Didier Cabrillac

Valérie Delorme

Jerome Garin

Véronique Ruffio- Châble

Jean-Loïc Giranton

Christian Dumas

Thierry Gaude

J Mark Cock

The S 15 Self-incompatibility Haplotype in Brassica oleracea Includes Three S Gene Family Members Expressed in Stigmas

Self-incompatibility in Brassica is controlled by a single, highly polymorphic locus which extends over several hundred kilobases and includes several expressed genes. Two stigma proteins, the S locus receptor kinase (SRK) and the S locus glycoprotein (SLG), are encoded by genes located at the S locus and are thought to be involved in the recognition of self-pollen by the stigma. We report here that two different SLG genes, SLGA and SLGB, are located at the S locus in the class II, pollen recessive S 15 haplotype. Both genes are interrupted by a single intron but SLGA encodes both soluble and membrane-anchored forms of SLG whereas SLGB only encodes soluble SLG proteins. Thus, including SRK, the S locus in the S 15 haplotype contains at least three members of the S gene family. The protein products of these three genes have been characterized and each SLG glycoform was assigned to an SLG gene.

Evidence is presented that the S 2 and S 5 haplotypes carry only one or the other of the SLG genes indicating either that they are redundant or that they are not required for the selfincompatibility response.

INTRODUCTION

Brassica spp possess hermaphroditic flowers with functional male and female reproductive organs in close proximity. Auto-fertilization is, however, rare in the majority of the members of this genus because of the widespread occurrence of a genetically-controlled selfincompatibility (SI) system which allows recognition and rejection of self-pollen by the stigma (reviewed in [START_REF] Nasrallah | Signalling the arrest of pollen tube development in self-incompatible plants[END_REF][START_REF] Mccormick | Self-incompatibility and other pollen-pistil interactions[END_REF]. SI in Brassica is controlled by a single, highly polymorphic locus, the S locus. The S locus has been shown to have a complex structure; a high level of polymorphism extends over several hundred kilobases and several different genes are located in this region of the genome.

Two of the genes that have been identified at the S locus, SLG (for S locus glycoprotein; [START_REF] Nasrallah | Amino-acid sequence of glycoproteins encoded by three alleles of the S-locus of Brassica oleracea[END_REF] and SRK (for S locus receptor kinase; [START_REF] Stein | Molecular cloning of a putative receptor protein kinase gene encoded at the selfincompatibility locus of Brassica oleracea[END_REF], are thought to be involved in the first step of the SI response, the recognition of self-pollen.

SRK is an integral membrane glycoprotein that closely resembles animal receptor protein kinases [START_REF] Stein | Molecular cloning of a putative receptor protein kinase gene encoded at the selfincompatibility locus of Brassica oleracea[END_REF]Delorme et al., 1995b;[START_REF] Stein | SRK, the stigmaspecific S locus receptor kinase of Brassica, is targeted to the plasma membrane in transgenic tobacco[END_REF]. It possess an extracellular receptor domain, a single membrane-spanning domain, and a cytosolic kinase domain. SLG is a secreted glycoprotein that closely resembles the extracellular domain of SRK and this similarity is often particularly marked within haplotype(s), indicating that there has been convergent evolution between linked SLG and SRK alleles [START_REF] Stein | Molecular cloning of a putative receptor protein kinase gene encoded at the selfincompatibility locus of Brassica oleracea[END_REF][START_REF] Goring | An S receptor kinase gene in self-compatible Brassica napus has a 1 bp deletion[END_REF][START_REF] Kusaba | Striking sequence similarity in inter-and intra-specific comparisons of class I SLG alleles from Brassica oleracea and Brassica campestris: Implications for the evolution and recognition mechanisms[END_REF]. The SLG gene also shares similarity with a number of other sequences both in Brassica spp and in other species and these genes are referred to collectively as the S gene family. A member of the S gene family is defined as a gene which encodes a protein that possesses an S domain with sequence similarity to the S locus glycoprotein.

Both SLG and SRK are expressed specifically in stigmas in a developmentally regulated manner. The accumulation of SLG and SRK transcripts and proteins coincides with the acquisition of SI by the maturing stigma [START_REF] Sato | Activity of an S locus gene promoter in pistils and anthers of transgenic Brassica[END_REF][START_REF] Stein | SRK, the stigmaspecific S locus receptor kinase of Brassica, is targeted to the plasma membrane in transgenic tobacco[END_REF]. The only other organ in which SLG and SRK transcripts have been detected is the anther, but transcripts of both genes are present at very low levels and the protein products of these genes have not been detected in this organ [START_REF] Sato | Activity of an S locus gene promoter in pistils and anthers of transgenic Brassica[END_REF][START_REF] Stein | Molecular cloning of a putative receptor protein kinase gene encoded at the selfincompatibility locus of Brassica oleracea[END_REF]Delorme et al., 1995b;[START_REF] Stein | SRK, the stigmaspecific S locus receptor kinase of Brassica, is targeted to the plasma membrane in transgenic tobacco[END_REF]. Moreover, recent evidence indicates that SRK transcripts in anthers are copied from the noncoding strand of the gene [START_REF] Cock | Natural antisense transcripts of the S locus receptor kinase gene and related sequences in Brassica oleracea[END_REF]. Both SLG and SRK are highly polymorphic and the alleles of both genes can be grouped into two classes based on the degree of sequence divergence. These two classes correlate closely with the two classes of S haplotype that have been defined based on their associated SI phenotype. Class I S haplotypes possess highly polymorphic SLG and SRK genes, confer a strong SI phenotype and tend to be dominant in interactions with other haplotypes. SLG and SRK genes of class II haplotypes, on the other hand, are less polymorphic and these haplotypes confer a weaker SI response and tend to be recessive to the class I haplotypes.

There is therefore a great deal of circumstantial evidence implicating SLG and SRK in the SI response but, despite a significant effort in several different laboratories, direct evidence linking these genes to the SI response has been difficult to obtain. A reduction in SLG expression due to antisense inhibition or cosuppression has been correlated with the loss of the SI response [START_REF] Toriyama | Transformation of Brassica oleracea with an S-locus gene from B. campestris changes the selfincompatibility phenotype[END_REF][START_REF] Shiba | Breakdown of self-incompatibility by the antisense RNA of the SLG gene[END_REF] but pleiotropic effects on SRK were not ruled out in these experiments. Whilst these observations suggested a role for SLG in the SI response, another study showed that the level of expression of SLG was not correlated with the strength of the SI response and indicated that, if SLG has a role in the SI response, it must be capable of carrying out its function even when expressed at a low level [START_REF] Gaude | Expression level of the SLG gene is not correlated with the self-incompatibility phenotype in the class II S haplotypes of Brassica oleracea[END_REF].

Several studies have demonstrated a correlation between reduced SRK expression and an impaired SI response [START_REF] Goring | An S receptor kinase gene in self-compatible Brassica napus has a 1 bp deletion[END_REF]Nasrallah et al., 1994a;[START_REF] Conner | Transgene-induced silencing of S-locus genes and related genes in Brassica[END_REF]. In two instances, the presence of mutant SRK alleles has been correlated with a self-compatible phenotype [START_REF] Goring | An S receptor kinase gene in self-compatible Brassica napus has a 1 bp deletion[END_REF]Nasrallah et al., 1994a), although in both cases it is possible that other genes were also affected. The recent observation that a mutant SRK gene induced partial self compatibility when transformed into a self-incompatible Brassica napus line strongly implicates SRK in the SI response, especially as the effect of the transgene was haplotype-specific [START_REF] Stahl | The selfincompatibility phenotype in Brassica is altered by the transformation of a mutant S locus receptor kinase[END_REF].

Based on the resemblance of SRK to animal receptor protein kinases, current models of self-pollen recognition in the SI response propose that SRK recognizes a pollen-borne ligand. Using a bioassay, [START_REF] Stephenson | The male determinant of self-incompatibility in Brassica oleracea is located in the pollen coating[END_REF] have recently shown that the pollen coat of self pollen carries a factor which can initiate the SI response. The gene encoding the male factor has not yet been identified. One possible candidate, the S locus anther (SLA) gene, an S locus linked gene expressed specifically in anthers [START_REF] Boyes | An anther-specific gene encoded by an S locus haplotype of Brassica produces complementary and differentially regulated transcripts[END_REF], has recently been shown not to be required for the SI response (Pastuglia et al., 1997b).

In the female part of the flower, S locus genes encode a complex mixture of proteins in addition to SLG and SRK. [START_REF] Tantikanjana | An alternative transcript of the S-locus glycoprotein gene in a class-II pollen-recessive self-incompatibility haplotype of Brassica oleracea encodes a membrane-anchored protein[END_REF] showed that the class II SLG 2 allele possesses two exons and that alternative transcripts encode SLG and a membrane-anchored form of SLG, mSLG. mSLG has not been found in stigmas of class I haplotypes (class I SLG genes lack the second exon), and it has been suggested that this protein modulates the SI response and is responsible for the recessive phenotype associated with the class II haplotypes [START_REF] Tantikanjana | An alternative transcript of the S-locus glycoprotein gene in a class-II pollen-recessive self-incompatibility haplotype of Brassica oleracea encodes a membrane-anchored protein[END_REF]. Similarly, the class I SRK 3 allele has been shown to encode, in addition to an integral SRK protein, a soluble truncated protein, eSRK, which corresponds to the SRK extracellular domain [START_REF] Giranton | The S locus receptor kinase gene encodes a soluble glycoprotein corresponding to the SRK extracellular domain in Brassica oleracea[END_REF]. eSRK is also thought to play a role in modulating the SI response.

In this study, we demonstrate that the class II S 15 haplotype carried by the B. oleracea line P57Sc includes alleles of three different stigma-expressed genes; SRK and two different SLG genes, SLGA and SLGB. Evidence is also presented that the S 5 haplotype contains SLGB but not SLGA whereas the S 2 haplotype contains SLGA but not SLGB. These data raise questions both about the functional roles SLG and mSLG and about how sequences at the S locus have evolved.

RESULTS

Identification of a third S gene family member at the S locus in the B. oleracea line

P57Sc (haplotype S 15 )

The self-compatible B. oleracea line P57Sc described by [START_REF] Gaude | Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea[END_REF] was shown to reject pollen from an S 15 homozygous tester line indicating that it carries the S 15 haplotype and that the SI system is functional on the female side (see Methods). A 116 kD protein, which is thought to correspond to SRK 15 , has been identified in stigma extracts of the P57Sc line (Delorme et al., 1995b). To further characterize this protein, we attempted to identify the corresponding gene.

Alleles corresponding to two S gene family members have been shown to be linked to the S locus in the P57Sc line. The first, SLG Sc is predicted to encode both a secreted and a membrane-anchored glycoprotein (Pastuglia et al., 1997b). SLG Sc is unlikely to also encode an SRK protein (with a cytosolic kinase domain) because this gene is located directly upstream of another S locus gene, SLA (Pastuglia et al., 1997b; see Figure 1). For the second gene, a cDNA (designated CG15) was isolated which also encoded a secreted glycoprotein [START_REF] Gaude | Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea[END_REF]. One possibility was that this cDNA corresponded to an alternative transcript of SRK. An analogous situation has been described for the S 3 haplotype in which SRK 3 has been shown to produce alternative transcripts that encode a truncated form of the SRK protein which resembles the SLG protein of the same haplotype [START_REF] Giranton | The S locus receptor kinase gene encodes a soluble glycoprotein corresponding to the SRK extracellular domain in Brassica oleracea[END_REF].

To determine whether the gene which encoded CG15 also encodes an SRK protein we isolated the corresponding region of genomic DNA and sequenced a region of 2.8 kb downstream of the region corresponding to CG15 (Figure 1). No similarity with the transmembrane or kinase domains of other SRK alleles was found within the 2.8 kb region suggesting that the gene corresponding to CG15 does not encode an SRK protein. However, we could not rule out the possibility that the S domain and transmembrane domain were separated by a large intron which spanned the sequenced region.

As an alternative approach to identify the SRK allele of the P57Sc line, oligonucleotides were designed to amplify SRK cDNA using the the polymerase chain reaction (PCR). The nucleotide sequences of the kinase domains of two class II SRK alleles, SRK 5 (see Methods) and SRK 2 (JMC, unpublished data), were aligned with the corresponding regions of the closely related genes SLR3 (Cock et al., 1995), SFRI (accession number Y14285), SFR2 (Pastuglia et al., 1997a) and SFR3 (accession number Y14286). Sequences conserved in the class II SRK alleles but diverged or absent in the other genes were selected for oligonucleotide synthesis. These oligonucleotides were used either in combination with PS3, an oligonucleotide that hybridizes near the ATG initiation codon of several class II S genes [START_REF] Nishio | Registration of S alleles in Brassica campestris L by the restriction fragment sizes of SLGs[END_REF], to amplify 5' sequences or with a oligo(dT) adapter in a rapid amplification of cDNA ends (RACE)-PCR protocol [START_REF] Frohman | Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer[END_REF] to amplify 3' sequences from stigma cDNA. These experiments allowed the reconstruction of a cDNA of 2883 bp. The cDNA is predicted to encode a polypeptide which consists of a signal peptide, an S domain, a membrane-spanning domain and a kinase domain. The predicted mass of the mature polypeptide is 94.0 kD. The S domain region of the amplified cDNA was highly similar to SLG Sc and CG15 (89.5% and 87.3% respectively, Table 1) but was not identical, indicating that the cDNA was derived from a third gene.

Because P57Sc carries the S 15 haplotype, the gene corresponding to the amplified cDNA was designated as SRK 15 , SLG Sc was renamed SLGA 15 and the allele corresponding to CG15 was designated as SLGB 15 (Figure 1). To confirm that the P57Sc line carried the S 15 haplotype, part of the S domain of each of the three genes was amplified from genomic DNA of the S 15 tester line. DNA sequence analysis of the amplified products showed that they were identical to the corresponding regions of the three genes carried by P57Sc (apart from a number of errors introduced during the PCR, each of which was only found in one of several clones analysed for each gene; data not shown).

Comparison of SLGA 15 and SRK 15 revealed a region of 502 bp with 100% sequence identity (Figure 1). The presence of this region of identity indicates that there has been concerted evolution between the two genes probably involving gene conversion. A similar region was not found when SLG 2 and SRK 2 were compared indicating that gene conversion occurred after the divergence of these two S haplotypes.

Genetic mapping and expression patterns of SRK 15 , SLGA 15 and SLGB 15

To demonstrate that SRK 15 , SLGA 15 and SLGB 15 were linked to the S locus, we used the PCR to analyse their segregation in an F 2 population resulting from a cross between the P57Sc line (homozygous for the S 15 haplotype) and a line homozygous for the S 3a haplotype. Figure 2A shows that SRK 15 , SLGA 15 and SLGB 15 sequences were only amplified from genomic DNA of F 2 progeny carrying the S 15 haplotype, indicating that all three genes are linked to the S locus.

The expression patterns of SLGA 15 and SLGB 15 were analysed by ribonuclease protection analysis of mRNA abundance in a range of different organs. Figure 2B shows that SLGA 15 and SLGB 15 transcripts were detected only in stigma RNA. No transcripts were detected in the other organs tested even after a long exposure. Analysis of autoradiographs by densitometry indicated that SLGA 15 transcripts were approximately four fold less abundant than SLGB 15 transcripts. Reverse transcriptase (RT)-PCR was used to analyse the organspecific expression pattern of SRK 15 . Figure 2C shows that SRK 15 transcripts were detected only in stigmas and, at a much lower abundance, in anthers at the trinucleate pollen stage.

Analysis of S locus-encoded glycoproteins in stigmas of Brassica plants homozygous for the S 15 haplotype

Comparison of the sequences of SRK 15 , SLGA 15 and SLGB 15 indicated that these three genes encoded mature protein products with identical amino-termini. The first 10 amino acid residues of each protein are predicted to be identical to the synthetic peptide that was used to raise the monoclonal antibody MAb 157-35-50 [START_REF] Giranton | The S locus receptor kinase gene encodes a soluble glycoprotein corresponding to the SRK extracellular domain in Brassica oleracea[END_REF] and this antibody should therefore recognize the protein products of all three genes. MAb 157-35-50 was used to detect proteins in stigma extracts from plants homozygous for five different S haplotypes, including S 15 , following separation of the proteins by SDS-PAGE. Figure 3A shows that MAb 157-35-50 detected both a large protein of between 102 and 120 kD, depending on the S haplotype, and a number of smaller proteins of between 43 and 68 kD in stigmas of four of the five lines.

The molecular masses and polymorphic nature of the 102 to 120 kD proteins was consistent with their corresponding to the SRK proteins of each S haplotype. This was supported by the fact that, following deglycosylation, there was a shift in the mobilities of these proteins and a polypeptide of 94 kD was detected in extracts from lines homozygous for S 2 , S 15 and S 3 (Figure 3B). The method was not sensitive enough to detect deglycosylated SRK 5 . The molecular mass of the deglycosylated proteins corresponded to the predicted molecular mass of the polypeptide backbone of SRK. The mobility of the protein detected in stigma extracts of the S 15 homozygous line indicated a molecular mass of 105 kD, slightly lighter than the previous estimate of 116 kD (Delorme et al., 1995b). Further evidence that this protein corresponds to SRK 15 was obtained by analysing 24 plants of the F 2 population segregating the S 15 and S 3a haplotypes (Figure 3C). The 105 kD protein was expressed only in plants which possessed the S 15 haplotype and is therefore encoded by a gene linked to the S locus in the S 15 haplotype. This result was confirmed by analysing additional F 2 populations (data not shown).

In addition to the larger SRK proteins, MAb 157-35-50 detected smaller proteins of between 43 and 68 kD in the various S haplotypes (Figure 3A). Several of these proteins, particularly those from S 3 and S 2 have been characterized previously [START_REF] Giranton | The S locus receptor kinase gene encodes a soluble glycoprotein corresponding to the SRK extracellular domain in Brassica oleracea[END_REF][START_REF] Tantikanjana | An alternative transcript of the S-locus glycoprotein gene in a class-II pollen-recessive self-incompatibility haplotype of Brassica oleracea encodes a membrane-anchored protein[END_REF]. The 59.4 and 69.8 kD proteins detected in the stigma extract from the S 3 line are glycoforms of a soluble, truncated form of SRK 3 (eSRK 3 ) which have a polypeptide backbone of 46.8 kD [START_REF] Giranton | The S locus receptor kinase gene encodes a soluble glycoprotein corresponding to the SRK extracellular domain in Brassica oleracea[END_REF]Figure 3B). SLG 3 is not detected by MAb 157-35-50. On the other hand, the 55 to 68 kD proteins detected in the S 2 line have previously been shown to include both the soluble SLG 2 protein and a membrane anchored form of SLG 2 called mSLG 2 [START_REF] Tantikanjana | An alternative transcript of the S-locus glycoprotein gene in a class-II pollen-recessive self-incompatibility haplotype of Brassica oleracea encodes a membrane-anchored protein[END_REF]. This is consistent with the fact that proteins of 52 and 48 kD were detected in deglycosylated S 2 stigma extracts, presumably corresponding to the polypeptide backbones of mSLG 2 and SLG 2 respectively.

The pattern of bands detected by MAb 157-35-50 in stigma extracts of the S 15 line was more similar to that observed with the S 2 line than with the S 3 line. The mobilities of the proteins of between 54 and 65 kD was altered following deglycosylation to give two proteins of 48 and 52 kD (Figure 3B). By analogy to S 2 , we suggest that these correspond to soluble and membrane anchored forms of SLG, respectively. Interestingly, following deglycosylation of stigma proteins from the S 5 homozygous line a single major protein of 48 kD was detected with MAb 157-35-50 (the size predicted for soluble SLG protein) suggesting that stigmas of this line do not contain significant levels of mSLG (Figure 3B).

The above interpretation of the results presented in Figures 3A and3B predicts that the 105 kD protein corresponds to SRK and is anchored in the membrane whereas the smaller 54 to 65 kD proteins include both soluble and membrane-anchored proteins. To investigate further the sub-cellular location of the S locus-encoded proteins of the S 15 homozygous line, we separated stigma extracts into soluble and membrane fractions and then further fractionated the membrane preparation by aqueous two-phase partitioning to enrich for plasma membrane. An antibody against the plasma membrane-localised ATPase was used to verify that the fractions had been enriched in plasma membrane (data not shown). MAb 157-35-50 was then used to detect S locus-encoded proteins in the different fractions. Figure 3D shows that the 105 kD protein was most abundant in the fraction enriched in plasma membrane. The group of proteins of between 54 and 65 kD could be separated into, on the one hand, a number of proteins of between 54 and 65 kD, which partitioned into the soluble fraction, and, on the other, a major protein of 65 kD, which was associated with the plasma membrane (Figure 3D). These results are consistent with the 105 kD protein corresponding to a plasma membrane-anchored SRK, the 65 kD protein corresponding to plasma membraneanchored mSLG and the 54 to 65 kD proteins being different soluble glycoforms of SLG.

Analysis of the DNA sequences of SRK 15 , SLGA 15 and SLGB 15 indicated that the soluble glycoproteins detected by MAb 157-35-50 in stigma extracts of the S 15 homozygous line could have been encoded by any of the three genes. These soluble glycoproteins can be resolved by isoelectric focusing (IEF) gel electrophoresis into four major glycoproteins which have been designated a,_b,_b' and b" [START_REF] Gaude | Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea[END_REF]. Figure 4 shows that these glycoproteins vary in pI from 6.1 to 7.3 and in relative abundance from 16.5% to 41.5%. To characterize them further, they were each purified by selective, two dimensional gel electrophoresis [START_REF] Gaude | Use of a fast protein electrophoretic purification procedure for N-terminal sequence analysis to identify S-locus related proteins in stigmas of Brassica oleracea[END_REF]. The purified proteins were then digested with either endopepidase Lys-C or endopeptidase Gluc-C and HPLC-purified peptides were sequenced by Edman degradation. The protein sequence data indicated that a_and_b were encoded by SLGB 15 (Figure 4). No peptide sequence data was obtained for b" and the sequence obtained for_b' was conserved in SRK 15 , SLGA 15 and SLGB 15 . In order to confirm these results and to assign a gene to the b' and b" proteins, the IEF-resolved a,_b,_b' and b" proteins were digested 'in-gel' with trypsin and the tryptic peptides analysed by matrix-assisted laser desorption ionisation time-of-flight mass spectroscopy (MALDI-TOF-MS). This analysis demonstrated that a,_b and_b' were encoded by SLGB 15 and that b" was encoded by SLGA 15 (Table 2). None of the four proteins were encoded by SRK 15 .

SLGB 15 encodes 79.5% of the SLG protein in the stigma, only 20.5% being encoded by SLGA 15 (Figure 4). This is consistent with the fact that SLGB 15 transcripts are approximately 4 fold more abundant than those of SLGA 15 (Figure 2), bearing in mind that a proportion of the SLGA 15 transcripts presumably encode an mSLG protein. Expression of these two genes therefore appears to be regulated principally at the mRNA level.

Transcripts of the S locus genes of the S 15 haplotype

Based on the above analysis of proteins detected in stigma extracts from the S 15 homozygous line, SRK 15 is predicted to encode a membrane-anchored, 105 kD protein (SRK), SLGA 15 to encode both a soluble protein (SLG b") and a membrane-anchored protein of 65 kD (mSLG) and SLGB 15 has been shown to encode several soluble proteins (SLGs a,_b and_b'). If this is the case then each gene would be expected to produce transcripts capable of encoding the corresponding protein products. This has been demonstrated for SRK 15 and SLGB 15 , which were both initially identified as cDNA clones which encode SRK and SLG proteins, respectively (Figure 5). However, SLGA 15 was identified as a genomic clone and SLGA 15 transcription has not yet been analysed. Therefore, we used RACE-PCR to characterize transcripts of this gene. Figure 5 shows that two different transcripts were identified, one which terminates within the single intron and therefore is predicted to encode a secreted SLG protein and a second which includes both exon 1 and exon 2 and hence is predicted to encode a membrane-anchored mSLG protein. Therefore, analysis of RNA transcripts of the SRK 15 , SLGA 15 and SLGB 15 indicates that these three genes potentially encode all the proteins detected in stigma extracts with the MAb 157-35-50 antibody.

Interestingly, none of the soluble, S-locus-encoded proteins present in S 15 stigma extracts corresponded to a truncated form of the SRK protein (Figure 4). This contrasts with the situation in the S 3 haplotype where significant levels of such a protein, eSRK, have been detected [START_REF] Giranton | The S locus receptor kinase gene encodes a soluble glycoprotein corresponding to the SRK extracellular domain in Brassica oleracea[END_REF]. eSRK is encoded by transcripts which retain sequences corresponding to the first intron of the SRK gene with the result that a stop codon occurs at the end of the reading frame encoding the S domain [START_REF] Giranton | The S locus receptor kinase gene encodes a soluble glycoprotein corresponding to the SRK extracellular domain in Brassica oleracea[END_REF]. This stop codon is found in a conserved position just downstream of the 5' splice site of intron 1 in all the SRK alleles that have been analysed to date. We isolated the first intron of SRK 15 by PCR amplification from genomic DNA and sequence analysis showed that the stop codon is also present in SRK 15 (data not shown). Furthermore, RACE-PCR analysis identified short SRK 15 transcripts which possessed sequences corresponding to the 5' end of intron 1 and which are therefore predicted to encode an eSRK protein (Figure 5). The absence of an eSRK protein amongst the soluble S 15 stigma proteins suggests, however, that these transcripts are not translated or that they are only present in very low abundance.

Comparison of the sequences of the S 15 alleles of SLGA, SLGB and SRK with those of other haplotypes.

The deduced amino acid sequences of the S domains of SRK 15 , SLGA 15 and SLGB 15 were compared with SRK and SLG proteins encoded by other class I and class II S haplotypes and with two proteins, SLR2 and SFR2, which are also encoded by members of the S gene family in Brassica but whose genes are not linked to the S locus. Table 1 shows that SLGA 15 is most similar to SLG 2 whereas SLGB 15 is most similar to SLG 5 . This confirms a previous observation that, whilst there is evidence for convergent evolution of SLG and SRK alleles within a particular S haplotype [START_REF] Stein | Molecular cloning of a putative receptor protein kinase gene encoded at the selfincompatibility locus of Brassica oleracea[END_REF], alleles within a haplotype are not necessarily more similar to each other than to alleles from other haplotypes [START_REF] Kusaba | Striking sequence similarity in inter-and intra-specific comparisons of class I SLG alleles from Brassica oleracea and Brassica campestris: Implications for the evolution and recognition mechanisms[END_REF].

The similarities between SLGA 15 and SLG 2 and between SLGB 15 and SLG 5 are interesting because these two pairs of genes share similar structures. Both SLGA 15 and SLG 2 possess a single intron and are capable of encoding both soluble and membrane-anchored forms of SLG (Figure 5; [START_REF] Tantikanjana | An alternative transcript of the S-locus glycoprotein gene in a class-II pollen-recessive self-incompatibility haplotype of Brassica oleracea encodes a membrane-anchored protein[END_REF]. Comparison of the sequences of the introns of these two alleles [START_REF] Tantikanjana | An alternative transcript of the S-locus glycoprotein gene in a class-II pollen-recessive self-incompatibility haplotype of Brassica oleracea encodes a membrane-anchored protein[END_REF]Pastuglia et al., 1995b) revealed that they were 95.9% similar (data not shown). As for SLGB 15 , it also possesses an intron but does not encode a membrane-anchored form of SLG (Figure 1). The predicted C-terminus of SLG 5 (-threonine-cysteine-threonine-glycine-COOH) is identical to the C-terminus of SLGB 15 which is encoded by the second exon. We showed, by PCR amplification from genomic DNA, that the last four residues of SLG 5 are also encoded by a second exon. Comparison of the nucleotide sequence of the intron of SLG 5 (accession number Y18256) showed that it was 97.7 % similar to that of SLGB 15 (data not shown). [START_REF] Hatakeyama | Molecular Characterization of S Locus Genes, SLG and SRK, in a Pollen-Recessive Self-Incompatibility Haplotype of Brassica rapa L[END_REF] The deduced amino acid sequences of different alleles of SLG and SRK exhibit a high level of polymorphism but polymorphic residues are not distributed evenly along the gene sequence. In particular, two hypervariable regions have been identified (residues 217 to 224 and 273 to 292 in SLGA 15 ) and it has been suggested that these hypervariable regions may be involved in determining the identity of each allele [START_REF] Nasrallah | Amino-acid sequence of glycoproteins encoded by three alleles of the S-locus of Brassica oleracea[END_REF]. In a survey of class I SLG alleles from B. oleracea and B. campestris, [START_REF] Kusaba | Striking sequence similarity in inter-and intra-specific comparisons of class I SLG alleles from Brassica oleracea and Brassica campestris: Implications for the evolution and recognition mechanisms[END_REF] identified two pairs of alleles that shared identical hypervariable regions. One pair included an allele from each species, and may represent alleles with the same SI-specificity in the two species but the second pair were both from B. oleracea and were derived from two genetically distinct S haplotypes, S 8 and S 46 . Comparison of SLGA 15 with SLG 2 showed that these two alleles are even more similar than the SLG 8 /SLG 46 pair, with only five amino acid differences between the mature proteins (compared with nine for SLG 8 /SLG 46 ), none of which are located in the hypervariable regions (Figure 6). Moreover, none of the polymorphisms in the SLGA 15 /SLG 2 pair are predicted to alter sites of N-linked glycosylation. These observations suggest that, if SLG plays a role in the recognition step of the SI response, the hypervariable domains are not always involved in determining allele-specificity.

Intron structure and evolution of S locus genes in the S 15 haplotype

To better understand the evolutionary relationship between the three S locus genes, the DNA sequences of the first intron of SRK 15 and the introns of SLGA 15 and SLGB 15 were compared.

Figure 7A shows that the intron of each allele contained regions that were highly similar to sequences in introns of each the two other alleles. However, none of the three introns contained sequences which were significantly similar to both of the other introns. Figure 7B shows two possible models for the evolution of these intron sequences based on these similarities. In the first model, three copies of an ancestral gene would have been generated by triplication and each of the three copies would then have undergone a specific deletion of intron sequences to generate the intron sequences of SRK 15 , SLGA 15 and SLGB 15 . In the second model, multiple copies of an ancestral gene generated by gene duplication would have undergone a series of recombination events involving intron sequences to generate the patchwork of sequences shared between the three genes. In both cases, only major steps in the evolution of the sequences are shown, additional mutations would have lead to the divergence of the sequences from one another.

The high level of sequence similarity between the introns of SLGA 15 and SLG 2 and between the those of SLGB 15 and SLG 5 suggests that the major structural rearrangements of the introns indicated in Figure 7B would have occurred before the divergence of these class II S alleles. This suggests a relatively ancient origin for the intron sequences and we were interested in determining whether the introns of SRK 15 , SLGA 15 and SLGB 15 resembled those of other members of the S gene family in Brassica. The S domain of SFR2 shares 55.5% similarity with that of SLGB 15 at the amino acid level (Table 1). We isolated the first intron from SFR2 (accession number Y18257) and compared its sequence with those of SRK 15 , SLGA 15 and SLGB 15 . No significant similarity was detected (data not shown). The S domain of SLR2 shares greater similarity with those of the S locus genes and is most similar to SLGB (85.6% amino acid similarity; Table 1). SLR2 is also similar to SLGB at the structural level, both genes contain an intron and the C-terminal end of SLR2 (glycine-threonine-glycine-COOH) is encoded by a second exon [START_REF] Tantikanjana | The Brassica S gene family: molecular characterisation of the SLR2 gene[END_REF]. These similarities suggest that SLR2 may have diverged from SLGB after the divergence of the three S locus genes and it will be interesting to compare the introns of these two genes to test this hypothesis.

Do other S haplotypes contain three S gene family members?

The above data indicated that the S 15 haplotype includes three members of the S gene family.

We were interested in determining whether this was also the case for other S haplotypes. A pair of oligonucleotides corresponding to conserved sequences within the S domains of the three S 15 alleles were therefore used to amplify sequences from cDNA and genomic DNA of B. oleracea lines homozygous for two other class II S haplotypes, S 5 and S 2 . No novel SLG sequences were detected using this approach (data not shown). To investigate this question further, fragments from the introns of SLGA 15 and SLGB 15 were used as gene-specific probes to detect alleles of these two genes in blots of genomic DNA. Figure 8 shows that a probe from intron 1 of SLGA 15 hybridized to a single fragment in BamHI and EarI restriction digested DNA from S 15 and S 2 homozygous lines. A similar result was obtained with a number of different restriction enzymes and the fragments were identical size in each case, confirming the observation (see above) that SLGB 15 and SLG 2 share a high degree of similarity of both exon and intron sequences. No corresponding fragment was detected in digests of S 5 DNA despite the fact that the S 5 alleles of the two other S locus genes SLGA (i.e. SLG 5 ) and SRK are also highly similar to those of the S 15 haplotype. This result suggests that the SLGA gene has been lost from the S 5 haplotype. When stigma proteins from the S 5 homozygous line were deglycosylated and S locus proteins identified with MAb 157-35-50, a single major band of 48 kD, the size expected for soluble SLG, was detected (Figure 3B). In contrast, two major proteins of 48 and 52 kD, which probably correspond to SLG and mSLG respectively, were detected in extracts from the S 2 and S 15 homozygous lines (Figure 3B).

Again, these results are consistent with the S 5 haplotype lacking a functional SLGA allele and, therefore, being unable to encode mSLG.

A similar experiment was carried out with a probe from the SLGB 15 intron. In this case a single, conserved fragment hybridized to the probe in DNA from S 15 and S 5 DNA but no corresponding fragment was detected in S 2 DNA (Figure 8). The probe did, however, hybridize weakly to a fragment of 2.9 kb in an BamHI digest of S 5 DNA and it was not clear whether this represented hybridization to SLGB or to another member of the S gene family.We favour the latter interpretation because a weakly hybridizing fragment was also detected in DNA of plants carrying the class I S 3 haplotype. The SLGB gene, therefore, is probably absent from (or present but significantly rearranged in) the S 2 haplotype. Gaude et al. (1995) have shown that the S 2 homozygous line expresses a very low level of SLG whereas SLG levels are significantly higher, and comparable with those of S 15 , in the S 5 homozygous line. Figure 4 shows that, in the S 15 haplotype, SLGA is strongly expressed and accounts for 79.5% of stigma SLG whilst SLGB only encodes 20.5%. The hypothesis that the S 5 and S 2 haplotypes have lost SLGA and SLGB respectively is therefore attractive because it may explain the significant difference in levels of SLG in stigmas of the two haplotypes.

DISCUSSION

Three members of the S gene family are linked to the S locus in the S 15 haplotype

The results presented here demonstrate that three, closely-related members of the S gene family, SRK 15 , SLGA 15 and SLGB 15 , are linked to the S locus in the S 15 haplotype. In a similar study, Suzuki et al. (1997) showed that three members of the S gene family are closely linked to the S locus in the B. campestris S 9 haplotype. However, the genes identified in the S 9 haplotype share less than 70% similarity with SLG 9 and SRK 9 at the nucleotide level and are expressed in both vegetative and floral tissues suggesting that they form a phylogenetic group distinct from SLG/SRK and that they have (a) different funtional role(s). Furthermore, two of the genes identified by Suzuki et al. (1997) probably encode non-functional proteins. In contrast, the three genes identified in the B. oleracea S 15 haplotype share several characteristics which have been associated with a function in self-incompatibility.

SRK 15 , SLGA 15 and SLGB 15 encode four soluble glycoproteins (glycoforms of SLG) and two membrane-anchored glycoproteins (SRK and mSLG). These proteins accumulate specifically in stigmas. Both SRK and mSLG were shown to be localised to the plasma membrane fraction in Brassica stigmas. These data are consistent with the observation that SRK is targeted to the plasma membrane when expressed ectopically in transgenic tobacco (Stein et al., 1995). Peptide sequence and MALDI-TOF-MS analysis was used to assign each of the four soluble glycoproteins to an S locus gene. MALDI-TOF-MS was particularly useful for discriminating between the different products of the three genes and this technique is likely to have wide applications in distinguishing between gene products of other gene families and, coupled with genome information in model species, in correlating genes and gene products in general.

Functional roles of SLG, mSLG and eSRK

Considerable heterogeneity was observed when the S-locus-encoded proteins present in stigmas of plants homozygous for the S 15 haplotype were compared with those of other class II haplotypes. The abundance of soluble SLG proteins varied between haplotypes and, whilst putative mSLG proteins were detected in both S 15 and S 2 homozygous plants, mSLG was not detected in an S 5 homozygous plant. We present evidence that these differences reflect differences in the number of S gene family members at the S locus. DNA blot analysis indicated that the S 2 haplotype contains SRK and SLGA but not SLGB, whereas the S 5 haplotype contains SRK and SLGB but not SLGA. If these two lines each lack one of the two SLG genes it follows that (1) neither gene is required for the SI response or (2) the two genes are functionally redundant with respect to the SI response. Moreover, SLGA 15 and SLGB 15 are more similar to proteins encoded by closely related S haplotypes (for example, there are only five amino acid differences between SLG 2 and SLGA 15 ) than they are to each other (45 amino acid differences, Figure 6). This argues against functional redundance of SLGA and SLGB and, therefore, suggests that SLG is unlikely to be required for the recognition step of the SI response.

The absence of an SLGA gene in the S 5 haplotype also raises questions about the functional role of mSLG. [START_REF] Tantikanjana | An alternative transcript of the S-locus glycoprotein gene in a class-II pollen-recessive self-incompatibility haplotype of Brassica oleracea encodes a membrane-anchored protein[END_REF] showed that mSLG was present in stigmas of the class II S 2 haplotype but absent from stigmas of the class I S 6 haplotype. They suggested that mSLG may interfere with SRK function and that this may account for the recessive nature of the S 2 haplotype in heterozygotes. The absence of mSLG from stigmas of the S 5 line, which is also a recessive class II haplotype, suggests that either mSLG is not responsible for this phenotype or that other factors can also have an influence. Comparative analysis of S 5 and S 2 homozygous lines may help to elucidate the role of the mSLG protein.

The four soluble glycoproteins recognised by MAb 157-35-50 in extracts of stigmas of the S 15 line were all attributed to one or other of the SLG genes. Therefore, unlike S 3 [START_REF] Giranton | The S locus receptor kinase gene encodes a soluble glycoprotein corresponding to the SRK extracellular domain in Brassica oleracea[END_REF], this haplotype does not express a detectable level of eSRK. This suggests that either eSRK is not required for the SI response or that it is able to function at very low abundance (below the detection level of this antibody). It is interesting that eSRK was detected in plants carrying the class I S 3 haplotype but not in plants with the class II S 15 haplotype despite the fact that both SRK alleles possess a stop codon at the 5' end of the intron 1. One possibility, which merits further investigation, is that the phenotypic differences between class I and class II haplotypes may be due to the presence or absence of eSRK.

Another important observation is that SLGA 15 and SLG 2 differ at only five amino acid positions, none of which are in the hypervariable domains which have been implicated in allele-specificity. Hence, if SLG does play a role in recognition of self-pollen, the hypervariable domains do not determine allele-specificity in these haplotypes. As pointed out by [START_REF] Nasrallah | Evolution of the Brassica self-incompatibility locus: A look into Slocus gene polymorphisms[END_REF], the high level of polymorphism of the hypervariable domains may indicate a role in recognition or may merely be due to these being regions of minor functional importance that are more free to vary than other parts of the gene.

Recombination and gene conversion between S locus alleles

Despite the large physical size of the S locus in Brassica spp, no recombination event between the different genes at the S locus has been reported. Recombination is thought to be suppressed at the S locus because of extensive sequence rearrangements and divergence which have resulted in structural heteromorphisms between S haplotypes [START_REF] Boyes | The selfincompatibility (S) haplotypes of Brassica contain divergent and rearranged sequences of ancient origin[END_REF].

However, there is accumulating evidence that different sequences within the S locus have evolved independently, at least to some extent, suggesting that recombination or gene conversion events have occurred between haplotypes during evolution [START_REF] Kusaba | Striking sequence similarity in inter-and intra-specific comparisons of class I SLG alleles from Brassica oleracea and Brassica campestris: Implications for the evolution and recognition mechanisms[END_REF][START_REF] Charlesworth | Flowering plant self-incompatibility: the molecular genetics of Brassica S-loci[END_REF]. The fact that the similarity between SLG and SRK alleles within a haplotype is often greater than between many alleles of each of these two genes [START_REF] Stein | Molecular cloning of a putative receptor protein kinase gene encoded at the selfincompatibility locus of Brassica oleracea[END_REF][START_REF] Goring | An S receptor kinase gene in self-compatible Brassica napus has a 1 bp deletion[END_REF]Delorme et al., 1995b;[START_REF] Kusaba | Striking sequence similarity in inter-and intra-specific comparisons of class I SLG alleles from Brassica oleracea and Brassica campestris: Implications for the evolution and recognition mechanisms[END_REF] indicates that sequence information may also have been exchanged between genes within the same S haplotype. This hypothesis is supported by two features which were identified when S locus genes of the S 15 haplotype were compared.

Firstly, comparison of intron sequences of SRK 15 , SLGA 15 and SLGB 15 showed that they included a patchwork of conserved sequence motifs each of which was found in the introns of only two of the three genes. The structure of the introns suggests that the three genes have evolved by a series of gene duplications followed by either deletion or recombination events (Figure 7). The hypothesis that recombination may have occurred between intron sequences of S locus genes is interesting in the light of data presented here which indicates that the number of S gene family members at the S locus may vary between haplotypes. Recombination events, occurring for example as a result of unequal sister chromatid exchange or intrachromasomal recombination, could lead to the loss or gain of alleles. In addition, recombination within intron sequences would provide a mechanism for the acquisition or loss of exons encoding transmembrane and kinase domains by or from genes resembling SLG and SRK respectively [START_REF] Tantikanjana | An alternative transcript of the S-locus glycoprotein gene in a class-II pollen-recessive self-incompatibility haplotype of Brassica oleracea encodes a membrane-anchored protein[END_REF]. Moreover, when kinase domains of SRKs from different haplotypes are compared they exhibit fewer nonsynonymous differences, fewer non-conservative amino acid differences and a generally lower level of variability than SRK S domains [START_REF] Charlesworth | Flowering plant self-incompatibility: the molecular genetics of Brassica S-loci[END_REF]. This suggests that some recombination has occurred between the S and kinase domains of SRK genes from different haplotypes. Hence sequence information may have been exchanged by recombination both between alleles of different haplotypes and between genes of the same haplotype at the S locus.

The second sequence of interest is a 502 nucleotide region of 100% sequence identity which is shared by SRK 15 and SLGA 15 (Figure 1). The presence of this shared sequence indicates that the two genes have been involved in a gene conversion event. Similar regions of 100% nucleotide sequence identity are also shared by the SLG and SRK alleles of both the B. napus S A10 and the B. rapa S 9 haplotypes [START_REF] Goring | An S receptor kinase gene in self-compatible Brassica napus has a 1 bp deletion[END_REF]Suzuki et al., 1997). The positions of the identical regions are different in each SLG/SRK pair indicating that the events occurred independently after divergence of the S alleles. Hence, there is evidence that gene conversion has occurred in three out of the eleven SLG/SRK haplotype pairs for which sequence data is currently available. Similar regions of sequence identity are significantly less common between SLG alleles (JMC, unpublished results) suggesting that gene conversion events between genes of the same haplotype have occurred more frequently than between alleles of the same S locus gene. In humans, homogenisation of tandemly repeated genes occurs primarily by intrachromosomal events and exchanges of sequences between chromosomes are less frequent [START_REF] Liao | Concerted evolution of the tandemly repeated genes encoding human U2 snRNA (the RNU2 locus) involves rapid intrachromasomal homogenization and rare interchromasomal gene conversion[END_REF]. Moreover, in yeast, intrachromasomal homogenisation of tandem repeats occurs primarily as a result of gene conversion [START_REF] Gangloff | Gene conversion plays the major role in controlling the stability of tandem repeats in yeast[END_REF]. Evolution of tandemly repeated disease resistance genes at the Cf-4/9 locus in tomato is thought to have occurred by a similar mechanism involving shuffling of sequences between genes within the locus [START_REF] Parniske | Novel disease resistance specificities result from sequence exchange between tandemly repeated genes at the Cf-4/9 locus of tomato[END_REF]. The above evidence that there has been a higher frequency of sequence exchange between alleles of genes within the same haplotype than between haplotypes suggests that more dispersed gene clusters such as the S gene family members at the Brassica S locus may evolve in a similar manner.

The concerted evolution of SLG and SRK alleles within a particular haplotype has been put forward as evidence of a shared function for these two genes [START_REF] Stein | Molecular cloning of a putative receptor protein kinase gene encoded at the selfincompatibility locus of Brassica oleracea[END_REF].

Although this may be the case, it is also possible that their similarity may be merely the result of general homogenisation mechanisms operating on linked genes with sequence homology. Indeed, theoretical considerations indicate that, under certain conditions, it may be difficult for duplicated genes to avoid the homogenising effects of gene conversion [START_REF] Walsh | Sequence-Dependant Gene Conversion: Can Duplicated Genes Diverge Fast Enough to Escape Gene Conversion?[END_REF].

METHODS

Plant Material, Genetic Crosses, and Determination of Incompatibility Phenotype

The Brassica oleracea var acephala line P57Sc was shown to be self-compatible by [START_REF] Gaude | Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea[END_REF] and the S haplotype carried by this line was therefore arbitrarily designated S Sc .

Genetic crosses with closely related B. oleracea var botrytis lines have indicated that the selfcompatible phenotype of P57Sc is due to a defect in the pollen (Pastuglia et al., 1997b). We were therefore able to identify the S haplotype carried by P57Sc by measuring seed set after application of pollen from tester lines from the collection at Horticulture Research International, Wellesbourne, UK. Pollen from an S 15 homozygous line was incompatible on P57Sc stigmas indicating that P57Sc carries the S 15 haplotype (data not shown). This was subsequently confirmed by comparing the sequences of P57Sc S locus genes with partial sequences of the corresponding genes from the S 15 tester line (see results). The S 3 homozygous line has been described (Delorme et al., 1995b), the S 2 and S 5 homozygous lines were a gift from D. J. Ockendon (Horticulture Research International, Wellesbourne, UK).

An F 2 population obtained by self-fertilisation of an S 3a /S 15 F 1 hybrid (the parental lines are described in Delorme et al., 1995a, andGaude et al., 1993, respectively where they are referred to as S 3a and S Sc ) was used to demonstrate linkage of SRK 15 , SLGA 15 and SLGB 15 to the S locus. The incompatibility phenotypes of the F 2 progeny were determined by selfpollination and by crosses to tester plants using previously described procedures (Delorme et al., 1995b) and aniline blue staining of pollinated pistils was carried out as previously described [START_REF] Ruffio-Châble | Distribution of Shaplotypes and its relationship with self-incompatibility in Brassica oleracea. Part 1. In inbred lines of cauliflower (B. oleracea var 'botrytis')[END_REF].

Cloning of S locus Gene Sequences and DNA Sequence Analysis

The isolation of a genomic clone corresponding to SLGA 15 (previously called SLG Sc ;Pastuglia et al., 1997b) and a cDNA corresponding to SLGB 15 (CG15; [START_REF] Gaude | Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea[END_REF] has been described previously. A fragment of genomic DNA carrying the SLGB 15 gene was isolated from a genomic library (Pastuglia et al., 1997b) constructed with DNA of the P57Sc line.

Restriction endonuclease digested fragments of isolated l clones were subcloned into pBluescript II SK + (Stratagene, La Jolla, CA) for sequence analysis.

An SRK 5 cDNA was reconstructed by RT-PCR (reverse transcriptase-PCR) amplification of overlapping fragments from cDNA prepared from stigmas of a plant homozygous for the S 5 haplotype. Oligonucleotide pairs used were 5'-GCTATTGCGGATGTTCG-3' (SG57) and a degenerate oligonucleotide 5'-AAIATICKIGCCATICCRAARTC-3' (RK2), 5'-AATCTCTGGCAACAATC-3' (SG46) and 5'-AGCTTTGCAATTAGCCTGACTTCGT-3' (SK26), PS3 [START_REF] Nishio | Registration of S alleles in Brassica campestris L by the restriction fragment sizes of SLGs[END_REF] and 5'-CTCCTCACTGTTCTCCG-3' (SG48). The 3' end of the SRK 5 cDNA was isolated by RACE-PCR [START_REF] Frohman | Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer[END_REF] using 5'-AATCTCTGGCAACAATC-3' (SK27) followed by 5'-AGCTTTGCAATTAGCCTGACTTCGT-3' (SK29) in a nested PCR with the adapter oligonucleotide 5'-GACTCGAGTCGACATCG-3' (RA2).

An SRK 15 cDNA was reconstructed in a similar manner from PCR products amplified with the following oligonucleotide pairs: PS3 and 5'-TTCTCGCCCTCATAAACACAACAG-3' (SK30), 5'-ATTAAGCATTACTACTCGAGTCGCAGAAGC-3' (SK31) and 5'-ATGCAACACCTATTGTGGGAAATCAAGTTC-3' (SK32), and a nested RACE-PCR reaction using SK27 and SK29 in combination with RA2. The 3' ends of transcripts which terminate within the first intron of SRK 15 were amplified by a nested RACE-PCR reaction using oligonucleotides SG46 and 5'-AACAGGTTCCTTACATCATGGAGATCCTAT-3' (SG62) with RA2. Similarly, RACE-PCR and oligonucleotides SG46 and 5'-GGATTGCCAGAGTTTATTCTTAATCAAGGAC-3' (SG64) with RA2 were used to amplify the 3' ends of SLGA 15 transcripts. The first intron of SRK 15 was amplified from genomic DNA of the P57Sc line by the PCR using oligonucleotides SG62 and 5'-CTCCTCCAAAAGCAGAACACGATAACACTC-3' (SG66). The first intron of SFR2 was amplified from the cloned SFR2 gene (Pastuglia et al., 1997a) by the PCR using oligonucleotides 5'-AATGTGAAGAGAAGTGC-3' (fK4) and 5'-CATCAGTCCCTTGTACG-3' (fK28). Part of the S domains of each of the three S genes (1020 bp from SLGA, 1019 bp from SLGB and 997 bp from SRK) were amplified from the S 15 homozygous tester line using two oligonucleotides, 5'-TGGAACCCTCAAAATCT-3' (SG22)

and 5' GGCCTGCAGCAGCATTCAATCTGAC-3' (SG2), which correspond to sequences conserved in the three genes.

The method used for extraction of genomic DNA has been described previously (Pastuglia et al., 1997b). For RT and RACE PCR, first strand cDNA was prepared from 1 µg of total stigma RNA using the Superscript Kit (Gibco BRL) and oligonucleotide RA1 [START_REF] Frohman | Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer[END_REF]. PCR conditions were 2 min at 94°C followed by 35 cycles of 30 sec at 94°C, 30 sec at 55°C, and 2 min at 72°, with a final extension for 10 min at 72°C. The same conditions were used for nested PCR reactions except that 25 cycles were used for each reaction. RT-PCR and RACE-PCR products were cloned into pGEM-T easy (Promega).

DNA sequencing was carried out using the dideoxynucleotide chain termination method (Sanger et al., 1977) either on an automatic sequencer (Applied Biosystems, Foster City, CA) or using a T7 DNA polymerase sequencing kit (Pharmacia Biotech, Uppsala, Sweden).

Sequence data was analysed using Lasergene sequence analysis software (DNASTAR, London, UK).

Genetic Mapping of S Locus Genes

Segregation of SLGB 15 in an F 2 population was followed by PCR amplification of a 510 kb sequence from genomic DNA with oligonucleotides 5'-CTACTCCAGATTGACAATCAGTGAGTTG-3' (SG63) and SG2. Segregation of both SRK 15 and SLGA 15 was followed using oligonucleotides SG66 and 5'-TTCTGTTGGAACAACTAAATAAAAT-3' (SK34) for which give two different products of 424 bp and 526 bp respectively for the two genes.

Ribonuclease Protection and Reverse Transcriptase PCR analysis of Gene Transcription

Ribonuclease protection analysis of mRNA abundance was carried out as described previously [START_REF] Cock | Natural antisense transcripts of the S locus receptor kinase gene and related sequences in Brassica oleracea[END_REF]. Radiolabeled probes were hybridized to 2 µg of total RNA. An SLGA 15 specific probe was synthesised by subcloning a 419-bp EcoRI-XhoI fragment into pBluescript II SK + (Stratagene, La Jolla, CA), linearising with SalI, and transcribing with T3

RNA polymerase. This probe included bases 553 to 360 of SLGA 15 (relative to the first base of the ATG initiation codon). For SLGB 15 , the 3' end of the cDNA was sub-cloned as a BamHI fragment into pBluescript II SK + , linearised with BglII and transcribed with T3 RNA polymerase. This probe included bases 1287 to 860 of SLGB 15 (relative to the first base of the ATG initiation codon). Reverse transcriptase PCR analysis of SRK 15 expression was carried out on single-stranded cDNA, prepared as described above, using oligonucleotides SG62 and 5'-CATGAACTCATCGGTACCTTGAGC-3' (SK28). The region between these two oligonucleotide sequences in the SRK 15 gene includes at least one intron. The expected size of the RT-PCR product is 1175 bp. PCR amplification conditions were as follows: 2 min at 94°C followed by 35 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 1 min and a final elongation step of 10 min at 72°C.

DNA blot analysis

Total genomic DNA was extracted from either young leafs or floral buds as described by [START_REF] Vallejos | A molecular marker-based linkage map of Phaseolus vulgaris L[END_REF]. DNA blot analysis was carried out using standard procedures [START_REF] Sambrook | Molecular Cloning: A Laboratory Manual[END_REF]. Intron probes were obtained by PCR amplification from plasmid DNA using 5'-GTAATCTTCTAAAACTAAACACATCGG-3' (SK35) with 5'-TATTGTTCAATGCTAAAGTATGTGC-3' (SK36) to amplify a 415 bp fragment of the SLGA 15 intron and SK35 with 5'-ATATTAGCCGACCCGTTTCA3' (SK33) to amplify a 474 bp fragment of the SLGB 15 intron.

Protein Extraction, Deglycosylation, Fractionation of Membrane Proteins,

Electrophoretic Analysis and Immunodetection of Proteins

Protein extraction, separation of proteins by isoelectric focusing or SDS-PAGE, electrotransfer onto nitrocellulose membranes and detection of antigen with antibodies were as described previously [START_REF] Gaude | Use of a fast protein electrophoretic purification procedure for N-terminal sequence analysis to identify S-locus related proteins in stigmas of Brassica oleracea[END_REF][START_REF] Gaude | Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea[END_REF]. Deglycosylation of proteins was carried out as described previously (Delorme et al., 1995b). Stigma plasma membranes were prepared as described by [START_REF] Larsson | Preparation of high purity plasma membranes[END_REF]. Enrichment for plasma membranes was followed using an antibody specific for the plasma membrane proton ATPase [START_REF] Morsomme | Single point mutations in various domains of a plant plasma membrane H + -ATPase expressed in Saccharomyces cerevisiae increase H + -pumping and permit yeast growth at low pH[END_REF]. Monoclonal antibody (MAb) 157-35-50, which was raised against a peptide corresponding to the amino-terminal end of a soluble stigma glycoprotein from the P57Sc line, has been described previously [START_REF] Giranton | The S locus receptor kinase gene encodes a soluble glycoprotein corresponding to the SRK extracellular domain in Brassica oleracea[END_REF].

Purification of Stigma Glycoproteins, Amino Acid Sequence Analysis and MALDI-TOF-MS Analysis of Purified Proteins

Soluble stigma glycoproteins were purified by two successive electrophoresis steps as described by [START_REF] Gaude | Use of a fast protein electrophoretic purification procedure for N-terminal sequence analysis to identify S-locus related proteins in stigmas of Brassica oleracea[END_REF]. Purified proteins were electroblotted onto polyvinylidene difluoride membranes (Immobilon P, Millipore) and digested with endoproteinase Lys-C or endoproteinase Gluc-C (Promega, France) as described by [START_REF] Fernandez | Internal protein sequence analysis: Enzymatic digestion for less than 10µg of protein bound to polyvinylidene difluoride or nitrocellulose membrane[END_REF]. Peptides were purified by reverse phase-HPLC using a 400 solvent delivery system (Applied Biosystem, Roissy, France) chromatograph apparatus equipped with a C18 column (Vidac, Hesperia, CA). The gradient was from 0,095% (v/v) trifluoroacetic acid (TFA) in 4% (v/v) acetonitrile to 0,01% TFA / 48% acetonitrile for 60 min at a flow rate of 300µl/min. Collected peptides were sequenced using the Edman degradation method on an automatic sequencer (Procise 473A protein sequencer, Applied Biosystems).

For mass spectrometry analysis, purified proteins were directly cut from the Imidazole-Zinc stained SDS-PAGE gel (Matsui et al., 1991). The stained protein spots were digested in-gel essentially as desribed by [START_REF] Shevchenko | Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels[END_REF]. Briefly, spots were excised from the stained gel, and destained with citric acid. After washing with 50% acetonitrile, which was then removed, gel pieces were dried in a vacuum centrifuge. The (Pastuglia et al., 1997b); a cDNA corresponding to SLGB 15 (CG15) has been described [START_REF] Gaude | Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea[END_REF]. Accession numbers for the SLGB 15 and SRK 15 sequences are Y18261 and Y18260 respectively. S dom, S domain; end S dom, end of the S domain; kin, kinase domain. Question marks indicate that the peptide sequence data did not permit the unequivocal assignment of a protein to a particular S locus gene. 58.0

a The region compared is actually slightly shorter than the S domain because the sequence of the amino-terminal end of SLG 5 was not available. The region compared runs from the 21st residue of the mature S domain (for SLGB 15 ) to the end of the S domain. The results of the comparisons are expressed as percentage similarity which is calculated as follows: 100 times the number of matched amino acids divided by the sum of the length in amino acids of the aligned region, plus the number of gaps introduced to optimise the alignment.

b SLGA 15 was previously SLG Sc (Pastuglia et al, 1997b), SLG 2 [START_REF] Chen | A new class of S sequences defined by a pollen recessive self-incompatibility allele of Brassica oleracea[END_REF], SLGB 15 corresponds to the cDNA CG15 [START_REF] Gaude | Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea[END_REF], SLG 5 [START_REF] Scutt | An S5 self-incompatibility allele-specific cDNA sequence from Brassica oleracea shows high homology to the SLR2 gene[END_REF], SRK 15 (this study, accession number Y18260), SRK 2 [START_REF] Stein | Molecular cloning of a putative receptor protein kinase gene encoded at the selfincompatibility locus of Brassica oleracea[END_REF], SRK 5 (this study, accession number Y18259), SLR2-2 [START_REF] Boyes | Isolation of a Second S-Locus-Related cDNA from Brassica oleracea: Genetic Relationships Between the S Locus and Two Related Loci[END_REF], SLG 3 and SRK 3 (Delorme et al., 1995b), SFR2 (Pastuglia et al., 1997a). a Numbers refer to the position of a peptide in the sequence of the corresponding protein.

b M, methionine.

  have recently described an SLG allele with a similar structure to SLGB 15 and SLG 5 in the class II S 29 haplotype of B. rapa. Although there are significant differences between the introns of SLGB 15 and B. rapa SLG 29 (for instance, the intron of B. rapa SLG 29 lacks the direct repeat found in the SLGB 15 intron, Figure 7), they share extensive regions of sequence similarity indicating that they are diverged alleles of the same genes. Taken together, these observations indicate that SLG 2 is an allele of the SLGA gene whereas SLG 5 and B. rapa SLG 29 are alleles of SLGB. Thus alleles of SLGA and SLGB are present in other class II S haplotypes.

  dried gel fragments were re-swollen in 20 µl of 20 mM NH 4 HCO 3 , containing 0.5 µg of trypsin (Promega, Sequencing grade), and incubated for 3 hours at 37°C. A 0.4 µl volume of the digest solution was removed for mass spectrometric analysis. These samples were mixed on the sample probe with 0.4 µl of a saturated solution of a-cyano-4-hydroxy-trans-cinnamic acid prepared in 40% v/v acetonitrile, 0.1% v/v trifluoroacetic acid (TFA). Samples were rinsed by placing a 5 µl volume of 0.1% TFA on the matrix surface after the analyte solution had dried completely. After 2 min, the liquid was blown off with pressurized air. MALDI mass spectra of peptide mixtures were obtained using a Bruker Biflex mass spectrometer (Bruker-Franzen Analytik, Bremen, Germany) equipped with a SCOUT multiprobe inlet and a gridless delayed extraction ion source. The ion acceleration voltage was 19.5 kV and the reflectron voltage was 20.0 kV. For delayed ion-extraction, a 6.2 kV potential difference between the probe and the extraction lens was applied. Mass spectra were acquired as the sum of ion signals generated by irradiation of the target with 100 laser pulses. They were calibrated using ion signals from trypsin autodigestion peptides (MH + 842.50, MH + 1045.55, and MH + 2211.09 where MH + is the protonated molecular mass).

Figure 2 .

 2 Figure 2. Genetic linkage to the S locus and expression patterns of SLGA 15 , SLGB 15 , and

Figure 3 .

 3 Figure 3. Analysis of molecular mass, glycosylation status and subcellular location of S-

Figure 4 .

 4 Figure 4. Biochemical analysis of soluble S-locus-encoded proteins in stigmas of the S 15

Figure 5 .

 5 Figure 5. RNA transcripts of SLGA 15 , SLGB 15 , and SRK 15 .

Figure 6 .

 6 Figure 6. Alignment of the deduced amino acid sequences of the S domain of SRK 15 , SLG 2 ,

Figure 7 .

 7 Figure 7. Conserved sequences within the introns of SLGA 15 , SLGB 15 and SRK 15 suggest a

Figure 8 .

 8 Figure 8. Detection of SLGA and SLGB alleles in different class II haplotypes.

  1. Structure of three S gene family members, SLGA 15 , SLGB 15 , and SRK 15 , from the B. oleracea S 15 self-incompatibility haplotype. Schematic representation of the sequenced region of each gene. Transcribed sequences are represented by narrow boxes and coding regions by thick boxes. The hatched box represents a region of 502 bp which is 100% identical at the nucleotide level between SLGA 15 and SRK 15 . Membrane-spanning domains (TM) are represented by black boxes. The exon/intron structure of the SRK 15 kinase domain has not been determined in detail and this region is therefore represented by dashed lines. The transcribed region of the SLA gene downstream of SLGA 15 is represented by dotted lines. SLGA 15 was previously called SLG Sc

Table 1 .

 1 TABLES Percent amino acid similarity a between the S domain sequences of SLGA 15 , SLGB 15 and SRK 15 and those of selected members of the S gene family in Brassica b .

	SLGA 15 SLG 2 SLGB 15 SLG 5 SRK 15 SRK 2 SRK 5 SLR2 SLG 3 SRK 3	SLG 2 SLGB 15 SLG 5 SRK 15 SRK 2 98.6 90.4 87.3 89.5 90.2 90.4 87.1 88.6 90.2 94.7 87.3 94.3 86.3 92.1 87.3	SRK 5 SLR2 90.4 85.1 90.4 85.1 94.6 85.6 97.2 83.0 88.9 84.3 96.4 84.1 84.5	SLG 3 63.8 63.6 63.8 61.9 63.8 62.6 63.6 60.3	SRK 3 63.7 63.7 62.5 60.7 63.2 62.3 62.5 60.9 80.3	SFR2 54.8 54.3 55.5 56.3 54.3 55.0 55.7 52.7 55.7

Table 2 .

 2 MALDI-TOF-MS analysis of soluble S-locus-encoded proteins in stigmas of the S 15 haplotype.

	Protein Experimental Peptide Mass (D) a 980.55 1056.55 1240.77 1374.70 1772.93 b 980.55 1056.67 1128.76 1240.74 1374.80 980.55 969.59 1773.09 1243.73 b' 1056.61 1240.71 1224.68 b'' 1056.50	Expected Peptide Mass (D) 980.10 1056.10 1240.50 1374.60 1773.20 980.10 1056.10 1128.50 1240.50 1374.60 980.10 969.20 1773.20 1243.60 1056.10 1240.50 1224.50 1056.10	Peptide Sequence 53 TYAWVANR 60 a 304 NPQQWDLR 311 393 YAVGGQDLYVR 403 160 SSDDPSSGNFAYK 172 100 SPVIAELLPNGNFVMR 1 53 TYAWVANR 60 53 TYAWVANR 60 250 LTWIPPSR 257 304 NPQQWDLR 311 40 WYLGIWYK 47 393 YAVGGQDLYVR 403 15 160 SSDDPSSGNFAYK 172 100 SPVIAELLPNGNFVMR 1 178 GLPEFILNQGR 188 304 NPQQWDLR 311 393 YAVGGQDLYVR 403 389 FAVGGQDLYVR 399 300 NPQQWDLR 307	Modifications oxidized M oxidized M	b Corresponding Gene SLGB 15 SLGB 15 SLGB 15 SLGA 15
	1598.03	1598.10	15 19 TLVSPGGVFELGFFK 33
	1773.07	1773.20	100 SPVIAELLPNGNFVMR 1	oxidized M
			15

ACKNOWLEDGMENTS

We thank Marc Boutry (University of Louvain, Belgium) for the kind gift of an antibody against the plasma membrane proton ATPase and Monique Estienne, Anne-Marie Thierry, Richard Blanc, Hervé Leyral and Fabienne Deguerry for technical assistance. We would also like to thank Ioan Negrutiu, Peter Rogowsky and two anonomous reviewers for helpful comments on the manuscript. V.R.-C., and J.M.C. are members of the Institut National de la Recherche Agronomique, J.G. and T.G. are members of the Centre National de la Recherche Scientifique. This work was supported jointly by the Institut National de la Recherche Agronomique, the Centre National de la Recherche Scientifique and the Ecole Normale Supérieure de Lyon.