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SRK Region of the S Locus in Brassica Species
Annals of Botany 85 (Supplement A): 155±160, 2000 doi:10.1006/anbo.1999.1035, available online at http://www.idealibrary.com on
Genomic Organization of the SLG/SRK Region of the S Locus in Brassica Species M A S AO WATA N A B E * {, G O S U Z U K I{, S EI JI TA K AYA M A } , A K IR A I SO GA I} and KO KI C HI H IN ATA k {Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan, {Division of Natural Science, Osaka Kyoiku University, Kashiwara 582-8582, Japan, }Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma 630-0101, Japan and kResearch Institute of Seed Production Co., Ltd, Sendai 989-3204, Japan Received: 21 July 1999
Accepted: 9 September 1999
Self-incompatibility in Brassica species is controlled by a single S locus with multiple alleles. Recent experimental data suggest that SLG (S locus glycoprotein) and/or SRK (S receptor kinase) are the stigmatic S determinants of selfincompatibility in Brassica. A 76-kb genomic fragment containing both SLG and SRK was isolated and characterized in the S 9 haplotype of B. campestris. This fragment contains at least 14 expressed genes and three putative open reading frames (ORFs), suggesting that the S locus is embedded in a gene-rich region. Within this fragment, both anther and/or stigma-speci®c genes are clustered around the SLG and SRK genes. Some members of the S multigene family are also closely linked to the S locus. In this review we discuss the complex genomic structure of the S locus in # 2000 Annals of Botany Company Brassica species. Key words: Anther-expressed gene, Brassica campestris (syn. rapa) L., Brassica oleracea L., gene cluster, genome structure, self-incompatibility, SLG, S locus, SRK.
I N T RO D U C T I O N Many ¯owering plants have self-incompatibility (SI) systems that promote outbreeding and maintain genetic diversity. These systems prevent self-fertilization by rejecting pollen derived from plants with the same genotype (Fig. 1A, B). The SI system in Brassica species is controlled sporophytically by multiple alleles at a single locus called the S locus (Bateman, 1955). The genus Brassica contains about 40 species (Schulz, 1936). Two species, B. campestris (syn. rapa) and B. oleracea, have been used extensively in studies of SI, although SI lines of B. napus are now also being used. B. napus is an amphidiploid hybrid between B. campestris and B. oleracea, and is usually selfcompatible; however, self-incompatible B. napus lines have been synthesized by introgression of self-incompatibility from B. campestris (Goring et al., 1992a). In Japan, hybrid varieties of commercial Brassica vegetable crops have been produced using an SI trait since the 1950s by growing two selected self-incompatible genotypes, A and B, in alternate rows in an isolated ®eld, and harvesting hybrid F1 seeds between A and B (Fig. 1). To date, more than 30 S alleles have been identi®ed in B. campestris (Nou et al., 1993) and more than 50 S alleles in B. oleracea (Brace et al., 1994). Because the activity of S alleles is controlled sporophytically, dominant/recessive relationships in¯uence the ultimate phenotype of stigma and pollen. Thus, the phenotype of the pollen and stigma of heterozygous plants depends on the outcome of complex dominant/recessive allelic interactions. In B. campestris, * For correspondence. Fax 81-(0)19-621-6177, e-mail nabe@ iwate-u.ac.jp
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dominant/recessive relationships have been determined between 24 S alleles for almost all combinations (Hatakeyama et al., 1998a), whereas in B. oleracea dominant/ recessive relationships among 28 S alleles have been determined for one third of all combinations (Thompson and Taylor, 1966). Based on these studies, the characteristic features of dominance relationships in Brassica species include: (1) common codominant relationships; (2) dominant/recessive relationships occurring more frequently in pollen than in the stigma; (3) dominant/recessive relationships diering between stigma and pollen; (4) non-linear dominant/recessive relationships (Thompson and Taylor, 1966; Hatakeyama et al., 1998a). The expression and mechanisms of these dominant/recessive relationships will eventually be elucidated at a molecular level using the wellcharacterized plant material described above. Here we review the molecular characterization of the SI genes, SLG and SRK, and the genomic structure of the S locus, focusing mainly on B. campestris, which includes many important vegetable crops (Chinese cabbage, turnip, oil seed and leaf vegetables) grown in the Far East. M O L E C U L A R C H A R AC T E R I Z AT I O N O F S L G A N D S R K GE N E S Linkage between the S locus and S-locus glycoprotein (SLG) was ®rst demonstrated by isoelectric focusing analysis of stigmatic proteins (Nishio and Hinata, 1977) and provided a basis for subsequent molecular analyses of SI in Brassica species. In 1985, a cDNA clone of SLG was successfully isolated using dierential screening in B. oleracea (Nasrallah et al., 1985). In 1987, the amino # 2000 Annals of Botany Company
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Watanabe et al.ÐGenomic Organization of S Locus Gene in Brassica
FIG. 1. Commerical seed-production ®eld of a single-cross hybrid leaf vegetable of B. campestris. Alternate rows of self-incompatible, yet cross-compatible, S homozygotes are transplanted into an isolated ®eld (Matsushima island). Cross pollination (A) among the S homozygotes will produce hybrid seed; selfed and sibbed seed production will be largely prevented by SI (B).
acid sequences of SLGs, isolated from three S homozygotes of B. campestris, were determined (Isogai et al., 1987; Takayama et al., 1987). Following these experiments, over 30 SLG clones were isolated from Brassica species (Nasrallah et al., 1987; Lalonde et al., 1989; Trick and Flavell, 1989; Chen and Nasrallah, 1990; Goring et al., 1992a, 1992b; Scutt and Croy, 1992; Watanabe et al., 1994; Yamakawa et al., 1994; Delorme et al., 1995; Matsushita et al., 1996; Kusaba et al., 1997; Hatakeyama et al., 1998b, 1998c). These data revealed three characteristic features of SLG. Firstly, the amino acid sequence deduced from SLG cDNA contains a hydrophobic signal peptide at the amino terminal (Nasrallah et al., 1987). Secondly, each sequence contains 12 conserved cysteine residues at the carboxyl terminal. Thirdly, the S speci®city is thought to be determined by the protein portions of SLGs (Isogai et al., 1987; Takayama et al., 1987), because no discernible dierences were found in the N-glycosidic carbohydrate chains from three dierent SLGs (Takayama et al., 1989). The SLGs cloned so far have been classi®ed into two groups, Class I and Class II, based on their respective homologies. This classi®cation is correlated with dominant/ recessive relationships in the pollen, even though SLG is expressed speci®cally in stigmatic tissues. This correlation might have crucial implications for the identi®cation of the pollen S determinant. In 1991, a second S-linked gene, SRK (S receptor kinase), was identi®ed in B. oleracea and interestingly, the
extracellular domain of SRK was found to be highly homologous to SLG (Stein et al., 1991). It has been suggested that during the SI reaction, the S domain of SRK accepts a pollen S determinant and transduces the signal into papilla cells via a protein phosphorylation cascade. To date, over 10 sets of SLG and SRK genes from dierent S alleles of Brassica species have been isolated (Chen and Nasrallah, 1990; Stein et al., 1991; Goring and Rothstein, 1992; Glavin et al., 1994; Kumar and Trick, 1994; Watanabe et al., 1994; Delorme et al., 1995; Suzuki et al., 1995; Yamakawa et al., 1995; Hatakeyama et al., 1998b, 1998c). The homology between SLG and the S domain of SRK derived from the same S allele is about 90%, and in some cases over 98% (Watanabe et al., 1994; Suzuki et al., 1997a; Hatakeyama et al., 1998c). The transcript of SRK is expressed mainly in stigmatic tissue, where expression levels are extremely low relative to SLG (Stein et al., 1991; Glavin et al., 1994; Watanabe et al., 1994; Delorme et al., 1995). Complete conservation of 12 cysteine residues at the carboxyl terminal in the S domain of SRK should be important for the establishment of the tertiary con®guration of this protein. Transformation experiments with SLG or SRK have been performed in an attempt to elucidate the role of SLG and/ or SRK in SI. In transgenic B. campestris transformed with antisense SLG driven by the SLG promoter, the transcripts of SLG and SRK decreased, and the transformant plants became self-compatible (Shiba et al., 1995). When selfincompatible B. campestris was transformed with SLG derived from B. campestris, the transformants again became self-compatible because the SI phenotype of the stigma was changed but the pollen SI phenotype remained the same. This change from an SI phenotype to a self-compatible phenotype was due to co-suppression of the SLG-transgene and the endogenous SLG gene (Takasaki et al., 1999). When self-incompatible B. napus was transformed with an inactive copy of the SRK gene, the transformants also showed a change from an SI phenotype to a self-compatible phenotype. This eect was due to co-suppression and dominant-negative eects, whereby the transgenes induced a dramatic reduction in the expression of the endogenous S locus and S locus-related genes, indicating homologydependent silencing (Stahl et al., 1998). The silencing of SLG and/or SRK genes in self-incompatible host plants also resulted in the breakdown of SI (Conner et al., 1997). In each case, the change of S phenotype was only observed in the stigma, and not in pollen. When an SLG gene from self-incompatible B. campestris was introduced into selfcompatible B. napus under the control of a tapetum-speci®c promoter, pollination tests indicated that the pollen of the transgenic B. napus did not gain the SI phenotype (Sasaki et al., 1998). These experiments show that SLG and/or SRK are, at least, the stigmatic determinants of SI; however, gain-of-function experiments without co-suppression are required before the function of SLG and SRK in SI recognition can be established. G E NO M I C A N A LY S I S O F T H E S LO C U S As discussed above, the pollen S determinant(s) must be located at the S locus because SI in Brassica species is
Watanabe et al.ÐGenomic Organization of S Locus Gene in Brassica controlled by a single locus. Thus, there must be at least three genes (SLG, SRK and the pollen S determinant gene) at the S locus. Because the S locus comprises multiple genes within one segregational unit, an `S allele' is now referred to as an `S haplotype' (Nasrallah and Nasrallah, 1993). The term `haplotype' is generally used in the context of the major histocompatibility complex (MHC) in animal immune systems. The genes related to the MHC, which functions in the recognition of self and non-self antigens, form a large multigene family and occur in tandem (Trowsdale et al., 1991). In the Brassica SI system, several SLG- and SRK-like genes of the S multigene family are closely linked to the S locus (Oldknow and Trick, 1995; Suzuki et al., 1997b; Cabrillac et al., 1999). For instance, in the S 9 haplotype of B. campestris, the physical distance between SLG, SRK and three S-related genes (BcRK1, BcRL1 and BcSL1), which are linked to the S locus, was estimated to be less than 610 kb. BcRK1 is highly homologous to ARK1, which is located tandemly with another receptor-like kinase (ARK2) (Dwyer et al., 1994). Both the expression pattern and the nucleotide sequence in BcRK1 are quite similar to ARK1, suggesting that BcRK1, like ARK1, might be expected to be involved in cell expansion (Suzuki et al., 1997b). The genomic organization of the three S-related genes (BcRK1, BcRL1 and BcSL1) in B. campestris is similar to that of ARK1 and ARK2 in Arabidopsis. Comparative mapping between B. campestris and Arabidopsis demonstrated that the homoeologous region to the S locus in Arabidopsis was located in the vicinity of ETR1 on chromosome 1. ARK1 and ARK2 were also found to be located on chromosome 1, but about 1 centimorgan away from the S locus homoeologous region (Conner et al., 1998). Based on these results, BcRK1 would be an orthologous gene to ARK1. The expression pattern and genomic organization of these three S-related genes (BcRK1, BcRL1 and BcSL1) indicate that none of these S-related genes function in sexual reproduction processes, so they do not re¯ect further genetic diversity of SLG and SRK at the S locus. It may therefore be that these three S-related genes and the S locus became linked to each other by chance during the course of genomic evolution in the Brassicaceae. Assuming that the pollen S determinant of the SI recognition reaction is linked to the S locus, determination of the extent of the genomic region that co-segregates with the S locus may aid in its isolation. The physical distance between SLG and SRK, both of which co-segregate with the S locus, has been estimated as less than 200 kb in B. oleracea (Boyes and Nasrallah, 1993), and less than 20 kb in B. campestris (Yu et al., 1996; Boyes et al., 1997; Suzuki et al., 1999). The tendency for a shorter physical distance between SLG and SRK in B. campestris than in B. oleracea seems to be consistent for several S haplotypes in B. campestris and B. oleracea (Suzuki et al., 2000). These results indicate that the S locus is smaller in B. campestris than it is in B. oleracea. Isolation of genomic fragments between the SLG and SRK genes, using a chromosome walking strategy, has proved dicult for the S locus region (Boyes et al., 1997; Conner et al., 1998). Only two strategies have so far
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succeeded in cloning the genomic region between SLG and SRK. One strategy is the long PCR method with a speci®c primer set of SLG and SRK (Yu et al., 1996), and the other is the direct-cloning of an MluI genomic fragment (76 kb) containing both SLG and SRK genes, using the P1-derived arti®cial chromosome (PAC) vector (Suzuki et al., 1997c). The MluI genomic fragment (SLG/SRK region) of the S 9 homozygote of B. campestris has been screened for expressed sequences (Watanabe et al., 1999; Suzuki et al., 1999) and shown to contain at least 14 expressed genes including SLG and SRK (Fig. 2A). We also found three long putative ORFs (ORF-a, b and c) within the genomic sequence of the 76-kb region (Suzuki et al., 1999). The average gene density in this region is one gene for every 5.4 kb, suggesting that the S locus is embedded within a gene-rich region. Because SI genes reside within the S locus complex, together with many other genes not involved with SI (described below), it is necessary to determine very carefully which S-linked genes have a function in SI. Anther and/or stigma-speci®c genes located at the S locus are not always associated with SI recognition, as demonstrated for the SLA gene (Boyes and Nasrallah, 1995; Pastuglia et al., 1997). Functional analyses, such as gain-of-function and loss-of-function experiments, are needed to prove that genes linked to the S locus are indeed genes involved in SI, as stressed by McCubbin and Kao (1999). Because the eciency of Brassica transformation is very low, identifying the unknown pollen S determinant gene by genome analysis is a laborious process. We have investigated the expression pattern of all 14 genes identi®ed in the 76-kb region (Suzuki et al., 1999). SP1, SP2, SP3, SP4, SP7, SP10 and SLL2 genes were found to be expressed in both ¯oral and vegetative tissues. Other genes (SLG, SRK, SP5, SP6, SP8, SP11 and SAE1) were found to be anther and/or stigma-speci®c genes. With the exception of SP8, these anther and/or stigma genes were clustered with SLG and SRK (Suzuki et al., 1999; Fig. 2). We have also identi®ed ORF-a, a gene expressed speci®cally in anther tissues (G. Suzuki et al., unpubl. res.) and ORF-c which is highly homologous to a Brassica EST clone, which is expressed in ¯oral buds (M. Watanabe and G. Suzuki, unpubl. res.). Comparative mapping around the region of the S locus in self-incompatible B. campestris and selfcompatible Arabidopsis demonstrated that a genomic region of approx. 30 kb, which contains both the SLG and SRK genes in B. campestris, was completely deleted in Arabidopsis (Conner et al., 1998). The region containing the anther and/or stigma speci®c genes (SP6 to SAE1), identi®ed by Suzuki et al. (1999), was very similar to the 30-kb genomic region identi®ed by Conner et al. (1998) at this map position. Therefore, the region from SP6 to SAE1 might be important for the SI recognition reaction. Among the 14 genes detected, two (SAE1 and SP11) are speci®cally expressed in anther tissues, and are located downstream of the SLG 9 or SRK 9 genes (Fig. 2A; Suzuki et al., 1999; Watanabe et al., 1999). The extent of allelic polymorphism and the function of SAE1 and SP11 are currently under investigation. In the S 9 haplotype of B. campestris, genes homologous to SLL2 (Yu et al., 1996) and ClpP (Letham and Nasrallah,
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FIG. 2. Genomic structure of the S locus complex region. A, A map of the 76-kb SLG/SRK region of S 9 haplotype of B. campestris (Suzuki et al., 1999). Coloured boxes represent 14 S-linked genes expressed in stigmas (green), anthers (yellow), both stigmas and anthers (light green), and both reproductive and vegetative tissues (light blue). The SLG 9 gene was located near the middle of the 76-kb region. SRK 9 was located approx. 13 kb upstream of SRK 9 , resulting in the 76-kb region containing approx. 15 kb upstream of SRK 9 and approx. 37 kb downstream of SLG 9 . White boxes indicate the putative ORFs (ORF-a, b and c) estimated from genomic DNA sequence. B, Directions of transcriptions of the SLG and SRK genes in three S haplotypes analysed to date. In the S 9 haplotype of B. campestris, SLG is located at the downstream region of SRK and the directions of the genes are the same (Suzuki et al., 1999). In the S 910 haplotype of B. napus, SRK is located at the downstream region of SLG in the same direction, which was con®rmed by long PCR between them (Yu et al., 1996). In the S8 haplotype of B. campestris, SLG and SRK are located in the opposite direction, which was estimated by a partial long-range restriction map of the S locus (Boyes et al., 1997). The region between SLG8 and SRK8 has not yet been cloned.
1998) were found downstream of SLG (Suzuki et al., 1999; Watanabe et al., 1999). Although the locations of these three genes were reported in only the S8 and S 9 haplotypes of B. campestris, the gene arrangement of SLG, SLL2 and the ClpP homologue is the same in both haplotypes (Boyes et al., 1997; Conner et al., 1998; Suzuki et al., 1999). Yu et al. (1996) reported that another S-linked gene, termed SLL1, is located between SLG and SLL2 in the S 910 haplotype of B. napus. In the S 9 haplotypes of B. campestris, a sequence similar to SLL1 was also found in the region between SLG and SLL2; however, this SLL1-like sequence was fragmented and appeared to be a pseudogene (Suzuki et al., 1999). The SLL1 gene was not considered to function in SI recognition because sequence diversity was not observed in dierent S haplotypes (Yu et al., 1996). Our observation that SLL1 is fragmented supports this prediction. Putative amino acid sequences of SLL2 showed 96.3% identity between the S 9 and S 910 haplotypes (Watanabe et al., 1999). Similarly, putative amino acid sequences of ClpP homologues showed 97.5% identity between the S8 and S 9 haplotypes (Suzuki et al., 1999). Because SLL2 and ClpP are not involved in SI recognition, their amino acid sequence identities are much higher (above 95%) than those observed in the SI-related genes, SLGs and SRKs. The S locus complex contains non-polymorphic regions including the gene cluster of SLL1, SLL2 and the ClpP homologue, which are unlikely to be involved in SI, suggesting that dierent regions of the S locus complex have evolved independently (Charlesworth and Awadalla, 1998; MacCubbin and Kao, 1999). In the B. campestris S 9 haplotype, both SLG and SRK were found to be transcribed in the same direction which,
interestingly, was the opposite direction to SLG and SRK transcription in the S8 haplotype (Boyes et al., 1997). However, in the S 910 haplotype of B. napus, SLG and SRK were shown to be transcribed divergently (Yu et al., 1996; Fig. 2B). These results indicate that the direction of transcription of the SLG and SRK genes varies between dierent S haplotypes. As reported by Boyes et al. (1997), sequence rearrangement and a lack of sequence similarity appear to contribute to suppression of recombination at the S locus. Notwithstanding this, the dierent orientations of SLG and SRK at the S locus in dierent S haplotypes must also contribute to reduced rates of recombination. In general, a low level of recombination is observed around centromeric regions of chromosomes, but the Brassica S locus is positioned at the terminus of the chromosome (Iwano et al., 1998; Kamisugi et al., 1998). Thus, the low level of recombination observed at the Brassica S locus must be independent of its chromosomal location, in contrast to the situation in Petunia species, which possess a gametophytic SI system, where the subcentromeric location of the S locus (Entani et al., 1999) is expected to contribute signi®cantly to the suppression of recombination. In summary, the complexity of the Brassica S locus is characterized by the following important features: (1) two similar genes, SLG and SRK, are tightly linked; (2) gene conversion has occurred between SLG and SRK (Suzuki et al., 1997a); (3) highly polymorphic SLG and SRK genes are located close to a cluster of highly conserved genes (described above); (4) sequence rearrangements have occurred (Boyes et al., 1997); and (5) recombination is suppressed within the S locus complex. The evolution and
Watanabe et al.ÐGenomic Organization of S Locus Gene in Brassica maintenance of the S locus complex is one of the most interesting problems in the plant genomics. F U T U R E P RO S P E C T S Comparative mapping of the S locus among several haplotypes is an important strategy for understanding the extent of sequence variation and diversity at the S locus. The direct cloning system based on the PAC vector (Suzuki et al., 1997c) is proving useful in this study. Fluorescence in situ hybridization (FISH) on the extended DNA ®bre (EDF) allows the physical mapping of cloned DNA fragments with a resolution of a few kilobases (de Jong et al., 1999), and this technique has allowed the physical mapping of the SLG 9 and SRK 9 genes on EDFs using EDF-FISH analysis and the DNA combing technique (Suzuki et al., 1999). This analysis will also be applicable to other S locus genes and S haplotypes, which will prove to be a powerful tool for structural analysis and physical mapping of the complex S locus. Genome analysis in a broad area at the S locus will ultimately lead to the identi®cation of the pollen S determinant gene(s). After identi®cation of the pollen S determinant(s), several strategies, such as structural biology, biochemistry and biophysics, will be applicable to elucidate the mechanisms of recognition during the SI reaction in Brassica so as to complement the traditional strategies for studying SI, namely genetics and molecular biology. N OT E A D D E D I N P RO O F A gene identical to SP11, termed SCR, has recently been shown by loss-of-function and gain-of-function experiments to encode the pollen S determinant of SI (Schopfer et al., 1999). From a pollination bioassay we have also found that the SP11 gene has a function as the male determinant (Takayama et al., 2000). Directions of SLG 910 and SRK 910 genes in Fig. 2B were corrected to be the same as those in the S8 haplotype by Cui et al. (1999). AC K N OW L E D GE M E N T S This work was supported in part by Grants-in-Aids for Special Research on Priority Area (07281102 and 07281103, Genetic Dissection of Sexual Dierentiation and Pollination Process in Higher Plants) from the Ministry of Education, Science, Culture and Sport, Japan. We are grateful to Professor Kiichi Fukui, Osaka University, and Dr Katsunori Hatakeyama, Research Institute of Seed Production, for many interesting discussions on this manuscript. L I T E R AT U R E C I T E D Bateman AJ. 1955. Self-incompatibility systems in angiosperms. III. Cruciferae. Heredity 9: 52±68. Boyes DC, Nasrallah JB. 1993. Physical linkage of the SLG and SRK genes at the self-incompatibility locus of Brassica oleracea. Molecular and General Genetics 236: 369±373.
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encoded at the self-incompatibility locus of Brassica oleracea. Proceedings of the National Academy of Science, USA 88: 8816±8820. Suzuki G, Watanabe M, Nishio T. 2000. Physical distances between S-locus genes in various S haplotypes of Brassica rapa and B. oleracea. Theoretical and Applied Genetics (in press). Suzuki G, Watanabe M, Isogai A, Hinata K. 1997a. Highly conserved 50 -¯anking regions of two self-incompatibility genes, SLG 9 and SRK 9. Gene 191: 123±126. Suzuki G, Watanabe M, Toriyama K, Isogai A, Hinata K. 1995. Molecular cloning of members of the S-multigene family in selfincompatible Brassica campestris L. Plant and Cell Physiology 36: 1273±1280. Suzuki G, Watanabe M, Toriyama K, Isogai A, Hinata K. 1997c. Direct cloning of the Brassica S locus by using a P1-derived arti®cial chromosome (PAC) vector. Gene 199: 133±137. Suzuki G, Watanabe M, Kai N, Matsuda N, Takayama S, Isogai A, Hinata K. 1997b. Three repeated members of the S-multigene family are linked to the S-locus in self-incompatible Brassica campestris L. Molecular and General Genetics 26: 257±265. Suzuki G, Kai N, Hirose T, Fukui K, Nishio T, Takayama S, Isogai A, Watanabe M, Hinata K. 1999. Genomic organization of the S locus: Identi®cation and characterization of genes in SLG/SRK region of an S 9 haplotype of Brassica campestris (syn. rapa). Genetics 152: 391±400. Takasaki T, Hatakeyama K, Watanabe M, Toriyama K, Isogai A, Hinata K. 1999. Introduction of SLG (S-locus glycoprotein) alters the phenotype of endogenous S-haplotype, but confers no new S-haplotype speci®city in Brassica rapa L. Plant Molecular Biology 40: 659±668. Takayama S, Isogai A, Tsukamoto C, Ueda Y, Hinata K, Okazaki K, Suzuki A. 1987. Sequences of S-glycoproteins, products of the Brassica campestris self-incompatibility locus. Nature 326: 102±104. Takayama S, Isogai A, Tsukamoto C, Shiozawa H, Ueda Y, Hinata K, Okazaki K, Koseki K, Suzuki A. 1989. Structure of N-glycosidic saccharide chains in S-glycoproteins, products of S-genes associated with self-incompatibility in Brassica campestris. Agricultural Biochemical Chemistry 53: 713±722. Takayama S, Shiba H, Iwano M, Shimosato H, Che F-S, Kai N, Watanabe M, Suzuki G, Hinata K, Isogai A. 2000. The pollen determinant of self-incompatibility in Brassica campestris. Proceedings of the National Academy of Science (USA) 97 (in press). Thompson KF, Taylor JP. 1966. Non-linear dominance relationships between S alleles. Heredity 21: 345±362. Trick M, Flavell RB. 1989. A homologous S genotype of Brassica oleracea expresses two S-like genes. Molecular and General Genetics 218: 112±117. Trowsdale J, Ragoussis J, Campbell RD. 1991. Map of the human MHC. Immunology Today 12: 443±446. Watanabe M, Suzuki G, Toriyama K, Takayama S, Isogai A, Hinata K. 1999. Two anther-expressed genes downstream of SLG 9: identi®cation of a novel S-linked gene speci®cally expressed in anthers at the uninucleate stage of Brassica campestris (syn. rapa) L. Sexual Plant Reproduction 12: 129±134. Watanabe M, Takasaki T, Toriyama K, Yamakawa S, Isogai A, Suzuki A, Hinata K. 1994. A high degree of homology exists between the protein encoded by SLG and the S receptor domain encoded by SRK in self-incompatible Brassica campestris L. Plant and Cell Physiology 35: 1221±1229. Yamakawa S, Watanabe M, Hinata K, Suzuki A, Hinata K. 1995. The sequences of S-receptor kinases (SRK) involved in selfincompatibility and their homologies to S-locus glycoproteins of Brassica campestris. Bioscience, Biotechnology and Biochemistry 59: 161±162. Yamakawa S, Shiba H, Watanabe M, Shiozawa H, Takayama S, Hinata K, Isogai A, Suzuki A. 1994. The sequence of S-glycoproteins involved in self-incompatibility of Brassica campestris and their distribution among Brassicaceae. Biosci. Biotech. Biochem. 58: 921±925. Yu K, Schafer U, Glavin TL, Goring DR, Rothstein SJ. 1996. Molecular characterization of the S locus in two self-incompatible Brassica napus lines. Plant Cell 8: 2369±2380.
Genomic Organization of the SLG/SRK Region of the S Locus in Brassica Species M A S AO WATA N A B E * {, G O S U Z U K I{, S EI JI TA K AYA M A } , A K IR A I SO GA I} and KO KI C HI H IN ATA k {Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan, {Division of Natural Science, Osaka Kyoiku University, Kashiwara 582-8582, Japan, }Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma 630-0101, Japan and kResearch Institute of Seed Production Co., Ltd, Sendai 989-3204, Japan Received: 21 July 1999
Accepted: 9 September 1999
Self-incompatibility in Brassica species is controlled by a single S locus with multiple alleles. Recent experimental data suggest that SLG (S locus glycoprotein) and/or SRK (S receptor kinase) are the stigmatic S determinants of selfincompatibility in Brassica. A 76-kb genomic fragment containing both SLG and SRK was isolated and characterized in the S 9 haplotype of B. campestris. This fragment contains at least 14 expressed genes and three putative open reading frames (ORFs), suggesting that the S locus is embedded in a gene-rich region. Within this fragment, both anther and/or stigma-speci®c genes are clustered around the SLG and SRK genes. Some members of the S multigene family are also closely linked to the S locus. In this review we discuss the complex genomic structure of the S locus in # 2000 Annals of Botany Company Brassica species. Key words: Anther-expressed gene, Brassica campestris (syn. rapa) L., Brassica oleracea L., gene cluster, genome structure, self-incompatibility, SLG, S locus, SRK.
I N T RO D U C T I O N Many ¯owering plants have self-incompatibility (SI) systems that promote outbreeding and maintain genetic diversity. These systems prevent self-fertilization by rejecting pollen derived from plants with the same genotype (Fig. 1A, B). The SI system in Brassica species is controlled sporophytically by multiple alleles at a single locus called the S locus (Bateman, 1955). The genus Brassica contains about 40 species (Schulz, 1936). Two species, B. campestris (syn. rapa) and B. oleracea, have been used extensively in studies of SI, although SI lines of B. napus are now also being used. B. napus is an amphidiploid hybrid between B. campestris and B. oleracea, and is usually selfcompatible; however, self-incompatible B. napus lines have been synthesized by introgression of self-incompatibility from B. campestris (Goring et al., 1992a). In Japan, hybrid varieties of commercial Brassica vegetable crops have been produced using an SI trait since the 1950s by growing two selected self-incompatible genotypes, A and B, in alternate rows in an isolated ®eld, and harvesting hybrid F1 seeds between A and B (Fig. 1). To date, more than 30 S alleles have been identi®ed in B. campestris (Nou et al., 1993) and more than 50 S alleles in B. oleracea (Brace et al., 1994). Because the activity of S alleles is controlled sporophytically, dominant/recessive relationships in¯uence the ultimate phenotype of stigma and pollen. Thus, the phenotype of the pollen and stigma of heterozygous plants depends on the outcome of complex dominant/recessive allelic interactions. In B. campestris, * For correspondence. Fax 81-(0)19-621-6177, e-mail nabe@ iwate-u.ac.jp
0305-7364/00/0A0155+06 $35.00/00
dominant/recessive relationships have been determined between 24 S alleles for almost all combinations (Hatakeyama et al., 1998a), whereas in B. oleracea dominant/ recessive relationships among 28 S alleles have been determined for one third of all combinations (Thompson and Taylor, 1966). Based on these studies, the characteristic features of dominance relationships in Brassica species include: (1) common codominant relationships; (2) dominant/recessive relationships occurring more frequently in pollen than in the stigma; (3) dominant/recessive relationships diering between stigma and pollen; (4) non-linear dominant/recessive relationships (Thompson and Taylor, 1966; Hatakeyama et al., 1998a). The expression and mechanisms of these dominant/recessive relationships will eventually be elucidated at a molecular level using the wellcharacterized plant material described above. Here we review the molecular characterization of the SI genes, SLG and SRK, and the genomic structure of the S locus, focusing mainly on B. campestris, which includes many important vegetable crops (Chinese cabbage, turnip, oil seed and leaf vegetables) grown in the Far East. M O L E C U L A R C H A R AC T E R I Z AT I O N O F S L G A N D S R K GE N E S Linkage between the S locus and S-locus glycoprotein (SLG) was ®rst demonstrated by isoelectric focusing analysis of stigmatic proteins (Nishio and Hinata, 1977) and provided a basis for subsequent molecular analyses of SI in Brassica species. In 1985, a cDNA clone of SLG was successfully isolated using dierential screening in B. oleracea (Nasrallah et al., 1985). In 1987, the amino # 2000 Annals of Botany Company
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FIG. 1. Commerical seed-production ®eld of a single-cross hybrid leaf vegetable of B. campestris. Alternate rows of self-incompatible, yet cross-compatible, S homozygotes are transplanted into an isolated ®eld (Matsushima island). Cross pollination (A) among the S homozygotes will produce hybrid seed; selfed and sibbed seed production will be largely prevented by SI (B).
acid sequences of SLGs, isolated from three S homozygotes of B. campestris, were determined (Isogai et al., 1987; Takayama et al., 1987). Following these experiments, over 30 SLG clones were isolated from Brassica species (Nasrallah et al., 1987; Lalonde et al., 1989; Trick and Flavell, 1989; Chen and Nasrallah, 1990; Goring et al., 1992a, 1992b; Scutt and Croy, 1992; Watanabe et al., 1994; Yamakawa et al., 1994; Delorme et al., 1995; Matsushita et al., 1996; Kusaba et al., 1997; Hatakeyama et al., 1998b, 1998c). These data revealed three characteristic features of SLG. Firstly, the amino acid sequence deduced from SLG cDNA contains a hydrophobic signal peptide at the amino terminal (Nasrallah et al., 1987). Secondly, each sequence contains 12 conserved cysteine residues at the carboxyl terminal. Thirdly, the S speci®city is thought to be determined by the protein portions of SLGs (Isogai et al., 1987; Takayama et al., 1987), because no discernible dierences were found in the N-glycosidic carbohydrate chains from three dierent SLGs (Takayama et al., 1989). The SLGs cloned so far have been classi®ed into two groups, Class I and Class II, based on their respective homologies. This classi®cation is correlated with dominant/ recessive relationships in the pollen, even though SLG is expressed speci®cally in stigmatic tissues. This correlation might have crucial implications for the identi®cation of the pollen S determinant. In 1991, a second S-linked gene, SRK (S receptor kinase), was identi®ed in B. oleracea and interestingly, the
extracellular domain of SRK was found to be highly homologous to SLG (Stein et al., 1991). It has been suggested that during the SI reaction, the S domain of SRK accepts a pollen S determinant and transduces the signal into papilla cells via a protein phosphorylation cascade. To date, over 10 sets of SLG and SRK genes from dierent S alleles of Brassica species have been isolated (Chen and Nasrallah, 1990; Stein et al., 1991; Goring and Rothstein, 1992; Glavin et al., 1994; Kumar and Trick, 1994; Watanabe et al., 1994; Delorme et al., 1995; Suzuki et al., 1995; Yamakawa et al., 1995; Hatakeyama et al., 1998b, 1998c). The homology between SLG and the S domain of SRK derived from the same S allele is about 90%, and in some cases over 98% (Watanabe et al., 1994; Suzuki et al., 1997a; Hatakeyama et al., 1998c). The transcript of SRK is expressed mainly in stigmatic tissue, where expression levels are extremely low relative to SLG (Stein et al., 1991; Glavin et al., 1994; Watanabe et al., 1994; Delorme et al., 1995). Complete conservation of 12 cysteine residues at the carboxyl terminal in the S domain of SRK should be important for the establishment of the tertiary con®guration of this protein. Transformation experiments with SLG or SRK have been performed in an attempt to elucidate the role of SLG and/ or SRK in SI. In transgenic B. campestris transformed with antisense SLG driven by the SLG promoter, the transcripts of SLG and SRK decreased, and the transformant plants became self-compatible (Shiba et al., 1995). When selfincompatible B. campestris was transformed with SLG derived from B. campestris, the transformants again became self-compatible because the SI phenotype of the stigma was changed but the pollen SI phenotype remained the same. This change from an SI phenotype to a self-compatible phenotype was due to co-suppression of the SLG-transgene and the endogenous SLG gene (Takasaki et al., 1999). When self-incompatible B. napus was transformed with an inactive copy of the SRK gene, the transformants also showed a change from an SI phenotype to a self-compatible phenotype. This eect was due to co-suppression and dominant-negative eects, whereby the transgenes induced a dramatic reduction in the expression of the endogenous S locus and S locus-related genes, indicating homologydependent silencing (Stahl et al., 1998). The silencing of SLG and/or SRK genes in self-incompatible host plants also resulted in the breakdown of SI (Conner et al., 1997). In each case, the change of S phenotype was only observed in the stigma, and not in pollen. When an SLG gene from self-incompatible B. campestris was introduced into selfcompatible B. napus under the control of a tapetum-speci®c promoter, pollination tests indicated that the pollen of the transgenic B. napus did not gain the SI phenotype (Sasaki et al., 1998). These experiments show that SLG and/or SRK are, at least, the stigmatic determinants of SI; however, gain-of-function experiments without co-suppression are required before the function of SLG and SRK in SI recognition can be established. G E NO M I C A N A LY S I S O F T H E S LO C U S As discussed above, the pollen S determinant(s) must be located at the S locus because SI in Brassica species is
Watanabe et al.ÐGenomic Organization of S Locus Gene in Brassica controlled by a single locus. Thus, there must be at least three genes (SLG, SRK and the pollen S determinant gene) at the S locus. Because the S locus comprises multiple genes within one segregational unit, an `S allele' is now referred to as an `S haplotype' (Nasrallah and Nasrallah, 1993). The term `haplotype' is generally used in the context of the major histocompatibility complex (MHC) in animal immune systems. The genes related to the MHC, which functions in the recognition of self and non-self antigens, form a large multigene family and occur in tandem (Trowsdale et al., 1991). In the Brassica SI system, several SLG- and SRK-like genes of the S multigene family are closely linked to the S locus (Oldknow and Trick, 1995; Suzuki et al., 1997b; Cabrillac et al., 1999). For instance, in the S 9 haplotype of B. campestris, the physical distance between SLG, SRK and three S-related genes (BcRK1, BcRL1 and BcSL1), which are linked to the S locus, was estimated to be less than 610 kb. BcRK1 is highly homologous to ARK1, which is located tandemly with another receptor-like kinase (ARK2) (Dwyer et al., 1994). Both the expression pattern and the nucleotide sequence in BcRK1 are quite similar to ARK1, suggesting that BcRK1, like ARK1, might be expected to be involved in cell expansion (Suzuki et al., 1997b). The genomic organization of the three S-related genes (BcRK1, BcRL1 and BcSL1) in B. campestris is similar to that of ARK1 and ARK2 in Arabidopsis. Comparative mapping between B. campestris and Arabidopsis demonstrated that the homoeologous region to the S locus in Arabidopsis was located in the vicinity of ETR1 on chromosome 1. ARK1 and ARK2 were also found to be located on chromosome 1, but about 1 centimorgan away from the S locus homoeologous region (Conner et al., 1998). Based on these results, BcRK1 would be an orthologous gene to ARK1. The expression pattern and genomic organization of these three S-related genes (BcRK1, BcRL1 and BcSL1) indicate that none of these S-related genes function in sexual reproduction processes, so they do not re¯ect further genetic diversity of SLG and SRK at the S locus. It may therefore be that these three S-related genes and the S locus became linked to each other by chance during the course of genomic evolution in the Brassicaceae. Assuming that the pollen S determinant of the SI recognition reaction is linked to the S locus, determination of the extent of the genomic region that co-segregates with the S locus may aid in its isolation. The physical distance between SLG and SRK, both of which co-segregate with the S locus, has been estimated as less than 200 kb in B. oleracea (Boyes and Nasrallah, 1993), and less than 20 kb in B. campestris (Yu et al., 1996; Boyes et al., 1997; Suzuki et al., 1999). The tendency for a shorter physical distance between SLG and SRK in B. campestris than in B. oleracea seems to be consistent for several S haplotypes in B. campestris and B. oleracea (Suzuki et al., 2000). These results indicate that the S locus is smaller in B. campestris than it is in B. oleracea. Isolation of genomic fragments between the SLG and SRK genes, using a chromosome walking strategy, has proved dicult for the S locus region (Boyes et al., 1997; Conner et al., 1998). Only two strategies have so far
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succeeded in cloning the genomic region between SLG and SRK. One strategy is the long PCR method with a speci®c primer set of SLG and SRK (Yu et al., 1996), and the other is the direct-cloning of an MluI genomic fragment (76 kb) containing both SLG and SRK genes, using the P1-derived arti®cial chromosome (PAC) vector (Suzuki et al., 1997c). The MluI genomic fragment (SLG/SRK region) of the S 9 homozygote of B. campestris has been screened for expressed sequences (Watanabe et al., 1999; Suzuki et al., 1999) and shown to contain at least 14 expressed genes including SLG and SRK (Fig. 2A). We also found three long putative ORFs (ORF-a, b and c) within the genomic sequence of the 76-kb region (Suzuki et al., 1999). The average gene density in this region is one gene for every 5.4 kb, suggesting that the S locus is embedded within a gene-rich region. Because SI genes reside within the S locus complex, together with many other genes not involved with SI (described below), it is necessary to determine very carefully which S-linked genes have a function in SI. Anther and/or stigma-speci®c genes located at the S locus are not always associated with SI recognition, as demonstrated for the SLA gene (Boyes and Nasrallah, 1995; Pastuglia et al., 1997). Functional analyses, such as gain-of-function and loss-of-function experiments, are needed to prove that genes linked to the S locus are indeed genes involved in SI, as stressed by McCubbin and Kao (1999). Because the eciency of Brassica transformation is very low, identifying the unknown pollen S determinant gene by genome analysis is a laborious process. We have investigated the expression pattern of all 14 genes identi®ed in the 76-kb region (Suzuki et al., 1999). SP1, SP2, SP3, SP4, SP7, SP10 and SLL2 genes were found to be expressed in both ¯oral and vegetative tissues. Other genes (SLG, SRK, SP5, SP6, SP8, SP11 and SAE1) were found to be anther and/or stigma-speci®c genes. With the exception of SP8, these anther and/or stigma genes were clustered with SLG and SRK (Suzuki et al., 1999; Fig. 2). We have also identi®ed ORF-a, a gene expressed speci®cally in anther tissues (G. Suzuki et al., unpubl. res.) and ORF-c which is highly homologous to a Brassica EST clone, which is expressed in ¯oral buds (M. Watanabe and G. Suzuki, unpubl. res.). Comparative mapping around the region of the S locus in self-incompatible B. campestris and selfcompatible Arabidopsis demonstrated that a genomic region of approx. 30 kb, which contains both the SLG and SRK genes in B. campestris, was completely deleted in Arabidopsis (Conner et al., 1998). The region containing the anther and/or stigma speci®c genes (SP6 to SAE1), identi®ed by Suzuki et al. (1999), was very similar to the 30-kb genomic region identi®ed by Conner et al. (1998) at this map position. Therefore, the region from SP6 to SAE1 might be important for the SI recognition reaction. Among the 14 genes detected, two (SAE1 and SP11) are speci®cally expressed in anther tissues, and are located downstream of the SLG 9 or SRK 9 genes (Fig. 2A; Suzuki et al., 1999; Watanabe et al., 1999). The extent of allelic polymorphism and the function of SAE1 and SP11 are currently under investigation. In the S 9 haplotype of B. campestris, genes homologous to SLL2 (Yu et al., 1996) and ClpP (Letham and Nasrallah,
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FIG. 2. Genomic structure of the S locus complex region. A, A map of the 76-kb SLG/SRK region of S 9 haplotype of B. campestris (Suzuki et al., 1999). Coloured boxes represent 14 S-linked genes expressed in stigmas (green), anthers (yellow), both stigmas and anthers (light green), and both reproductive and vegetative tissues (light blue). The SLG 9 gene was located near the middle of the 76-kb region. SRK 9 was located approx. 13 kb upstream of SRK 9 , resulting in the 76-kb region containing approx. 15 kb upstream of SRK 9 and approx. 37 kb downstream of SLG 9 . White boxes indicate the putative ORFs (ORF-a, b and c) estimated from genomic DNA sequence. B, Directions of transcriptions of the SLG and SRK genes in three S haplotypes analysed to date. In the S 9 haplotype of B. campestris, SLG is located at the downstream region of SRK and the directions of the genes are the same (Suzuki et al., 1999). In the S 910 haplotype of B. napus, SRK is located at the downstream region of SLG in the same direction, which was con®rmed by long PCR between them (Yu et al., 1996). In the S8 haplotype of B. campestris, SLG and SRK are located in the opposite direction, which was estimated by a partial long-range restriction map of the S locus (Boyes et al., 1997). The region between SLG8 and SRK8 has not yet been cloned.
1998) were found downstream of SLG (Suzuki et al., 1999; Watanabe et al., 1999). Although the locations of these three genes were reported in only the S8 and S 9 haplotypes of B. campestris, the gene arrangement of SLG, SLL2 and the ClpP homologue is the same in both haplotypes (Boyes et al., 1997; Conner et al., 1998; Suzuki et al., 1999). Yu et al. (1996) reported that another S-linked gene, termed SLL1, is located between SLG and SLL2 in the S 910 haplotype of B. napus. In the S 9 haplotypes of B. campestris, a sequence similar to SLL1 was also found in the region between SLG and SLL2; however, this SLL1-like sequence was fragmented and appeared to be a pseudogene (Suzuki et al., 1999). The SLL1 gene was not considered to function in SI recognition because sequence diversity was not observed in dierent S haplotypes (Yu et al., 1996). Our observation that SLL1 is fragmented supports this prediction. Putative amino acid sequences of SLL2 showed 96.3% identity between the S 9 and S 910 haplotypes (Watanabe et al., 1999). Similarly, putative amino acid sequences of ClpP homologues showed 97.5% identity between the S8 and S 9 haplotypes (Suzuki et al., 1999). Because SLL2 and ClpP are not involved in SI recognition, their amino acid sequence identities are much higher (above 95%) than those observed in the SI-related genes, SLGs and SRKs. The S locus complex contains non-polymorphic regions including the gene cluster of SLL1, SLL2 and the ClpP homologue, which are unlikely to be involved in SI, suggesting that dierent regions of the S locus complex have evolved independently (Charlesworth and Awadalla, 1998; MacCubbin and Kao, 1999). In the B. campestris S 9 haplotype, both SLG and SRK were found to be transcribed in the same direction which,
interestingly, was the opposite direction to SLG and SRK transcription in the S8 haplotype (Boyes et al., 1997). However, in the S 910 haplotype of B. napus, SLG and SRK were shown to be transcribed divergently (Yu et al., 1996; Fig. 2B). These results indicate that the direction of transcription of the SLG and SRK genes varies between dierent S haplotypes. As reported by Boyes et al. (1997), sequence rearrangement and a lack of sequence similarity appear to contribute to suppression of recombination at the S locus. Notwithstanding this, the dierent orientations of SLG and SRK at the S locus in dierent S haplotypes must also contribute to reduced rates of recombination. In general, a low level of recombination is observed around centromeric regions of chromosomes, but the Brassica S locus is positioned at the terminus of the chromosome (Iwano et al., 1998; Kamisugi et al., 1998). Thus, the low level of recombination observed at the Brassica S locus must be independent of its chromosomal location, in contrast to the situation in Petunia species, which possess a gametophytic SI system, where the subcentromeric location of the S locus (Entani et al., 1999) is expected to contribute signi®cantly to the suppression of recombination. In summary, the complexity of the Brassica S locus is characterized by the following important features: (1) two similar genes, SLG and SRK, are tightly linked; (2) gene conversion has occurred between SLG and SRK (Suzuki et al., 1997a); (3) highly polymorphic SLG and SRK genes are located close to a cluster of highly conserved genes (described above); (4) sequence rearrangements have occurred (Boyes et al., 1997); and (5) recombination is suppressed within the S locus complex. The evolution and
Watanabe et al.ÐGenomic Organization of S Locus Gene in Brassica maintenance of the S locus complex is one of the most interesting problems in the plant genomics. F U T U R E P RO S P E C T S Comparative mapping of the S locus among several haplotypes is an important strategy for understanding the extent of sequence variation and diversity at the S locus. The direct cloning system based on the PAC vector (Suzuki et al., 1997c) is proving useful in this study. Fluorescence in situ hybridization (FISH) on the extended DNA ®bre (EDF) allows the physical mapping of cloned DNA fragments with a resolution of a few kilobases (de Jong et al., 1999), and this technique has allowed the physical mapping of the SLG 9 and SRK 9 genes on EDFs using EDF-FISH analysis and the DNA combing technique (Suzuki et al., 1999). This analysis will also be applicable to other S locus genes and S haplotypes, which will prove to be a powerful tool for structural analysis and physical mapping of the complex S locus. Genome analysis in a broad area at the S locus will ultimately lead to the identi®cation of the pollen S determinant gene(s). After identi®cation of the pollen S determinant(s), several strategies, such as structural biology, biochemistry and biophysics, will be applicable to elucidate the mechanisms of recognition during the SI reaction in Brassica so as to complement the traditional strategies for studying SI, namely genetics and molecular biology. N OT E A D D E D I N P RO O F A gene identical to SP11, termed SCR, has recently been shown by loss-of-function and gain-of-function experiments to encode the pollen S determinant of SI (Schopfer et al., 1999). From a pollination bioassay we have also found that the SP11 gene has a function as the male determinant (Takayama et al., 2000). Directions of SLG 910 and SRK 910 genes in Fig. 2B were corrected to be the same as those in the S8 haplotype by Cui et al. (1999). AC K N OW L E D GE M E N T S This work was supported in part by Grants-in-Aids for Special Research on Priority Area (07281102 and 07281103, Genetic Dissection of Sexual Dierentiation and Pollination Process in Higher Plants) from the Ministry of Education, Science, Culture and Sport, Japan. We are grateful to Professor Kiichi Fukui, Osaka University, and Dr Katsunori Hatakeyama, Research Institute of Seed Production, for many interesting discussions on this manuscript. L I T E R AT U R E C I T E D Bateman AJ. 1955. Self-incompatibility systems in angiosperms. III. Cruciferae. Heredity 9: 52±68. Boyes DC, Nasrallah JB. 1993. Physical linkage of the SLG and SRK genes at the self-incompatibility locus of Brassica oleracea. Molecular and General Genetics 236: 369±373.
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