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.     

Genetic mapping and molecular characterization of the self-incompatibility (S) locus in Petunia inflata

Gametophytic self-incompatibility (SI) possessed by the Solanaceae is controlled by a highly polymorphic locus called the S locus. The S locus contains two linked genes, S-RNase, which determines female specificity, and the as yet unidentified pollen S gene, which determines male specificity in SI interactions. To identify the pollen S gene of Petunia inflata, we had previously used mRNA differential display and subtractive hybridization to identify 13 pollen-expressed genes that showed S -haplotype-specific RFLP. Here, we carried out recombination analysis of 1205 F2 plants to determine the genetic distance between each of these S -linked genes and S-RNase. Recombination was observed between four of the genes (3.16, G211, G212, and G221) and S-RNase, whereas no recombination was observed for the other nine genes (3.2, 3.15, A113, A134, A181, A301, G261, X9, and X11). A genetic map of the S locus was constructed, with 3.16 and G221 delimiting the outer limits. None of the observed crossovers disrupted SI, suggesting that all the genes required for SI are contained in the chromosomal region defined by 3.16 and G221. These results and our preliminary chromosome walking results suggest that the S locus is a huge multi-gene complex. Allelic sequence diversity of G221 and 3.16, as well as of 3.2, 3.15, A113, A134 and G261, was determined by comparing two or three alleles of their cDNA and/or genomic sequences. In contrast to S-RNase, all these genes showed very low degrees of allelic sequence diversity in the coding regions, introns, and flanking regions.     

Highly divergent sequences of the pollen self-incompatibility (S) gene in class-I S haplotypes of Brassica campestris (syn. rapa) L

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.     

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.     

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.     

Identification and classification of S haplotypes in Raphanus sativus by PCR-RFLP of the S locus glycoprotein (SLG) gene and the S locus receptor kinase (SRK) gene

Polymorphism of the S-locus glycoprotein (SLG) and S-locus receptor kinase (SRK) genes in Raphanus sativus was analyzed by PCR-RFLP using SLG- and SRK-specific primers. Twenty four inbred lines of R. sativus could be grouped into nine S haplotypes. DNA fragments of SLG alleles specifically amplified from five S haplotypes by PCR with Class-I SLG-specific primers showed different profiles upon polyacrylamide-gel electrophoresis after digestion with restriction endonucleases. The five R. sativus SLG alleles were determined for their nucleotide sequences of DNA fragments. Comparison of the amino-acid sequences with a reported Brassica SLG (S₆) showed 77-84% homology. Deduced amino-acid sequences showed 12-conserved cystein residues and three hypervariable regions which are characteristic of Brassicsa SLG. A DNA fragment was also amplified by PCR from two of each S haplotype with Class-II SLG-specific primers, and showed polymorphism when cleaved with restriction endonucleases. The nucleotide sequences of amplified DNA fragments of the Class-II SLG revealed about 60% similarity with those of the Class-I SLG. It is concluded that there exist both Class I and Class II S alleles in R. sativus, as in Brassica campestris and Brassica oleracea. PCR using SRK-specific primers amplified a DNA fragment of about 1.0 kb from seven of each S haplotype out of 24 tested. These DNA fragments showed high polymorphism in polyacrylamide-gel electrophoresis after digestion with restriction endonucleases. Nucleotide sequences of the DNA fragments amplified from the seven S haplotypes showed that the fourth and the fifth exons of SRK are highly conserved, and that there is high variation in the fifth intron, the sixth intron and seventh exon of the SRK which may be responsible for the polymorphic band patterns in PCR-RFLP analysis. The PCR-RFLP method has proven useful for the identification of S alleles in inbred lines and for listing S haplotypes in R. sativus. Phylogenic analysis of the SLG and SRK sequences from Raphanus and Brassica revealed that the Raphanus SLGs and SRKs did not form an independent cluster, but were dispersed in the tree, clustering together with Brassica SLGs and SRKs. Furthermore, SLGs and SRKs from Raphanus were both grouped into Class-I or Class-II S haplotypes. Therefore, these results suggest that the diversification of the SLG and SRK alleles occurred prior to the differentiation of the two genera Brassica and Raphanus.     

The self-incompatibility (S) haplotypes of Brassica contain highly divergent and rearranged sequences of ancient origin.

In Brassica, the recognition of self-related pollen by the stigma is controlled by the highly polymorphic S locus that encodes several linked and coadapted genes and can span several hundred kilobases. We used pulsed-field gel electrophoresis to analyze the structure of different S haplotypes. We show that the S2 and S13 haplotypes of Brassica oleracea contain extensive sequence divergence and rearrangement relative to each other. In contrast, haplotypic configuration is more conserved between B. oleracea S13 and B. campestris S8, two haplotypes that have been proposed to be derived from a common ancestral haplotype based on sequence comparisons. These results support the view that extensive restructuring of the S locus preceded speciation in Brassica. This structural heteromorphism, together with haplotype-specific sequences, may suppress recombination within the S locus complex, potentially providing a mechanism for maintaining the linkage of coadapted allelic combinations of genes over time.     

The self-incompatibility locus (S) and quantitative trait loci for self-pollination and seed dormancy in sunflower

Wild populations of common sunflower (Helianthus annuus L.) are self-incompatible and have deep seed dormancy, whereas modern cultivars, inbreds, and hybrids are self-compatible and partially-to-strongly self-pollinated, and have shallow seed dormancy. Self-pollination (SP) and seed dormancy are genetically complex traits, the number of self-compatibility (S) loci has been disputed, and none of the putative S loci have been genetically mapped in sunflower. We genetically mapped quantitative trait loci (QTL) for self-incompatibility (SI), SP, and seed dormancy in a backcross population produced from a cross between an elite, self-pollinated, nondormant inbred line (NMS373) and a wild, self-incompatible, dormant population (ANN1811). A population consisting of 212 BC₁ progeny was subsequently produced by backcrossing a single hybrid individual to NMS373. BC₁ progeny produced 0–838 seeds per primary capitula when naturally selfed and 0–518 seeds per secondary capitula when manually selfed and segregated for a single S locus. The S locus mapped to linkage group 17 and was tightly linked to a cluster of previously identified QTL for several domestication and postdomestication traits. Two synergistically interacting QTL were identified for SP among self-compatible (ss) BC₁ progeny (R²=34.6%). NMS373 homozygotes produced 271.5 more seeds per secondary capitulum than heterozygotes. Germination percentages of seeds after-ripened for 4 weeks ranged from 0% to 100% among self-compatible BC₁S₁ families. Three QTL for seed dormancy were identified (R²=38.3%). QTL effects were in the predicted direction (wild alleles decreased self-pollination and seed germination). The present analysis differentiated between loci governing SI and SP and identified DNA markers for bypassing SI and seed dormancy in elite × wild crosses through marker-assisted selection.Electronic Supplementary Material Electronic supplementary material is available for this article at     

An F-box gene linked to the self-incompatibility (S) locus of Antirrhinum is expressed specifically in pollen and tapetum

In many flowering plants, self-fertilization is prevented by an intraspecific reproductive barrier known as self-incompatibility (SI), that, in most cases, is controlled by a single multiallelic S locus. So far, the only known S locus product in self-incompatible species from the Solanaceae, Scrophulariaceae and Rosaceae is a class of ribonucleases called S RNases. Molecular and transgenic analyses have shown that S RNases are responsible for pollen rejection by the pistil but have no role in pollen expression of SI, which appears to be mediated by a gene called the pollen self-incompatibility or Sp gene. To identify possible candidates for this gene, we investigated the genomic structure of the S locus in Antirrhinum, a member of the Scrophulariaceae. A novel F-box gene, AhSLF-S ₂, encoded by the S ₂ allele, with the expected features of the Sp gene was identified. AhSLF-S ₂ is located 9 kb downstream of S ₂ RNase gene and encodes a polypeptide of 376 amino acids with a conserved F-box domain in its amino-terminal part. Hypothetical genes homologous to AhSLF-S ₂ are apparent in the sequenced genomic DNA of Arabidopsis and rice. Together, they define a large gene family, named SLF (S locus F-box) family. AhSLF-S ₂ is highly polymorphic and is specifically expressed in tapetum, microspores and pollen grains in an allele-specific manner. The possibility that Sp encodes an F-box protein and the implications of this for the operation of self-incompatibility are discussed.     

An F-box gene linked to the self-incompatibility (S) locus of Antirrhinum is expressed specifically in pollen and tapetum

In many flowering plants, self-fertilization is prevented by an intraspecific reproductive barrier known as self-incompatibility (SI), that, in most cases, is controlled by a single multiallelic S locus. So far, the only known S locus product in self-incompatible species from the Solanaceae, Scrophulariaceae and Rosaceae is a class of ribonucleases called S RNases. Molecular and transgenic analyses have shown that S RNases are responsible for pollen rejection by the pistil but have no role in pollen expression of SI, which appears to be mediated by a gene called the pollen self-incompatibility or Sp gene. To identify possible candidates for this gene, we investigated the genomic structure of the S locus in Antirrhinum, a member of the Scrophulariaceae. A novel F-box gene, AhSLF-S2, encoded by the S2 allele, with the expected features of the Sp gene was identified. AhSLF-S2 is located 9 kb downstream of S2 RNase gene and encodes a polypeptide of 376 amino acids with a conserved F-box domain in its amino-terminal part. Hypothetical genes homologous to AhSLF-S2 are apparent in the sequenced genomic DNA of Arabidopsis and rice. Together, they define a large gene family, named SLF (S locus F-box) family. AhSLF-S2 is highly polymorphic and is specifically expressed in tapetum, microspores and pollen grains in an allele-specific manner. The possibility that Sp encodes an F-box protein and the implications of this for the operation of self-incompatibility are discussed.     

Migration rates, frequency-dependent selection and the self-incompatibility locus in Leavenworthia (brassicaceae)

Loci subject to negative frequency-dependent selection are expected to exhibit higher effective migration rates compared to reference loci. Although the number of gene copies transferred between populations by migration is the same for all genes, those subject to negative frequency-dependent selection are more likely to be retained in the immigrant population because rare alleles are selectively favored. So far, evidence for this prediction has been indirect, based on summary statistics rather than on migration rate estimates. Here, we introduce an approximate Bayesian procedure to jointly estimate migration rates at two predefined sets of loci between two populations. We applied the procedure to compare migration rate estimates at the self-incompatibility locus (S-locus) with that at 10 reference loci in two plant species, Leavenworthia alabamica and L. crassa (Brassicaceae). The maximum likelihood estimate for the proportion of migrants (m) was four times higher at the S-locus than at reference loci, but the difference was not statistically significant. Lack of significance might be due to insufficient data, but perhaps also to the recent divergence of the two species (311 ka), because we also show using simulations that the effective migration rate at the S-locus is expected to increase with increasing divergence time. These findings aid in understanding the evolutionary dynamics of negative frequency-dependent selection and they suggest that divergence time should be accounted for when employing migration rates to help detect negative frequency-dependent selection.     

Breeding behaviour of Kunzea pomifera (Myrtaceae): self-incompatibility, intraspecific and interspecific cross-compatibility

To examine breeding system characteristics of the endemic Australian prostrate shrub Kunzea pomifera, artificial hybridisations were undertaken using thirteen different genotypes of K. pomifera, to elucidate: (1) self-incompatibility, (2) intraspecific cross-compatibility in the species and (3) interspecific cross-compatibility with each of K. ambigua and K. ericoides. K. pomifera exhibited very low self-compatibility, with the barrier to self-fertilisation being prevention of pollen-tube growth in the style or ovary. Following intraspecific pollination amongst a number of different genotypes of K. pomifera, 38.4% of pollinated flowers developed fruit; arrest of compatible pollen-tubes in the style, preventing fertilisation, contributes to the low fruit set in this species. Interspecific compatibility was examined between K. pomifera (pistillate parent) and K. ambigua (staminate parent) where seed set per pollinated flower (4.47) was not significantly different from intraspecific crosses (4.66). In crosses between K. pomifera (pistillate parent) and K. ericoides as staminate plant, 0.037% of pollinated flowers produced fruit, with 0.0075 seeds per pollinated flower. Reproductive barriers between these two species were evident in the style of K. pomifera, where the growing tips of the K. ericoides pollen-tubes swelled and ceased to grow.     

Intra- and interspecific variation in genome size in Lathyrus (Leguminosae)

2C nuclear DNA amounts for 24 species of Lathyrus (section Lathyrus ) were determined using flow cytometry. A greater than two-fold variation was observed, ranging from 10.2 pg in L. basalticus to 24.2 pg in L. latifolius. In general, the perennial species had greater DNA amounts than the annuals. Significant intraspecific variation was observed in five species of Lathyrus (from 10.1% in L. annuus to 28% in L. tingitanus). A positive correlation was observed between DNA values obtained by flow cytometry and those previously determined by microdensitometry. Finally, the distribution of DNA amounts in species within section Lathyrus appears to be continuous.     

Use of an interspecific hybrid in identifying a new allelic specificity generated at the self-incompatibility locus after inbreeding in Lycopersicon peruvianum

Summary An interspecific hybrid between Lycopersicon esculentum () and L. peruvianum has been raised by embryo rescue in vitro and used to confirm the presence of a new S-allelic specificity in its inbred L. peruvianum parent, a plant derived by enforced bud self-pollination of a self-incompatible clone with the genotype S ₁ S ₂. The inbred plant showed breeding behavior characteristic of both S ₂ and a second specificity which was not S ₁, S ₂, S ₃ or S _f. Two-dimensional gel electrophoresis of stylar proteins, however, showed only a single typical S-associated component with the M_r and pI characteristic of S₂. The alteration in specificity, therefore, was not associated with a detectable change in an S-associated protein. The F₁ interspecific hybrid showed intermediacy of vegetative and reproductive characters, relatively high fertility and full self-incompatibility. Backcrossing to L. esculentum produced only abortive seeds requiring embryo culture. Backcrosses to L. peruvianum produced a very low proportion of filled germinable seeds. Pollen of the hybrid showed superior viability and tube growth rate compared with pollen of the two parent plants.