Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea.

In cruciferous plants, self-pollination is prevented by the action of genes situated at the self-incompatibility locus or S-locus. The self-incompatibility reaction is associated with expression of stigma glycoproteins encoded by the S-locus glycoprotein (SLG) gene. Only a few cases of self-compatible plants derived from self-incompatible lines in the crucifer Brassica have been reported. In these cases, self-compatibility was generally ascribed to the action of single genes unlinked to the S-locus. In contrast, we report here a line of Brassica oleracea var acephala with a self-compatible phenotype linked to the S-locus. By means of both biochemical and immunochemical analyses, we showed that this self-compatible (Sc) line nonetheless possesses stigmatic SLGs (SLG-Sc) that are expressed with a similar spatial and temporal pattern to that described for the SLGs of self-incompatible Brassica plants. Moreover, the SLG-Sc products segregate with the self-compatibility phenotype in F2 progeny, suggesting that changes at the S-locus may be responsible for the occurrence of the self-compatibility character. A cDNA clone encoding the SLG-Sc product was isolated, and the deduced amino acid sequence showed this glycoprotein to be highly homologous to the pollen recessive S2 allele glycoprotein. Hence, self-compatibility in this Brassica Sc line correlates with the expression of a pollen recessive-like S allele in the stigma.     

Isolation of a Second S-Locus-Related Cdna from Brassica Oleracea: Genetic Relationships between the S Locus and Two Related Loci

Self-incompatibility in Brassica oleracea is controlled by the highly polymorphic S locus. Isolation and subsequent characterization of the S-locus-glycoprotein (SLG) gene, which encodes the S-locus-specific glycoprotein (SLSG), has revealed the presence of a self-incompatibility multigene family. One of these S-locus-related genes, SLR1, has been shown to be expressed. In this study we present the isolation and preliminary characterization of a second expressed S-locus-related sequence, SLR2. Through restriction fragment length polymorphism (RFLP) linkage analysis we demonstrate that the SLR1 and SLR2 loci reside approximately 18.5 map units apart in one linkage group that segregates independently of the S-locus. The identification of a second SLR gene expressed in stigmas suggests that loci unlinked to the S-locus may play a role in the self-incompatibility response, or in pollination in general.     

Genomic organization of the S locus: Identification and characterization of genes in SLG/SRK region of S(9) haplotype of Brassica campestris (syn. rapa).

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), SRK(9), 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.     

Molecular characterization of the S locus in two self-incompatible Brassica napus lines.

In Brassica species, self-incompatibility has been mapped genetically to a single chromosomal location. In this region, there are two closely linked genes coding for the S locus glycoprotein (SLG) and S locus receptor kinase (SRK). They appear to comprise the pistil component of the self-incompatibility reaction. SLG and SRK are thought to recognize an unknown pollen component on the incompatible pollen, and the gene encoding this pollen component must also be linked to the SLG and SRK genes. To further our understanding of self-incompatibility, the chromosomal region carrying the SLG and SRK genes has been studied. The physical region between the SLG-910 and the SRK-910 genes in the Brassica napus W1 line was cloned, and a search for genes expressed in the anther revealed two additional S locus genes located downstream of the SLG-910 gene. Because these two genes are novel and are conserved at other S alleles, we designated them as SLL1 and SLL2 (for S locus-linked genes 1 and 2, respectively). The SLL1 gene is S locus specific, whereas the SLL2 gene is not only present at the S locus but is also present in other parts of the genomes in both self-incompatible and self-compatible Brassica ssp lines. Expression of the SLL1 gene is only detectable in anthers of self-incompatible plants and is developmentally regulated during anther development, whereas the SLL2 gene is expressed in anthers and stigmas in both self-incompatible and self-compatible plants, with the highest levels of expression occurring in the stigmas. Although SLL1 and SLL2 are linked to the S locus region, it is not clear whether these genes function in self-incompatibility or serve some other cellular roles in pollen-pistil functions.     

Identification of a gene linked to the Brassica S (self-incompatibility) locus by differential display

Self-incompatibility in Brassica is controlled by a complex locus, the S locus, that includes several expressed genes. Two S locus genes, SLG and SRK, are expressed in the stigma and have been implicated in self-pollen recognition. The male component of this recognition system is also predicted to be encoded by a gene at the S locus but this gene has not been identified to date. In this study, we have used differential display to screen for polymorphic, S-locus-linked genes that are expressed in anthers. This approach has allowed the identification of a gene, named S5J, which was shown to segregate completely with the S locus. We discuss the possible role of this gene in the self-incompatibility response and evaluate the utility of differential display for the identification of genes at specific genetic loci.     

Genomic organization of the Papaver rhoeas self-incompatibility S(1) locus

The self-incompatibility (SI) response in Papaver rhoeas depends upon the cognate interaction between a pollen-expressed receptor and a stigmatically expressed ligand. The genes encoding these components are situated within the S-locus. In order for SI to be maintained, the genes encoded by the S-locus must be co-inherited with no recombination between them. Several hypotheses, including sequence heterogeneity and chromosomal position, have been put forward to explain the maintenance of the S-locus in the SI systems of the Brassicaceae and the Solanaceae. A region of the Papaver rhoeas genome encompassing part of the self-incompatibility S(1) locus has been cloned and sequenced. The clone contains the gene encoding the stigmatic component of the response, but does not contain a putative pollen S-gene. The sequence surrounding the S(1) gene contains several diverse repetitive DNA elements. As such, the P. rhoeas S-locus bears similarities to the S-loci of other SI systems. An attempt to localize the P. rhoeas S-locus using fluorescence in situ hybridization (FISH) has also been made. The potential relevance of the findings to mechanisms of recombination suppression is discussed.     

A pollen coat protein, SP11/SCR, determines the pollen S-specificity in the self-incompatibility of Brassica species

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 S9-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.     

Identification of self-incompatibility (S-) locus linked pollen cDNA markers in Petunia inflata.

Solanaceous type self-incompatibility (SI) is controlled by a single polymorphic locus, termed the S-locus. The only gene at the S-locus that has been characterized thus far is the S-RNase gene, which controls pistil function, but not pollen function, in SI interactions between pistil and pollen. One approach to identifying additional genes (including the pollen S-gene, which controls pollen function in SI) at the S-locus and to study the structural organization of the S-locus is chromosome walking from the S-RNase gene. However, the presence of highly repetitive sequences in its flanking regions has made this approach difficult so far. Here, we used RNA differential display to identify pollen cDNAs of Petunia inflata, a self-incompatible solanaceous species, which exhibited restriction fragment length polymorphism (RFLP) for at least one of the three S-haplotypes (S1, S2, and S3) examined. We found that the genes corresponding to 10 groups of pollen cDNAs are genetically tightly linked to the S-RNase gene. These cDNA markers will expedite the mapping and cloning of the chromosomal region of the Solanaceae S-locus by providing multiple starting points.     

Genomic organization of the S-locus region of Brassica

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 S12-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.     

Balancing selection and low recombination affect diversity near the self-incompatibility loci of the plant Arabidopsis lyrata

The self-incompatibility (S-) locus region of plants in the Brassica family is a small genome region. In Arabidopsis lyrata, the S-genes, SRK and SCR, encode the functional female and pollen recognition proteins, which must be coadapted to maintain correct associations between the two component genes, and thus self-incompatibility (SI). Recombinants would be self-compatible and thus probably disadvantageous in self-incompatible species. Therefore, tight linkage between the two genes in incompatibility systems is predicted to evolve to avoid producing such recombinant haplotypes. The evolution of low recombination in S-locus regions has not been rigorously tested. To test whether these regions' per-nucleotide recombination rates differ from those elsewhere in the genome, and to investigate whether the A. lyrata S-loci have the predicted effect on diversity in their immediate genome region, we studied diversity in genes that are linked to the S-loci but are not involved in incompatibility and are not under balancing selection. Compared with other A. lyrata loci, genes linked to the S-loci have extraordinarily high polymorphism. Our estimated recombination in this region, from fitting a model of the effects of S-allele polymorphism on linked neutral sites, supports the hypothesis of locally suppressed recombination around the S-locus.     

Characterization and mapping of non-S gametophytic self-compatibility in sweet cherry (Prunus avium L.)

Self-incompatibility in Prunus (Rosaceae) species, such as sweet cherry, is controlled by a multiallelic locus (S), in which two tightly linked genes, S-RNase and SFB (S haplotype-specific F-box), determine the specificity of the pollen and the style. Fertilization in these species occurs only if the S-specificities expressed in the pollen and the pistils are different. However, modifier genes have been proposed to be necessary for a full manifestation of the self-incompatibility response. 'Cristobalina' is a spontaneous self-compatible sweet cherry cultivar that originated in Eastern Spain. Previous studies with this genotype suggested that pollen modifier gene(s), not linked to the S-locus, may be the cause of self-incompatibility breakdown. In this work, an F(1) population from 'Cristobalina' that segregates for this trait was used to identify molecular markers linked to self-compatibility by bulked segregant analysis. One simple sequence repeat (SSR) locus (EMPaS02) was found to be linked to self-compatibility in this population at 3.2?cM. Two additional populations derived from 'Cristobalina' were used to confirm the linkage of this marker to self-compatibility. Since EMPaS02 has been mapped to the sweet cherry linkage group 3, other markers located on the same linkage group were analysed in these populations to confirm the location of the self-compatibility locus.     

Characterization of expressed genes in the SLL2 region of self-compatible Arabidopsis thaliana.

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.     

Self-compatibility of two apricot selections is associated with two pollen-part mutations of different nature

Loss of pollen-S function in Prunus self-compatible mutants has recently been associated with deletions or insertions in S-haplotype-specific F-box (SFB) genes. We have studied two self-compatible cultivars of apricot (Prunus armeniaca), Currot (S(C)S(C)) and Canino (S(2)S(C)), sharing the naturally occurring self-compatible (S(C))-haplotype. Sequence analysis showed that whereas the S(C)-RNase is unaltered, a 358-bp insertion is found in the SFB(C) gene, resulting in the expression of a truncated protein. The alteration of this gene is associated with self-incompatibility (SI) breakdown, supporting previous evidence that points to SFB being the pollen-S gene of the Prunus SI S-locus. On the other hand, PCR analysis of progenies derived from Canino showed that pollen grains carrying the S(2)-haplotype were also able to overcome the incompatibility barrier. However, alterations in the SFB(2) gene or evidence of pollen-S duplications were not detected. A new class of F-box genes encoding a previously uncharacterized protein with high sequence similarity (approximately 62%) to Prunus SFB proteins was identified in this work, but the available data rules them out of producing S-heteroallelic pollen and thus the cause of the pollen-part mutation. These results suggest that cv Canino has an additional mutation, not linked to the S-locus, which causes a loss of pollen-S activity when present in pollen. As a whole, these findings support the proposal that the S-locus products besides other S-locus independent factors are required for gametophytic SI in Prunus.     

Self-incompatibility in the genus Arabidopsis: characterization of the S locus in the outcrossing A. lyrata and its autogamous relative A. thaliana

As a starting point for a phylogenetic study of self-incompatibility (SI) in crucifers and to elucidate the genetic basis of transitions between outcrossing and self-fertilizing mating systems in this family, we investigated the SI system of Arabidopsis lyrata. A. lyrata is an outcrossing close relative of the self-fertile A. thaliana and is thought to have diverged from A. thaliana approximately 5 million years ago and from Brassica spp 15 to 20 million years ago. Analysis of two S (sterility) locus haplotypes demonstrates that the A. lyrata S locus contains tightly linked orthologs of the S locus receptor kinase (SRK) gene and the S locus cysteine-rich protein (SCR) gene, which are the determinants of SI specificity in stigma and pollen, respectively, but lacks an S locus glycoprotein gene. As described previously in Brassica, the S haplotypes of A. lyrata differ by the rearranged order of their genes and by their variable physical sizes. Comparative mapping of the A. lyrata and Brassica S loci indicates that the S locus of crucifers is a dynamic locus that has undergone several duplication events since the Arabidopsis--Brassica split and was translocated as a unit between two distant chromosomal locations during diversification of the two taxa. Furthermore, comparative analysis of the S locus region of A. lyrata and its homeolog in self-fertile A. thaliana identified orthologs of the SRK and SCR genes and demonstrated that self-compatibility in this species is associated with inactivation of SI specificity genes.     

Cloning and mapping multiple S-locus F-box genes in European pear (Pyrus communis L.)

European pear, as well as its close relatives Japanese pear and apple, exhibits S-RNase-based gametophytic self-incompatibility. The male determinant of this self-incompatibility mechanism is a pollen-expressed protein containing an F-box domain; in the genera Petunia (Solanaceae), Antirrhinum (Plantaginaceae), and Prunus (Rosaceae), a single F-box gene determines the pollen S. In apple and Japanese pear, however, multiple S-locus F-box genes were recently identified as candidates for the pollen S, and they were named S-locus F-Box Brothers. These genes were considered good candidates for the pollen S determinant since they exhibit S-haplotype-specific polymorphisms, pollen-specific expression, and linkage to the S-RNase. In the present study, S-locus F-Box Brothers homologs have been cloned from two of the most agronomically important European pear varieties, “Abbé Fétel” (S_104-2/S₁₀₅) and “Max Red Bartlett” (S₁₀₁/S₁₀₂), and they have been mapped on a genetic linkage map developed on their progeny. Our results suggest that the number of F-box genes linked to the S-locus of the European pear is higher than expected according with previous reports for apple and Japanese pear, since up to five genes were found to be linked to a single S-haplotype. Moreover, two of these genes exhibited an incomplete linkage to the S-RNase, allowing the identification of low-frequency recombinant haplotypes, generated by a crossing-over event between the two genes. These F-box genes are most likely placed in close proximity of the S-locus but do not belong to it, and they can thus be excluded from being responsible for the determination of pollen S function.     

芸薹属自交不亲和基因的分子生物学及进化模式

芸薹属的自交不亲和性是受单基因座、复等位基因控制的孢子体控制型。自交不亲和基因座位（S-locus）是由多个基因组成的复杂区域,称之为S多基因家族,其大多数成员分布于芸薹属的整个染色体组。目前已鉴定出100多个S等位基因,它们的起源分化始于一千万年前。S-座位上存在的多基因有3种：SRK,SLG和SCR/SP11;SRK和SLG在柱头中表达,SCR/SP11在雄蕊中表达。SRK蛋白在识别同类花粉的过程中起主要作用,而SLG蛋白增强了这种自交不亲和反应。SLG与SRK基因中编码S-结构域的核苷酸序列相似性程度高达85％～98%。基因转换可能是SLG和SRK的高度同源性能够得以保持的原因。SRK,SLG和SCR基因紧密相连,并表现出高水平的序列多样性。SRK与SLG基因间的距离很近,在20～25 kb之间。在柱头和花粉中,自交不亲和等位基因之间的共显性关系要比显性和隐性关系更加普遍,这是芸薹属自交不亲和性的一大特点。自交不亲和基因的进化模式存在两种假说：双基因进化模式和中性变异体进化模式;可能存在几种不同的进化方式,它们共同在自然群体中新的S等位基因进化过程中起作用。     

Evolution of the S-locus region in Arabidopsis relatives

The S locus, a single polymorphic locus, is responsible for self-incompatibility (SI) in the Brassicaceae family and many related plant families. Despite its importance, our knowledge of S-locus evolution is largely restricted to the causal genes encoding the S-locus receptor kinase (SRK) receptor and S-locus cysteine-rich protein (SCR) ligand of the SI system. Here, we present high-quality sequences of the genomic region of six S-locus haplotypes: Arabidopsis (Arabidopsis thaliana; one haplotype), Arabidopsis lyrata (four haplotypes), and Capsella rubella (one haplotype). We compared these with reference S-locus haplotypes of the self-compatible Arabidopsis and its SI congener A. lyrata. We subsequently reconstructed the likely genomic organization of the S locus in the most recent common ancestor of Arabidopsis and Capsella. As previously reported, the two SI-determining genes, SCR and SRK, showed a pattern of coevolution. In addition, consistent with previous studies, we found that duplication, gene conversion, and positive selection have been important factors in the evolution of these two genes and appear to contribute to the generation of new recognition specificities. Intriguingly, the inactive pseudo-S-locus haplotype in the self-compatible species C. rubella is likely to be an old S-locus haplotype that only very recently became fixed when C. rubella split off from its SI ancestor, Capsella grandiflora.     

Segregation distortion of T-DNA markers linked to the self-incompatibility (S) locus in Petunia hybrida

In plants with a gametophytic self-incompatibility system the specificity of the pollen is determined by the haploid genotype at the self-incompatibility (S) locus. In certain crosses this can lead to the exclusion of half the gametes from the male parent carrying a particular S-allele. This leads to pronounced segregation distortion for any genetic markers that are linked to the S-locus. We have used this approach to identify T-DNA insertions carrying a maize transposable element that are linked to the S-locus of Petunia hybrida. A total of 83 T-DNA insertions were tested for segregation distortion of the selectable marker used during transformation with Agrobacterium. Segregation distortion was observed for 12 T-DNA insertions and at least 8 of these were shown to be in the same linkage group by intercrossing. This indicates that differential transmission of a single locus (S) is probably responsible for all of these examples of T-DNA segregation distortion. The identification of selectable markers in coupling with a functional S-allele will allow the preselection of recombination events around the S-locus in petunia. Our approach provides a general method for identifying transgenes that are linked to gametophytic self-incompatibility loci and provides an opportunity for transposon tagging of the petunia S-locus.     

A Brassica S-locus gene promoter targets toxic gene expression and cell death to the pistil and pollen of transgenic Nicotiana

The S-locus glycoprotein gene of Brassica is derived from the genetic locus that controls the self-incompatibility response and the specific recognition between pollen and stigma. The promoter of this gene was used to direct expression of the diphtheria toxin A chain gene and the Escherichia coli beta-glucuronidase gene in transgenic Nicotiana tabacum. Expression of the promoter in cells of the pistil and in pollen suggests that a single gene may direct the self-incompatibility response in the two interacting cell types. Additionally, the fusion genes were expressed gametophytically in the heterologous host species, Nicotiana, rather than sporophytically as expected for Brassica. Thus, although the genes involved in self-incompatibility in Brassica and Nicotiana are not homologous in their coding regions, signals for expression of these genes are apparently conserved between the two genera. Our analysis of toxic gene fusion transformants shows that genetic ablation is useful for probing developmental processes and for studying temporal and spatial patterns of gene expression in plants.     

Effects of recombination on hitchhiking diversity in the Brassica self-incompatibility locus complex

In self-incompatibility, a number of S haplotypes are maintained by frequency-dependent selection, which results in trans-specific S haplotypes. The region of several kilobases (approximately 40-60 kb) from SP6 to SP2, including self-incompatibility-related genes and some adjacent genes in Brassica rapa, has high nucleotide diversity due to the hitchhiking effect, and therefore we call this region the "S-locus complex." Recombination in the S-locus complex is considered to be suppressed. We sequenced regions of >50 kb of the S-locus complex of three S haplotypes in B. rapa and found higher nucleotide diversity in intergenic regions than in coding regions. Two highly similar regions of >10 kb were found between BrS-8 and BrS-46. Phylogenetic analysis using trans-specific S haplotypes (called interspecific pairs) of B. rapa and B. oleracea suggested that recombination reduced the nucleotide diversity in these two regions and that the genes not involved in self-incompatibility in the S-locus complex and the kinase domain, but not the S domain, of SRK have also experienced recombination. Recombination may reduce hitchhiking diversity in the S-locus complex, whereas the region from the S domain to SP11 would disfavor recombination.