SLG-A10 protein contains several characteristic features of SLG proteins, such as the signal peptide at the 5' end, several potential sites for N-glycosylation, and the 12 conserved cysteine residues at the carboxyl end of the sequence. See Figure 1. The SLG-A10 sequence shows high levels of DNA homology to a majority of characterized SLG genes ranging ~rom 84 to 91% for DNA
and 79 to 86% similarity for the predicted amino acid sequences. Trick et al., Mol. Gen. Genet. 218: 112 (1989); Dwyer et al., Plant Mol. Biol. 16: 481 (1991);
Goring et al., Plant J. 2: 983 (1992); Goring et al., Mol. Gen. Genet. 234: 185 (1992). A number of these 212~
-alleles have been associated with phenotypically strong self-incompatibility reactions. Thus, the sequence predictions suggest that the SLG-A10 gene should be able to promote a strong self-incompatibility phenotype.
RNA samples from different tissues of the Wl line were examined to determine if the lack of a self-incompatibility phenotype was due to an altered expression pattern of the SLG-A10 gene. The SLG-A10 steady state mRNA levels were then compared to that of the SLG-910 allele, which is associated with Wl self-incompatibility. In these studies, total RNA was extracted from W1 tissues using the method of Jones et al., EMBO J. 4: 2411 (1985). Poly(A)+ RNA samples were extracted from Westar tissue using the MICRO-FASTRACK
mRNA isolation kits (Invitrogen; San Diego, CA). For the RNA blots, 10 ~g of Wl total RNA was fractionated through a 1.2% formaldehyde gel and transferred to Zetabind membranes. See, for example, Sambrook et al., MOLECULAR
CLONING: A LABORATORY MANUAL, 2nd ed. (Cold Spring Harbor Laboratory Press 1989). Hybridization conditions and washing conditions for specific hybridization are described above.
The results of these studies demonstrated that both SLG genes were predominantly expressed in the stigma tissue, with mRNA transcripts detected in samples before and after anthesis. When SLG mRNA levels were compared to plasmid controls, the steady state levels of SLG-A10 mRNA were found to be about four to eight times higher than the SLG-910 transcripts. Similar results were also detected in the Regent-2 line when the steady state SLG-A10 mRNA levels were compared with steady state SLG-A14 mRNA levels. The SLG-A14 allele is associated with self-incompatibility in Regent-2 line;
Moreover, the developmental profile of SLG-A10 mRNA
levels in stigma samples from developing Wl buds was found to be very similar to that observed for the SLG-910 allele, as described by Goring et al., Mol. Gen. Genet.
234: 185 (1992). Finally, loss of self-incompatibility was not associated with an absence of SLG-A10 gene ~23751 expression as high levels of SLG-A10 transcripts also were detected in stigma RNA samples from developing buds in the self-compatible Westar line. Thus, there are no indications that the absence of self-incompatibility in plants containing the A10 allele is due to an altered expression profile, unless the higher steady state levels of SLG-A10 RNA exerts a negative effect.
~xample 2 Isolation and Characterization of the SRR-A10 Gene A DNA molecule encoding the SRK-A10 protein was obtained using polymerase chain reaction (PCR) techniques described in Goring et al., Mol. Gen. Genet. 234: 185 (1992). Briefly, W1 genomic DNA was digested with HindIII and fractionated on an agarose gel. The HindIII
fragments were used to amplify an 800 base pair region using PCR primers to conserved regions in published SLG
sequences. The PCR products were cloned into pBLUESCRIPT KS+ (Stratagene Cloning Systems; La Jolla, CA) and identified by seguencing and genomic blot hybridization patterns. Specific primers derived from the genomic PCR clone were then used to amplify the 5' and 3' ends of a 2.7 kilobase SRK-A10 cDNA using the Rapid Amplification of cDNA Ends (RACE) procedure [Frohman et al., Proc. Natl. Acad. Sci. USA 85: 8998 (1988)] with modifications described in Goring et al., Mol. Gen. Genet. 234: 185 (1992).
To determine which tissues expressed the SRK-A10 gene, W1 and Westar RNA samples were analyzed by RNA-PCR.
In these studies, W1 total RNA samples and Westar poly(A)+ RNA samples were amplified with specific SRR-A10 primers that span the kinase domain, which contains several introns. Briefly, 2~/~g of total RNA and approximately 1 ~g of poly(A)+ RNA were treated with DNase I to remove any contaminating genomic DNA. The RNA
samples were then used to synthesize first strand cDNA
according to the method of Harvey et al., Nucl. Acid.
Res. 19: 4002 (1991). Controls without reverse 21237~1 transcriptase also were used for each sample. One-quarter of each cDNA sample was amplified for 30 cycles for the pistil samples and 35 cycles for the remaining samples and then subjected to gel electrophoresis and DNA
gel blot analysis.
The results of these studies demonstrated that the SRR-A10 gene is predominantly expressed in the pistils throughout bud development in both W1 and Westar lines.
The pistils are the primary site of expression for other SR~ genes in Brassica. Stein et al., Proc. Natl. Acad.
Sci. USA 88: 8816 (1991). Also see Goring et al., Plant Cell 4: 1273 (1992), the contents of which are hereby incorporated by reference. Although very weak expression of SRK genes also has been found in the anthers, SRR-A10 transcripts were not detected in this tissue.
The nucleotide sequence of the DNA molecule encoding the SRK-A10 protein was determined as described in Example 1. To avoid errors that may have been introduced during PCR, several cDNA molecules derived from separate PCR amplifications were sequenced.
SRR genes from different S alleles encode proteins with similar features, such as a region at the N-terminal end that is very similar in sequence to SLGs, a transmembrane domain, and a C-terminal kinase domain.
The SRR-A10 cDNA was found to be 86% homologous with the SRK-910 gene [Goring et al., Plant Cell 4: 1273 (1992)~
and 87% homologous with the SRR6 [ Stein et al., Proc.
Natl. Acad. Sci. USA 88: 8816 (1991)] gene. However, the SR~-A10 gene sequence was most similar to the SLG-A10 gene sequence in the SLG domain (92% homology). An unusual feature of this homology is the presence of a 590 base pair region with 100% sequence identity between the two A10 allele genes suggesting that a gene conversion event has occurred. See Figure 2. While SLG-SRR pairs at a particular locus are very similar to each other, the three S loci characterized to date do not have an analogous region of identical sequence.
A comparison of the SLG-A10 and SRR-A10 sequences revealed a few deletions or insertions occurring in multiples of three base pairs, which would result in the removal or addition of amino acids in the SLG domain.
This also has been detected in the SLG-SRK pairs at the S24 and S6 loci. However, just downstream of the region of 100% homology at position 948 in the SRK-A10 sequence, there is a one base pair deletion that causes a shift in the reading frame. Translation of the DNA sequence revealed that premature termination would occur at nucleotide 978 and a truncated protein would be produced (Figure 2, double underline). Except for this one base pair deletion, the SRR-A10 gene codes for the predicted structures of a receptor kinase, including the transmembrane and kinase domains. The predicted kinase domain contains all of the conserved amino acids found in kinases, and like the other S receptor kinase genes, it shows greatest sequence similarity to serine/threonine kinases. Hanks et al., Science 241: 42 (1988). Thus, the SRR-A10 gene does not encode a functional S locus receptor kinase due to the frame shift mutation.

Example 3 Characterization of the A10 Allele Since the A10 allele is not associated with self-incompatibility, it was possible that the A10 allele was not linked to the S locus. Potentially, there are two S loci in B. napus because the plant contains the genomes of both B. campestris and B. oleracea. In the B. napus W1 line, both the B. campestris 910 allele and the A10 allele are homozygous, suggesting that the A10 allele is not at the S locus position in the B. campestris component of the B. napus genome. Furthermore, in lines in which both the 910 and A10 alleles are present as heterozygotes, these alleles segregate randomly in the self-progeny. This observation is consistent with the suggestion that the 910 and A10 alleles are not linked.
The segregation pattern of the A10 allele was compared to a B. oleracea allele (S~) that is present in the canola cultivar, Karat. The self-incompatible S24Karat homozygote, which does not contain the A10 allele was crossed to the self-compatible Westar, which is homozygous for the A10 allele. To produce a segregating F2 population, the F~ progeny were self-pollinated using salt to break down self-incompatibility. The A10 allele was detected in seedlings by PCR amplification using primers specific to the SLG-A10 gene and by DNA blot hybridization. Surprisingly, all 30 of the tested F2 progeny carried the A10 allele.
Since a DNA probe for the S24 allele was not available, 19 of the F2 progeny were analyzed for the presence of the S24 allele by testing for self-incompatibility, and the S~ SLG gene was then identified by cross-hybridization to the SLG-A10 cDNA. In these studies, blots were washed at 50C in O.lX SSC, 0.1~ SDS.
Thirteen of the 19 tested plants were found to be self-incompatible. The A10 allele was present in the 524Karat/Westar Fl plants, the Westar parental line, and all of the F2 plants. The signal intensity of the A10 allele in the F~ and F2 populations suggested that there was one copy of the A10 allele in self-incompatible F~
and F2 plants and two copies of the A10 allele in the self-compatible F2 plants and the Westar parental line.
Although the S24 and A10 alleles appear to be segregating in a 0:2:1 ratio with no S~ homozygotes, there is a statistically significant probability that the actual ratio is 0:1:1. In summary, these observations are consistent with the theory that the A10 allele represents a B. oleracea S allele in the B. napus genome.

Example 4 Construction of t~e Corrected SR~-A10 cDNA

Three overlapping fragments wére produced during the cloning of the SRR-A10 gene. The first fragment was synthesized using the 5' RACE procedure and contained nucleotides 0 to 770. The second fragment was obtained from PCR using SLG-specific primers and contained nucleotides 510 to 1310. The third fragment was .

synthesized using the 3' RACE procedure and contained nucleotides 1280 to 2685. All three fragments were cloned in pBLUESCRIPT (pBS).
The 5' fragment containing nucleotides 0 to 770 was digested with BamHI and NcoI, producing a fragment containing nucleotides 0 to 719. The cloning vector that contained nucleotides 510 to 1310 also was digested with BamHI and NcoI and then ligated with the 719 base pair fragment. The resultant vector contained nucleotides o to 1310 of the SRR-A10 DNA in pBS.
The following PCR primer was designed to correct the deletion in SRR-A10 DNA by inserting an adenine residue at nucleotide 948: 5' TATGTGCAAGATGTGTGG 3' [SEQ ID
NO:7]. Adenine was used to correct the one base pair deletion because the SLG-A10 cDNA contains an adenine residue at the corresponding position. The PCR primer, representing nucleotides 941 to 968 of the SRR-A10 gene, was used in a PCR with a second primer containing nucleotides 420 to 440 of the SRK-A10 gene to produce a DNA fragment containing nucleotides 420 to 968 of the SRK-A10 gene in which the one base pair deletion had been corrected.
A second PCR product was obtained using different primers that amplified DNA from nucleotides 740 to 1230 of the SRR-A10 gene. The 1310 base pair construct was used as the template DNA in both PCR mixtures. The two PCR products were mixed, and the two outside primers were used in another round of PCR. The result of this PCR
reaction was the amplification of a DNA fragment from nucleotide 420 to nucleotide 1230 of the SRR-A10 gene.
Half of the fragments produced in this manner were expected to carry the additional adenine at position 948.
The PCR fragment was then cleaved at nucleotide 507 with EcoRI, and at nucleotide 1163 w'i~h SalI. The resultant fragment was cloned into pBS.
Several clones were sequenced to verify that the SRR-A10 gene had been corrected by the addition of adenine.
One of the corrected clones was then digested with EcoRI
and SalI and the DNA fragment containing nucleotides 507 ~2~7Si to 1163 was subcloned into the 0-1310 bp fragment of the SRR-A10 gene. This resulted in the correction of the deletion in SRR-A10 gene in a fragment from nucleotide 0 to nucleotide 1310.
To produce a DNA molecule encoding a complete and corrected SRR-A10 gene, a PCR product was synthesized from nucleotides 1000 to 1970. A 3' fragment from nucleotide 1422 to 2685 was produced by 3' RACE and was ligated with the PCR product in pBS. The resulting clones contained nucleotides 1000 to 2685 of the SRR-A10 gene.
A 5' fragment containing nucleotides 0 to 1163, including the correction at nucleotide 948, was then isolated from the corrected 1310 base pair fragment, described above and ligated to the remainder (i.e.
nucleotides 1163-2685) of the SRR-A10 gene. The resultant DNA fragment contained the full length DNA
(i.e., nucleotides 0-2685) encoding the SRK-A10 protein in pBS. The region from nucleotides 1163 to 1422, which was derived from PCR, was sequenced to ensure that no mistakes were introduced during amplification.

Example 5 Production of Transgenic Plants COntA ining a Corrected SR~-Al O Gene The corrected SRR-A10 gene was inserted in pBS behind the SRR-910 promoter, and the resulting construct was subcloned into the plant transformation vector, DP1741.
The vector was introduced into self-compatible B. napus cultivars using the method of Moloney et al., Plant Cell Reports 8: 238 (1989), the contents of which are hereby incorporated by reference.
(, Although the foregoing refers to particular preferred embodiments, it will be understood that the present 2~237Si invention is not so limited. It will occur to those of ordinary skill in the art that various modifications may be made to the disclosed embodiments and that such modifications are intended to be within the scope of the present invention, which is defined by the following claims.
All publications and patent applications mentioned in this specification are indicative of the level of skill of those in the art to which the invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference in its entirety.

Claims (25)

1. An isolated DNA molecule, comprised of the nucleotide sequence of SEQ ID NO:4, wherein said DNA
molecule encodes an SLG protein.

2. The DNA molecule of claim 1, wherein said DNA
molecule consists of the nucleotide sequence of SEQ ID
NO:2.

3. An expression vector comprising the DNA molecule of claim 1.

4. The expression vector of claim 3, further comprising a regulatory element, wherein said regulatory element controls the production of said SLG protein.

5. The expression vector of claim 4, wherein said regulatory element is selected from the group consisting of the SLG-910 regulatory element, the SLG-A10 regulatory element, the SRK-A10 regulatory element and the CaMV 35S
promoter.

6. The expression vector of claim 5, wherein said regulatory element is the SLG-A10 promoter having the nucleotide sequence of SEQ ID NO:8.

7. An isolated DNA molecule, comprised of the nucleotide sequence of SEQ ID NO:5, wherein said DNA
molecule encodes an SRK protein.

8. The DNA molecule of claim 7, wherein said DNA
molecule consists of the nucleotide sequence of SEQ ID
NO:6.

9. The DNA molecule of claim 8, wherein the nucleotide at position 948 of SEQ ID NO:6 is adenine.

10. An expression vector comprising the DNA molecule of claim 7.

11. The expression vector of claim 10, further comprising a regulatory element, wherein said regulatory element controls the production of said SRK protein.

12. The expression vector of claim 11, wherein said regulatory element is selected from the group consisting of the SLG-910 regulatory element, the SLG-A10 regulatory element, the SRK-A10 regulatory element and the CaMV 35S
promoter.

13. The expression vector of claim 12, wherein said regulatory element is the SLG-A10 promoter having the nucleotide sequence of SEQ ID NO:8.

14. A method of producing a self-incompatible plant, comprising the steps of:
(a) producing a first parent self-compatible plant comprising an expression vector that comprises the SRK-encoding DNA molecule of claim 7, wherein said first parent plant does not contain the A10 allele; and (b) cross-fertilizing said first parent plant with a second parent plant having the A10 allele to produce a progeny plant, wherein said progeny plant produces SLG-A10 protein and SRK-A10 protein, resulting in the self-incompatibility phenotype.

15. The method of claim 14, wherein said second parent plant is selected from the group consisting of the Ceres line, the Regent line, the Westar line and the W1 line.

16. A self-incompatible plant comprising the nucleotide sequence of SEQ ID NO:6.

17. A method of producing hybrid seed, comprising the steps of:
(a) producing a self-incompatible transgenic plant comprising the nucleotide sequence of SEQ ID
NO:6; and (b) cross-fertilizing said transgenic plant with a second plant.

18. A method for conferring the self-incompatible phenotype on a self-compatible plant, said method comprising the steps of:
(a) preparing a first expression vector comprising an SLG-encoding DNA molecule consisting of the nucleotide sequence of SEQ ID NO:2;
(b) preparing a second expression vector comprising an SRK-encoding DNA molecule consisting of the nucleotide sequence of SEQ ID NO:6; and (c) transferring said first expression vector and said second expression vector into said self-compatible plant to produce a transformed plant, wherein said transformed plant expresses SLG-A10 protein and SRK-A10 protein, resulting in a self-incompatibility phenotype.

19. The method of claim 18, wherein each of said first expression vector and said second expression vector further comprises a Ti plasmid.

20. The method of claim 19, wherein said transferring step is performed by (i) producing Agrobacterium tumefaciens comprising said first expression vector and said second expression vector and (ii) infecting said self-compatible plant with said Agrobacterium tumefaciens to produce said transformed plant.

21. The method of claim 20, wherein said self-compatible plant is from the genus of Brassica.

22. A transformed plant comprising an expression vector, wherein said expression vector comprises the nucleotide sequence of SEQ ID NO:6.

23. The transformed plant of claim 22, wherein said transformed plant is from the genus of Brassica.

24. An isolated DNA molecule comprising the nucleotide sequence of SEQ ID NO:8, wherein said DNA
molecule is the SLG-A10 promoter.

25. An expression vector comprising the SLG-A10 promoter of claim 24.