鈴木 剛

The 3-ketoacyl-ACP synthase (KAS) II is a fatty-acid-related enzyme which catalyzes the elongation of 16:0-acyl carrier protein (ACP) to 18:0-ACP in plastids. The fatty acid biosynthesis 1-1 (fab1-1) mutant of Arabidopsis thaliana is partially deficient in its activity of Arabidopsis thaliana 3-ketoacyl-ACP synthase 2 (AtKAS2), and its phenotype has been intensively studied in connection with the chilling resistance and fatty acid composition. In this study, we used the T-DNA insertion mutant of AtKAS2 to examine its possible role in plant development. Reverse transcription (RT)-PCR showed that the AtKAS2 gene was expressed in various plant organs, except for roots, and was highly expressed in siliques. The fusion of beta-glucuronidase (GUS) to the AtKAS2 promoter demonstrated that the promoter was active in various tissues such as embryos, stomatal guard cells, inflorescences and pollen grains. We were not able to identify atkas2 homozygous mutant adult plants in heterozygous mutant progeny. Phenotypic and genetic analyses showed that disruption of the AtKAS2 by T-DNA insertion caused embryo lethality, and the development of the embryos was arrested at the globular stage. Taken together, our results suggest that AtKAS2 is required for embryo development in Arabidopsis during the transition from the globular to the heart stage.

Self-incompatibility (SI) in Brassica species is an elaborate system preventing self-fertilisation. It is important for high-quality seed production in Brassica vegetables, such as cabbage, turnip and Chinese cabbage. In classical genetics, Brassica SI is sporophytically controlled by a single S-locus with multiple alleles. This concept of the S-locus enabled us to beginmolecular studies to determine how the SI system is regulated. Through vigorous molecular analysis by research groupsworldwide, female and male S determinants were identified as a receptor-type protein kinase (SRK) and its ligand (SP11/SCR). In this chapter, we highlight six milestones, which are historically important findings in Brassica SI research, especially with respect to identification of genes regulating the recognition of self-pollen.

Haploid induction of wheat by crossing with Imperata cylindrica pollen is an efficient method for doubled haploid breeding. We investigated the process of wheat haploid formation after crossing with L cylindrica. Our cytological observations of zygotes showed the successful fertilization of parental gametes. Wheat haploids were formed by complete elimination of I. cylindrica chromosomes. Missegregation of L cylindrica chromosomes was observed in the first cell division of zygote. At metaphase L cylindrica chromosomes did not congress onto the equatorial plate. The sister chromosomes did not move toward the poles during anaphase, though their cohesion was released normally. L cylindrica chromosomes were still in the cytoplasm at telophase and eliminated from daughter nuclei. After two-celled stage, we could find no L cylindrica chromosome in the nuclei but micronuclei containing L cylindrica chromatin in the cytoplasm. These observations indicate that L cylindrica chromosomes are completely eliminated from nuclei in the first cell division probably due to lack of functional kinetochores.

In the Brassica self-incompatibility (SI) system, a pollen determinant, SP11, is involved in dominance/recessive relationships in pollen SI phenotypes. In order to gain some insights into the genomic structure around the SP11 and the mechanisms that give dominance/recessive relationships, we characterized the genomic region containing SP11 and SRK genes in three pollen recessive class-II S haplotypes. The direction of transcription of S genes was completely conserved among class-II S haplotypes. However, the region between SP11 and SRK (S-intergenic region) was highly polymorphic without short repetitive sequences. In addition, we found a sequence similarity between the short repetitive sequence and 5'-upstream region of SP11. This sequence similarity was found to be potentially related to the expression of dominance relationships through the change of chromatin structure.

Introduction of large-DNA fragments into cereals by Agrobacterium-mediated transformation is a useful technique for map-based cloning and molecular breeding. However, little is known about the organization and stability of large fragments of foreign DNA introduced into plant genomes. In this study, we produced transgenic rice plants by Agrobacterium-mediated transformation with a large-insert T-DNA containing a 92-kb region of the wheat genome. The structures of the T-DNA in four independent transgenic lines were visualized by fluorescence in situ hybridization on extended DNA fibers (fiber FISH). By using this cytogenetic technique, we showed that rearrangements of the large-insert T-DNA, involving duplication, deletion and insertion, had occurred in all four lines. Deletion of long stretches of the large-insert DNA was also observed in Agrobacterium.

The perianths of Liliaceae plants, such as lily and tulip, have two whorls of almost identical petaloid organs, which are called tepals. According to the modified ABC model proposed in tulip, the class B genes are expressed in whorl 1 as well as whorls 2 and 3, so that the organs of whorls 1 and 2 have the same petaloid structure. The floral structure of asparagus (Asparagus officinalis L.) is similar to that of Liliaceae plants, however, the expression of B-class genes (AODEF, AOGLOA, AOGLOB) was not found in whorl 1, but was confined to whorls 2 and 3. This result does not support the modified ABC model in asparagus. In order to gain a better understanding of asparagus flower development, we have characterized a genomic clone of the AODEF gene. We compared the genomic organization and promoter sequence of AODEF with three well-studied DEF-like genes, DEFICIENS (Antirrhinum), APETALA3 (Arabidopsis), and OSMADS16 (rice). Exon-intron structures of these genes are well-conserved except for the large fifth intron in the AODEF gene and the OSMADS16 gene. Putative cis-elements including CArG-boxes were found in the promoter region and forty-two microsatellites were found in the AODEF genomic sequence.

Plants have evolved many systems to prevent inappropriate fertilization. Among them, incompatibility is a well-organized system in which pollen germination or pollen-tube growth is inhibited in pistils. Self-incompatibility (SI), rejecting self-pollen, promotes outbreeding in flowering plants. On the other hand, inter-species incompatibility, preventing gene flow among species to restrict outbreeding, usually occurs unilaterally, and is known as unilateral incompatibility (UI). In Brassicaceae, little is known about the molecular mechanism of UI, although S-locus genes involved in recognition of self-pollen have been characterized in the SI system. In the present study, we characterized novel UI observed between members of the same species, Brassica rapa; pollen of Turkish SI lines was specifically rejected by pistils of the Japanese commercial SI variety 'Osome'.. The incompatible phenotype of this intra-species UI closely resembled that of SI. Segregation analysis revealed that the pollen factor of this UI was not linked to the S-locus.

ISI 被引用数：3

Self-incompatibility (SI) discriminating self and non-self pollen is regulated by S-locus genes in Brassica. In most S haplotypes, a set of three highly polymorphic genes, SLG, SRK, and SP11, is located at the S-locus region. In the present study, we found duplicated SP11 genes, S-15-Sp11a, S-15-Sp11b, and S-15-Sp11b, in the self-incompatible S-15 haplotype of B. oleracea. RNA gel blot and reverse transcription polymerase chain reaction (RT-PCR) analyses showed that two different sizes of SP11 transcripts were specifically detected in anther tissues: a 0.65-kb transcript corresponded to S-15-Sp11a (an exon-1 region of S-15-Sp11b was also co-transcribed in some cases), and a 1.4-kb transcript contained the duplicated three genes, S-15-Sp11a, S-15-Sp11b, and an exon-1 region of S-15-Sp11b, all three of which were connected to intergenic spacer regions.

Alliinase operates in the biochemical pathway that produces the compounds responsible for the characteristic flavor of onion. We isolated and characterized the 86-kb BAC clone containing a novel onion alliinase gene, ALL]. Identity of deduced amino acid sequence of ALL1 with a bulb alliinase is 65.4% and with a root alliinase is 67.3%. The ALL1 gene is expressed specifically in onion roots and estimated pI value of mature ALL1 protein is similar to that of root alliinase isoform 1, which is an uncharacterized protein having alliinase activity. The highly repetitive sequences around the ALL1 gene was observed from sequence and DNA gel blot analyses. The 33.2% G + C content of the 35-kb ALL1 region is similar to that of dicot plants and lower than that of monocot cereal plants, although onion is classified into monocots. The present study shows the first evidence of the onion genomic sequences around genes differed from the cereal genome. (C) 2003 Elsevier B.V. All rights reserved.

Diploid Ipomoea trifida is an ancestral wild species of the cultivated hexaploid sweet potato, and displays a sporophytic self-incompatibility (SI) that is controlled by a single multiallelic S-locus. To characterize the genomic region of the S-locus using a map-based cloning method, a BAC library consisting of approximately 40,000 clones was constructed from genomic DNA of SI-homozygote, and screened using S-linked DNA markers which were mapped in our previous study. We constructed a contig covering the S-locus region with additional screening of fosmid and lambda phase libraries. RFLP analysis of recombinant plants using terminal end sequences of the BAC clones as probes indicated that the S-locus region was delimited within a map distance of 0.57 cM, spanning approximately 300 kb in physical distance. Remarkable suppression of genetic recombination was detected in the S-locus region. From sequence analysis of the 313-kp region, 43 ORFs, many repetitive sequences and 5 transposable elements were predicted. None of the ORFs, however, showed a high homology with the SI genes reported to date at the S-locus of other plant families, suggesting that a unique molecular mechanism is involved in the SI system of the Convolvulaceae family.

Self-incompatibility (SI) in Ipomoea trifida is regulated by a single S locus with multiple alleles. Identification of SI genes in the S-locus region by positional cloning is one of the most important goals for understanding sexual reproduction in this species. Despite our intensive efforts to construct bacterial artificial chromosome (BAC) contigs covering the S-locus region, a gap was observed in the core region of the potential S locus. In order to confirm the physical linkage of two non-overlapping BAC contigs in the S-locus region and to determine the size of the gap between them, fluorescence in-situ hybridization (FISH) was performed on mitotic chromosomes and extended DNA fibres using previously isolated S-linked BAC clones as probes. The information obtained from this work would be useful for molecular cloning of the SI genes by a chromosome walking approach. In addition, we showed that strong suppression of recombination in the S locus was not related to the centromere because the S locus was mapped to one end of a chromosome.

Self-incompatibility (SI) discriminating self-and non-self pollen is regulated by S-locus genes in Brassica. In most of the S haplotypes, a highly polymorphic S-locus glycoprotein (SLG) gene is tightly linked to genes for the SI determinants, S-receptor kinase (SRK) and SP11, although the precise function of SLG in SI has not been clarified. In the present study, we performed DNA gel blot analysis for S-32, S-33, and S-36 haplotypes of Brassica rapa showing normal SI phenotypes and concluded that there might be no SLG in their genome. RNA gel blot analysis of the SLG-less S haplotypes indicated the possible existence of eSRK transcripts in the stigma. These three S haplotypes are useful resources to discern the molecular mechanism of the SI reaction without SLG.

To gain some insights into the structure of the S-locus and the mechanisms that have kept its diversity, a 75-kb genomic fragment containing the self-incompatibility (S) locus region was isolated from the S-12-haplotype of Brassica rapa and compared with those of other S-haplotypes. The region around the S determinant genes was highly polymorphic and filled with S-haplotype-specific intergenic sequences. The diverse genomic structure must contribute to the suppression of recombination at the S-locus.

Analysis of DNA from Aegilops tauschii revealed that sequences hybridisable to the starch branching enzyme I (SBE I) gene were contained within a 53-kb fragment. There were at least four genes or gene fragments but of these only one appeared to encode the SBE I observed in the endosperm. Two large-insert DNA clones that encode SBE I from A. tauschii were isolated. Hybridisation analysis confirmed the presence of multiple SBE I gene type sequences within this DNA fragment of approximately 100 kb. Fluorescent in situ hybridisation (FISH) on extended DNA fibres provided further evidence of the close proximity of three of these genes. Sequence analysis was undertaken and this demonstrated that wSBE I-D3, wSBE I-D2 and wSBE I-D4 genes were clustered within 27 kb of DNA of these only wSBE I-D4 encodes the SBE I purified from the endosperm. Multiple but distinct cDNAs containing SBE I-related sequences have been reported and these could arise from the SBE I locus by different transcription/splicing regimes. © Springer-Verlag 2002.

Self-incompatibility in Brassica species is regulated by a set of S-locus genes: SLG, SRK, and SP11/SCR. In the vicinity of the S-locus genes, several expressed genes, SLL2 and SP2/ClpP, etc., were identified in B. campestris. Arabidopsis thaliana is a self-compatible Brassica relative, and its complete genome has been sequenced. From comparison of the genomic sequences between B. campestris and A. thaliana, microsynteny between gene clusters of Arabidopsis and Brassica SLL2 regions was observed, though the S-locus genes, SLG, SRK, and SP11/SCR were not found in the region of Arabidopsis. Almost all genes predicted in this region of Arabidopsis were expressed in both vegetative and reproductive organs, suggesting that the genes in the SLL2 region might not be related to self-incompatibility. Considering the recent speculation that the S-locus genes were translocated as a single unit between Arabidopsis and Brassica, the translocation might have occurred in the region between the SLL2 and SP7 genes.

Onion (Allium cepa L.; 1C=15,000 Mb) is an agriculturally important plant. The genome of onion has been extensively studied at the conventional cytogenetic level, but molecular analyses have lagged behind due to its large genome size. To overcome this bottleneck, a partial bacterial artificial chromosome (BAC) library of onion was constructed. The average insert size of the BAC library was about 100 kb. A total of 48,000 clones, corresponding to 0.32 genome equivalent, were obtained. Fluorescent in situ hybridization (FISH) screening resulted in identification of BAC clones localized on centromeric, telomeric, or several limited interstitial chromosomal regions, although most of the clones hybridized with entire chromosomes. The partial BAC library proved to be a useful resource for molecular cytogenetic studies of onion, and should be useful for further mapping and sequencing studies of important genes of this plant. BAC FISH screening is a powerful method for identification of molecular cytogenetic markers in large-genome plants.

Self-incompatibility (SI) systems prevent self-pollination and promote outbreeding. In Brassica, the SI genes SLG (for S-locus glycoprotein) and SRK (for S-receptor kinase) are members of the S multigene family, which share the SLG-like domain (S domain), which encodes a putative receptor. We have cloned members of the S multigene family from the S-9 haplotype of B. campestris (syn. rapa). In addition, eight distinct genomic regions harboring 10 SLG/SRK-like genes were characterized in the present study. Sequence analysis revealed two novel SRK-like genes, BcRK3 and BcRK6 (for B. campestris receptor kinases 3 and 6, respectively). Other genes that were characterized included SFR2 (for S gene family receptor 2), SLR2 (for S locus related gene 2), and a pseudogene. Based on phylogenetic analysis of the nucleotide sequences of the S domain regions, SLG and SRK appear to be distinct from other members of the S multigene family. Linkage analysis showed that most members of the S multigene family are dispersed in the Brassica genome, and that SLR1 (S Locus related gene 1) is not linked to the SLR2 in B. campestris.

Self-incompatibility (SI) in Brassica is sporophytically controlled by the multi-allelic S locus. SI phenotypes of the stigma and pollen in an S heterozygote are determined by the two S haplotypes it carries; the two haplotypes may be co-dominant or exhibit a dominant/recessive relationship. Because the S receptor kinase (SRK) gene of the S locus was recently shown to determine the S haplotype specificity of the stigma, we wished to investigate whether SRK also plays a role in the dominance relationships between S haplotypes. We crossed plants carrying an SRK28 transgene with plants homozygous for one of five S haplotypes that are either co-dominant with, or recessive to, S-28 haplotype in the stigma, and analyzed the SI phenotypes of the progeny. In all cases, the SI phenotype of the stigma of plants carrying the SRK28 transgene could be predicted by the known dominance relationships between the S haplotype(s) and the S-28 haplotype. Moreover, in the S-43 homozygote carrying the SRK28 transgene where the S-43 phenotype in the stigma was masked by the presence of the SRK28, the transcript level of SRK28 was found to be much lower than that of SRK43. All these results suggest that the dominance relationships between S haplotypes in the stigma are determined by SRK, but not by virtue of its relative expression level.

Many flowering plants have evolved self-incompatibility (SI) systems to prevent inbreeding. In the Brassicaceae, SI is genetically controlled by a single polymorphic locus, termed the S-locus. Pollen rejection occurs when stigma and pollen share the same S-haplotype. Recognition of S-haplotype specificity has recently been shown to involve at least two S-locus genes, S-receptor kinase (SRK) and S-locus protein 11 or S-locus Cys-rich (SP11/SCR). SRK encodes a polymorphic membrane-spanning protein kinase, which is the sole female determinant of the S-haplotype specificity. SP11/SCR encodes a highly polymorphic Cys-rich small basic protein specifically expressed in the anther tapetum and in pollen. In cauliflower (B. oleracea), the gain-of-function approach has demonstrated that an allele of SP11/SCR encodes the male determinant of S-specificity. Here we examined the function of two alleles of SP11/SCR of B. rapa by the same approach and further established that SP11/SCR is the sole male determinant of SI in the genus Brassica sp. Our results also suggested that the 522-bp 5'-upstream region of the S-9-SP11 gene used to drive the transgene contained all the regulatory elements required for the unique sporophytic/gametophytic expression observed for the native SP11 gene. Promoter deletion analyses suggested that the highly conserved 192-bp upstream region was sufficient for driving this unique expression. Furthermore, immunohistochemical analyses revealed that the protein product of the SP11 transgene was present in the tapetum and pollen, and that in pollen of late developmental stages, the SP11 protein was mainly localized in the pollen coat, a finding consistent with its expected biological role.

Cereal centromeres commonly contain many repetitive sequences that are derived from Ty3/gypsy retrotransposon, FISH analysis using a large DNA insert Library of wheat identified a 67-kb clone (R11H) that showed strong hybridization signals on the centromeres. The R11H clone contains Ty3/gypsy retrotransposon-related sequences; both integrase and CCS1 family sequences were identified, Subsequently, we isolated additional 23 large-insert clones which also contained the integrase and CCS1 sequences. Based on the number of the integrase repeats in the clones determined by DNA gel blot analysis, we concluded that the retrotransposon-like sequences are tandemly repeated in wheat centromeres in ca, 55-kb interval on average, This conclusion is consistent with the results of FISH analysis on the extended DNA fibers.

A 314-bp tandemly repeated DNA sequence, named pAc074, was characterized in Allium cepa by fluorescence in situ hybridization (FISH) analyses using random amplified fragment as probe. The nucleotide sequences of the clone pAc074 is partially homologous to the satellite DNA sequences, ACSAT1, ACSAT2, and ACSAT3, of A. cepa with 81%, 81% and 78% similarity, respectively. Our sequential C-banding and FISH with pAc074 probe also clearly showed a close relation between C-heterochromatin at telomeric region and pAc074 sequences on all the chromosomes except on chromosome 6. On the long arm of chromosome 7, pA074 sequences appeared as interstitial band which did not correspond to C-heterochromatin bands. Instead, the C-heterochromatin bands corresponded with the 5S rDNA signals. This is the first evidence of simultaneous banding of the 5S rDNA and C-band in A. cepa.

In Brassica, self-incompatibility genes SLG (for S-locus glycoprotein) and SRK (for S-receptor kinase) are located in the S-locus complex region with several other S-linked genes. The S locus is a highly polymorphic region: polymorphism has been observed not only in sequences of SLG and SRK but also in the location of the S-locus genes. In order to compare the physical location of the S-locus genes in various S haplotypes, we used six class-I S haplotypes of B. rapa and seven class-I S haplotypes of B. oleracea in this study. DNA gel blot analysis using pulsed-field gel electrophoresis (PFGE) showed that the physical distances between SLG and SRK in B. rapa are significantly shorter than those in B. oleracea and that the sizes of MluI and BssHII fragments harboring SLG and SRK are less variable within B. rapa than within B, oleracea. We concluded that several large genomic fragments might have been inserted into the S-locus region of B. oleracea after allelic differentiation of S-locus genes.

Self-incompatibility (SI) enables flowering plants to discriminate between self- and non-self-pollen. In Brassica, SI is controlled by the highly polymorphic S locus. The recently identified male determinant, termed SP11 or SCR, is thought to be the ligand of S receptor kinase, the female determinant. To examine functional and evolutionary properties of SP11, we cloned 14 alleles from class-I S haplotypes of Brassica campestris and carried out sequence analyses. The sequences of mature SP11 proteins are highly divergent, except for the presence of conserved cysteines. The phylogenetic trees suggest possible co-evolution of the genes encoding the male and female determinants. (C) 2000 Federation of European Biochemical Societies.

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 self-incompatibility in Brassica. A 76-kb genomic fragment containing both SLG and SRK was isolated and characterized in the Sg 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-specific 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 Brassica species. (C) 2000 Annals of Botany Company.

Many flowering plants possess self-incompatibility (SI) systems that prevent inbreeding. In Brassica, SI is controlled by a single polymorphic locus, the S locus. Two highly polymorphic S locus genes, SLC (S locus glycoprotein) and SRK (S receptor kinase), have been identified, both of which are expressed predominantly in the stigmatic papillar cell. We have shown recently that: SRK is the determinant of the S haplotype specificity of the stigma. SRK is thought to serve as a receptor for a pollen ligand, which presumably is encoded by another polymorphic gene at the S locus. We previously have identified an S locus gene, SP11 (S locus protein 11), of the S-9 haplotype of Brassica campestris and proposed that it potentially encodes the pollen ligand. SP11 is a novel member of the PCP (pollen coat protein) family of proteins, some members of which have been shown to interact with SLG. In this work, we identified the SP11 gene from three additional S haplotypes and further characterized the gene. We found that (i) SP11 showed an S haplotype-specific sequence polymorphism; (ii) SP11 was located in the immediate flanking region of the SRK gene of the four S haplotypes examined; (iii) SP11 was expressed in the tapetum of the anther, a site consistent with sporophytic control of Brassica SI; and (iv) recombinant SP11 of the S-9 haplotype applied to papillar cells of S-9 stigmas, but not of S-8 stigmas, elicited SI response, resulting in inhibition of hydration of cross-pollen. All these results taken together strongly suggest that SP11 is the pollen S determinant in SI.

The self-incompatibility possessed by Brassica is an intraspecific reproductive barrier by which the stigma rejects self-pollen but accepts non-self-pollen for fertilization. The molecular/biochemical bases of recognition and rejection have been intensively studied. Self-incompatibility in Brassica is sporophytically controlled by the polymorphic S locus(1). Two tightly linked polymorphic genes at the S locus, S receptor kinase gene (SRK) and S locus glycoprotein gene (SLG), are specifically expressed in the papillar cells of the stigma(2-4), and analyses of self-compatible lines(5-7) of Brassica have suggested that together they control stigma function in self-incompatibility interactions. Here we show, by transforming self-incompatible plants of Brassica rapa with an SRK28 and an SLG(28) transgene separately, that expression of SRK28 alone, but not SLG(28) alone, conferred the ability to reject self (S-28)-pollen on the transgenic plants. We also show that the ability of SRK28 to reject S-28 pollen was enhanced by SLG(28). We conclude that SRK alone determines S haplotype specificity of the stigma, and that SLG acts to promote a full manifestation of the self-incompatibility response.

ISI 被引用数：180

In Brassica, two self-incompatibility genes, encoding SLG (S locus glycoprotein) and SRK (S-receptor kinase), are located at the S locus and expressed in the stigma. Recent molecular analysis has revealed that the S locus is highly polymorphic and contains several genes, i.e., SLG, SRK, the as-yet-unidentified pollen S gene(s), and other linked genes. In the present study, we searched for expressed sequences in a 76-kb SLG/SRK region of the S-9 haplotype of Brassica campestris (syn. rapa) and identified 10 genes in addition to the four previously identified (SLG(9), SRK9 SAE1, and SLL2) in this haplotype. This gene density (1 gene/5.4 kb) suggests that the S locus is embedded in a gene-rich region of the genome. The average G + C content in this region is 32.6%. An En/Spm-type transposon-like element was found downstream of SLG(9). Among the genes we identified that had not previously been found to be linked to the S locus were genes encoding a small cysteine-rich protein, a J-domain protein, and an antisilencing protein (ASF1) homologue. The small cysteine-rich protein was similar to a pollen coat protein, named PCP-A1, which had previously been shown to bind SLG.

The sporophytic self-incompatibility (SI) system in Brassica campestris (syn. rapa) is controlled by multiple alleles of a single locus, S, in which SLG and SRK are located. SLG and/or SRK appear to comprise the female component of the recognition reaction of SI. The gene encoding the male component must also be located at the S locus but has not been identified to date. RNA gel blot analysis with regions flanking SLG(9) and SRK9 revealed two anther-expressed genes 3' of the SLG(9) gene. Two cDNA clones were isolated using the 3'-flanking region of SLG(9) as probe. Because of sequence similarity with the Brassica SLL2 gene, one cDNA clone was designated SLL2-S-9 (for S Locus-Linked 2-S-9 gene). The 5'-region of SLL2-S-9 was not present in other SLL2 genes examined. The second gene was novel and was designated SAE1-S-9 (for S locus Anther Expressed 1-S-9 gene). SAE1-S-9 and SLL2-S-9 were located approximately 2.5 kb and 5.5 kb, respectively, downstream of SLG(9). A 1.4-kb transcript for SLL2-S9 was detected in both vegetative and reproductive tissues, whereas 1.9-kb and 2.4-kb transcripts were specifically detected in the anther of S-9 haplotype. SAE1-S-9 was specifically expressed in the anther at the uninucleate stage. This gene encoded a novel polypeptide of 257 amino acids. Database analysis revealed a homologous sequence in the upstream region of SRK3 of B. oleracea. We discuss possible relationships between SLL2-S-9 and SAE1-S-9 and SI.

The physical localization of the S-glycoprotein (SLG) locus in the chromosome of Brassica campestris L. 'pekinensis' cv 'Kukai' was visualized by multi-color fluorescent in situ hybridization (McFISH). 'Kukai', which is an FI hybrid between two parental lines, T-17 and T-18, has two SLG genes from both T-17 and T-18. In this study, a 1.3-kb DNA fragment was amplified from the genomic DNA of T-17 by PCR using a set of primers specific to the class-I SLG. From the genomic DNA of T-18, no DNA fragment was amplified using these primers. In the genomic Southern hybridization, a cloned PCR product hybridized with the genomic DNA of T-17 or F-1 but not with that of T-18. The PCR product had a sequence homology of approximately, 85% to another class-I SLG gene, SLG-9. Therefore, the PCR product from T-17 was named SLG-17, as it is thought to be a member of the class-I SLG. Using SLG-17 as the probe, FISH was carried out to visualize the position of the SLG locus. McFISH was also carried out simultaneously using the SLG-17 and SLG-9 genes as probes. The SLG-17 gene was detected as a doublet signal at the interstitial region close to the end of a small chromosome, with the signal site being identical to that of SLG-9. Therefore, it is concluded that the SLG-17 gene is localized at the interstitial region close to the end of the chromosome derived from T-17 in Brassica campestris L. 'pekinensis' CV 'Kukai'.

S-locus glycoprotein (SLG) is known to be one of the proteins related to self-incompatibility in Brassica, and its transcripts are detected in anthers as well as stigmas. However, an SLG protein has not been detected in anthers so far. Because of sporophytic control of the self-incompatibility (SI) phenotype of pollen, an SLG gene is expected to be expressed in the sporophytic tissue of anthers, i.e., the tapetum. Overexpression of an SLG gene in the tapetum would enable us to predict the localization and function of an SLG protein in anthers. In this study, an SLG gene of self-incompatible B. campestris under the control of a tapetum-specific promoter was introduced into self-compatible B. napus. Immunoblot analysis using anti-SLG antiserum detected the exogenous SLG protein in the immature anthers, but not in the mature anthers. Immunoelectron microscopy showed the SLG protein to be localized in the tapetum and in the exine cell wall layer at the stage when the tapetum was degenerating. This result indicates the possible movement of the SLG protein from the tapetum to the pollen surface. A pollination test indicated that the pollen of the transgenic B. napus did not gain the SI phenotype.

Two self-incompatibility genes in Brassica, SLG and SRK (SLG encodes a glycoprotein; SRK encodes a receptor-like kinase), are included in the S multigene family. Products of members of the S multigene family have an SLG-like domain (S domain) in common, which may function as a receptor. In this study, three clustered members of the S multigene family, BcRK1, BcRL1 and BcSL1, were characterized. BcRK1 is a putative functional receptor kinase gene expressed in leaves, flower buds and stigmas, while BcRL1 and BcSL1 are considered to be pseudogenes because deletions causing frameshifts were identified in these sequences. Sequence and expression pattern of BcRK1 were most similar to those of the Arabidopsis receptor-like kinase gene ARK1, indicating that BcRK1 might have a function similar to that of ARK1, in processes such as cell expansion or plant growth. Interestingly, the region containing BcRK1, BcRL1 and BcSL1 is genetically linked to the S locus and the physical distance between SLG, SRK and the three S-related genes was estimated to be less than 610 kb. Thus the genes associated with self-incompatibility exist within a cluster of S-like genes in the genome of Brassica.

Self-incompatibility of Brassica is regulated by the S locus, which contains several genes including SLG and SRK. We found that both SLG and SRK genes were located at an approx. 80-kb MluI fragment in an S-9 haplotype of B. campestris. Therefore, we cloned this MluI fragment into a BssHII site of the Pl-derived artificial chromosome (PAC) vector. The utility of the direct cloning method is discussed in this study. (C) 1997 Elsevier Science B.V.

The nucleotide (nt) sequences of the 5'-flanking regions of two Brassica self-incompatibility genes, SLG(9) and SRK9, were determined. Their sequences were highly conserved: a region spanning 1.9 kb in the 5'-flanking region was completely identical except for a 1319-bp segment in SLG(9). These observations strongly suggest that SLG(9) and SRK9 together with their promoter regions were involved in a gene duplication or conversion event which occurred before the 1319-bp SLG(9)-specific sequence was inserted in SLG(9) or deleted in SRK9. (C) 1997 Elsevier Science B.V.

Genomic clones of SLG(9) and SRK(9), isolated from self-incompatible Brassica campestris, were separately introduced into tobacco plants. The presence of an SLG(9)-like transcript in SRK(9) transformants indicated the possibility of alternative polyadenylation sites in the SRK(9) gene.

We isolated 12 groups of genomic clones that contained SLG-homologous regions from a genomic library constructed from an S-9 homozygote of self-incompatible Brassica campestris. Both SLG(9) and SRK(9) genomic clones, which are located within the self-incompatibility (S) locus, were included in these groups. The promoter regions of SLG(9) and SRK(9) were completely identical for at least 200 bp upstream from their respective initiation codons (ATG). The five sequence elements (boxes I to V) that are conserved in the promoters of SLG and SRK genes were also found in the SLG(9) and SRK(9) clones. However, one conserved element (box III) unexpectedly lacked 7 of 11 bp, although box III has been considered necessary for expression in pistil. The other ten groups of genomic clones were classified into six SRK-like groups and four SLG-like groups. These results indicate that SLG, SRK, SLG-like, and SRK-like genes form a large S-multigene family in B. campestris.