Identification and characterization of a polymorphic receptor kinase gene linked to the self-incompatibility locus of Arabidopsis lyrata

We study the segregation of variants of a putative self-incompatibility gene in Arabidopsis lyrata. This gene encodes a sequence that is homologous to the protein encoded by the SRK gene involved in self-incompatibility in Brassica species. We show by diallel pollinations of plants in several full-sib families that seven different sequences of the gene in A. lyrata are linked to different S-alleles, and segregation analysis in further sibships shows that four other sequences behave as allelic to these. The family data on incompatibility provide evidence for dominance classes among the S-alleles, as expected for a sporophytic SI system. We observe no division into pollen-dominant and pollen-recessive classes of alleles as has been found in Brassica, but our alleles fall into at least three dominance classes in both pollen and stigma expression. The diversity among sequences of the A. lyrata putative S-alleles is greater than among the published Brassica SRK sequences, and, unlike Brassica, the alleles do not cluster into groups with similar dominance.     

Independent S-Locus Mutations Caused Self-Fertility in Arabidopsis thaliana

A common yet poorly understood evolutionary transition among flowering plants is a switch from outbreeding to an inbreeding mode of mating. The model plant Arabidopsis thaliana evolved to an inbreeding state through the loss of self-incompatibility, a pollen-rejection system in which pollen recognition by the stigma is determined by tightly linked and co-evolving alleles of the S-locus receptor kinase (SRK) and its S-locus cysteine-rich ligand (SCR). Transformation of A. thaliana, with a functional AlSRKb-SCRb gene pair from its outcrossing relative A. lyrata, demonstrated that A. thaliana accessions harbor different sets of cryptic self-fertility–promoting mutations, not only in S-locus genes, but also in other loci required for self-incompatibility. However, it is still not known how many times and in what manner the switch to self-fertility occurred in the A. thaliana lineage. Here, we report on our identification of four accessions that are reverted to full self-incompatibility by transformation with AlSRKb-SCRb, bringing to five the number of accessions in which self-fertility is due to, and was likely caused by, S-locus inactivation. Analysis of S-haplotype organization reveals that inter-haplotypic recombination events, rearrangements, and deletions have restructured the S locus and its genes in these accessions. We also perform a Quantitative Trait Loci (QTL) analysis to identify modifier loci associated with self-fertility in the Col-0 reference accession, which cannot be reverted to full self-incompatibility. Our results indicate that the transition to inbreeding occurred by at least two, and possibly more, independent S-locus mutations, and identify a novel unstable modifier locus that contributes to self-fertility in Col-0.     

Comparative analysis of the within-population genetic structure in wild cherry (Prunus avium L.) at the self-incompatibility locus and nuclear microsatellites

Gametophytic self-incompatibility (SI) systems in plants exhibit high polymorphism at the SI controlling S-locus because individuals with rare alleles have a higher probability to successfully pollinate other plants than individuals with more frequent alleles. This process, referred to as frequency-dependent selection, is expected to shape number, frequency distribution, and spatial distribution of self-incompatibility alleles in natural populations. We investigated the genetic diversity and the spatial genetic structure within a Prunus avium population at two contrasting gene loci: nuclear microsatellites and the S-locus. The S-locus revealed a higher diversity (15 alleles) than the eight microsatellites (4-12 alleles). Although the frequency distribution of S-alleles differed significantly from the expected equal distribution, the S-locus showed a higher evenness than the microsatellites (Shannon's evenness index for the S-locus: E = 0.91; for the microsatellites: E = 0.48-0.83). Also, highly significant deviations from neutrality were found for the S-locus whereas only minor deviations were found for two of eight microsatellites. A comparison of the frequency distribution of S-alleles in three age-cohorts revealed no significant differences, suggesting that different levels of selection acting on the S-locus or on S-linked sites might also affect the distribution and dynamics of S-alleles. Autocorrelation analysis revealed a weak but significant spatial genetic structure for the multilocus average of the microsatellites and for the S-locus, but could not ascertain differences in the extent of spatial genetic structure between these locus types. An indirect estimate of gene dispersal, which was obtained to explain this spatial genetic pattern, indicated high levels of gene dispersal within our population (sigma(g) = 106 m). This high gene dispersal, which may be partly due to the self-incompatibility system itself, aids the effective gene flow of the microsatellites, thereby decreasing the contrast between the neutral microsatellites and the S-locus.     

Transmission ratio distortion in Arabidopsis lyrata: effects of population divergence and the S-locus

We investigated transmission ratio distortion within an Icelandic population of Arabidopsis lyrata using 16 molecular markers unlinked to the S-locus. Transmission ratio distortion was found more often than expected by chance at the gametic level, but not at the genotypic or zygotic level. The gametic effect may be due to meiotic drive or selection acting postmeiotically. At the gametic level, 10.9% of the tests were significant, which is substantially lower than earlier observed in an interpopulation cross (allowing for differences in power)-suggesting that the high level of transmission ratio distortion in the interpopulation cross is due to population divergence. It is also substantially lower than previously observed in intrapopulation crosses at the self-incompatibility locus, suggesting inherent fitness differences of the self-incompatibility alleles. We discuss the possible role of deleterious alleles accumulating at loci under balancing selection. Zygotic effects play a larger role in the interpopulation cross than in the intrapopulation crosses suggesting that Dobzhansky-Muller incompatibilities may be accumulating between the widely diverged populations.     

Molecular mechanisms underlying the breakdown of gametophytic self-incompatibility

The breakdown of self-incompatibility has occurred repeatedly throughout the evolution of flowering plants and has profound impacts on the genetic structure of populations. Recent advances in understanding of the molecular basis of self-incompatibility have provided insights into the mechanisms of its loss in natural populations, especially in the tomato family, the Solanaceae. In the Solanaceae, the gene that controls self-incompatibility in the style codes for a ribonuclease that causes the degradation of RNA in pollen tubes bearing an allele at the S-locus that matches either of the two alleles held by the maternal plant. The pollen component of the S-locus has yet to be identified. Loss of self-incompatibility can be attributed to three types of causes: duplication of the S-locus, mutations that cause loss of S-RNase activity, and mutations that do not cause loss of S-RNase activity. Duplication of the S-locus has been well studied in radiation-induced mutants but may be a relatively rare cause of the breakdown of self-incompatibility in nature. Point mutations within the S-locus that disrupt the production of S-RNase have been documented in natural populations. There are also a number of mutants in which S-RNase production is unimpaired, yet self-incompatibility is disrupted. The identity and function of these mutations is not well understood. Careful work on a handful of model organisms will enable population biologists to better understand the breakdown of self-incompatibility in nature.     

Sheltered load associated with S-alleles in Solanum carolinense

Bud pollinations allowed me to examine the effects of homozygosity at loci in the area of suppressed recombination around the S-locus in Solanum carolinense, whose S-alleles show surprisingly low diversification rates. The total number of seeds produced was lower for incompatible than compatible pollinations, revealing that self-incompatibility was only somewhat overcome by bud pollination. However, low seed set in incompatible crosses was not due solely to the incompatibility response; crosses between distinct plants sharing the same alleles at the S-locus led to dramatically high seed abortion, nearly equal to that found upon selfing. An excess of heterozygotes in the surviving progeny supports the supposition that these high abortion rates are due to sheltered load, that is, previously unexpressed load accumulated due to enforced heterozygosity and recombination suppression around the S-locus. Of the seven alleles examined in total, two showed evidence of severe load and five did not. The magnitude of load was consistent with terminal branch length in some, but not all, cases.     

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.     

DOES FREQUENCY‐DEPENDENT SELECTION WITH COMPLEX DOMINANCE INTERACTIONS ACCURATELY PREDICT ALLELIC FREQUENCIES AT THE SELF‐INCOMPATIBILITY LOCUS IN ARABIDOPSIS HALLERI?

Frequency-dependent selection is a major force determining the evolutionary dynamics of alleles at the self-incompatibility locus (S-locus) in flowering plants. We introduce a general method using numerical simulations to test several alternative models of frequency-dependent selection on S-locus data from sporophytic systems, taking into account both genetic drift and observed patterns of dominance interactions among S-locus haplotypes (S-haplotypes). Using a molecular typing method, we estimated S-haplotype frequencies in a sample of 322 adult plants and of 245 offspring obtained from seeds sampled on 22 maternal plants, collected in a single population of Arabidopsis halleri (Brassicaceae). We found eight different S-haplotypes and characterized their dominance interactions by controlled pollinations. We then compared the likelihood of different models of frequency-dependent selection: we found that the observed haplotype frequencies and observed frequency changes in one generation best fitted a model with (1) the observed dominance interactions and (2) no pollen limitation. Overall, our population genetic models of frequency-dependent selection, including patterns of dominance interactions among S-haplotypes and genetic drift, can reliably predict polymorphism at the S-locus. We discuss how these approaches allow detecting additional processes influencing the evolutionary dynamics of the S-locus, such as purifying selection on linked loci.     

Learning from rejection: the evolutionary biology of single-locus incompatibility

The self-incompatibility (S-) locus of flowering plants is among the most polymorphic known. PCR methods can now be used to estimate both the number of alleles in natural populations and their sequence diversity. The number of alleles provides an estimate of recent effective population size, thus the S-locus provides a tool for examining how species characteristics affect population size. Sequence relationships among alleles provide another estimate of population size extending millions of years into the past. Relationships between S-alleles and related genes provide a means of dating the age of origin of incompatibility systems and determining which, if any, angiosperm families share incompatibility by homology.     

The inheritance of modifiers conferring self-fertility in the partially self-incompatible perennial, Campanula rapunculoides L. (Campanulaceae)

The role of partial self-incompatibility in plant breeding system evolution has received little attention. Here, we examine the genetic basis of modifiers conferring self-fertility in the creeping bellflower, Campanula rapunculoides L. (Campanulaceae), a partially self-incompatible herb. A survey of 35 individuals from two natural populations indicates that 45% of them are strongly self-incompatible, 40% intermediately self-incompatible, and 15% weakly self-incompatible and that some plants show a strong breakdown in self-incompatibility over floral age. We generated 101 F1 families by random crossing among 31 parental plants and estimated the heritability of self-fertility in day 1 and day 4 female-phase flowers, the genetic correlation between day 1 and day 4 self-fertility, and the coefficient of additive genetic variance of self-fertility. We use linear regression and data from additional crosses to examine whether there are significant maternal effects in the expression of self-fertility. We use Fain's test to determine if a major gene influences self-fertility and, finding no evidence, use data from additional crosses on an F2 generation to estimate the mean number and dominance of genes conferring self-fertility. These analyses indicate that the heritability (h2) of self-fertility is 0.24 in day 1 female-phase flowers and 0.44 in day 4 flowers, self-fertility is primarily additive but shows some recessive effects, and self-fertility is estimated to be controlled by four genetic factors. In addition, we have evidence that there may be maternal effects for self-fertility, especially for weakly self-incompatible plants. The significance of these results in the context of mating system evolution is discussed.     

Identification of an S-locus glycoprotein allele introgressed from B. napus ssp. rapifera to B. napus ssp. oleifera

Self-incompatible Brassica napus ssp. oleifera lines were generated by introgressing the S-locus from the self-incompatible B. napus ssp. rapifera Z line into the self-compatible cultivars, Topas and Regent, resulting in T2 and R2, respectively. Screening of a cDNA library made from R2 stigma RNA produced several candidate SLG (S-locus glycoprotein) cDNAs. One of the cDNAs, A14, was found to be represented in only the R2, T2 and Z lines. In addition, the corresponding A14 gene was demonstrated to segregate with the T2 self-incompatibility phenotype in an F2 population derived from a cross between T2 and Topas, and to exhibit high mRNA levels in the stigmas prior to anthesis. Sequence analysis of the A14 cDNA revealed close homology to B. oleracea SLG alleles associated with a Class I high activity self-incompatibility phenotype.     

COMPUTER PROGRAMS: nessi: a program for numerical estimations in sporophytic self-incompatibility genetic systems

nessi is a computer program generating predictions about allelic and genotypic frequencies at the S-locus in sporophytic self-incompatibility systems under finite and infinite populations. For any pattern of dominance relationships among self-incompatibility alleles, nessi computes deterministic equilibrium frequencies and estimates distributions in samples from finite populations of the number of alleles at equilibrium, allelic and genotypic frequencies at equilibrium and allelic and genotypic frequency changes in a single generation. These predictions can be used to rigorously test the impact of negative frequency-dependent selection on diversity patterns in natural populations.     

A 15-Myr-old genetic bottleneck

Balancing selection preserves variation at the self-incompatibility locus (S-locus) of flowering plants for tens of millions of years, making it possible to detect demographic events that occurred prior to the origin of extant species. In contrast to other Solanaceae examined, SI species in the sister genera Physalis and Witheringia share restricted variation at the S-locus. This restriction is indicative of an ancient bottleneck that occurred in a common ancestor. We sequenced 14 S-alleles from the subtribe Iochrominae, a group that is sister to the clade containing Physalis and Witheringia. At least 6 ancient S-allele lineages are represented among these alleles, demonstrating that the Iochrominae taxa do not share the restriction in S-locus diversity. Therefore, the bottleneck occurred after the divergence of the Iochrominae from the lineage leading to the most recent common ancestor of Physalis and Witheringia. Using cpDNA sequences, 3 fossil dates, and a Bayesian-relaxed molecular clock approach, the crown group of Solanaceae was estimated to be 51 Myr old and the restriction of variation at the S-locus occurred 14.0-18.4 Myr before present. These results confirm the great age of polymorphism at the S-locus and the utility of loci under balancing selection for deep historical inference.     

Loss of gametophytic self-incompatibility with evolution of inbreeding depression

Gametophytic self-incompatibility (SI) in plants is a widespread mechanism preventing self-fertilization and the ensuing inbreeding depression, but it often evolves to self-compatibility. We analyze genetic mechanisms for the breakdown of gametophytic SI, incorporating a dynamic model for the evolution of inbreeding depression allowing for partial purging of nearly recessive lethal mutations by selfing, and accounting for pollen limitation and sheltered load linked to the S-locus. We consider two mechanisms for the breakdown of gametophytic SI: a nonfunctional S-allele and an unlinked modifier locus that inactivates the S-locus. We show that, under a wide range of conditions, self-compatible alleles can invade a self-incompatible population. Conditions for invasion are always less stringent for a nonfunctional S-allele than for a modifier locus. The spread of self-compatible genotypes is favored by extremely high or low selfing rates, a small number of S-alleles, and pollen limitation. Observed parameter values suggest that the maintenance of gametophytic SI is caused by a combination of high inbreeding depression in self-incompatible populations coupled with intermediate selfing rates of the self-compatible genotypes and sheltered load linked to the S-locus.     

Recognition specificity of self-incompatibility maintained after the divergence of Brassica oleracea and Brassica rapa

The determinants of recognition specificity of self-incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen, respectively. In the pair of S haplotypes BrS46 (S46 in B. rapa) and BoS7 (S7 in B. oleracea), which have highly similar SRK alleles, the SP11 alleles were found to be similar, with 96.1% identity in the deduced amino acid sequence. Two other pairs of S haplotypes, BrS47 and BoS12, and BrS8 and BoS32, having highly similar SRK and SP11 alleles between the two species were also found. The haplotypes in each pair are considered to have been derived from a single S haplotype in the ancestral species. The allotetraploid produced by interspecific hybridization between homozygotes of BrS46 and BoS15 showed incompatibility with a BoS7 homozygote and compatibility with other B. oleracea S haplotypes in reciprocal crossings. This result indicates that BrS46 and BoS7 have maintained the same recognition specificity after the divergence of the two species and that amino acid substitutions found in such cases in both SRK alleles and SP11 alleles do not alter the recognition specificity. DNA blot analysis of SRK, SP11, SLG and other S-locus genes showed different DNA fragment sizes between the interspecific pairs of S haplotypes. A much lower level of sequence similarity was observed outside the genes of SRK and SP11 between BrS46 and BoS7. These results suggest that the DNA sequences of the regions intervening between the S-locus genes were diversified after or at the time of speciation. This is the first report demonstrating the presence of common S haplotypes in different plant species and presenting definite evidence of the trans-specific evolution of self-incompatibility genes.     

Breakdown of gametophytic self-incompatibility in subdivided populations

In many hermaphroditic flowering plants, self-fertilization is prevented by self-incompatibility (SI), often controlled by a single locus, the S-locus. In single isolated populations, the maintenance of SI depends chiefly on inbreeding depression and the number of SI alleles at the S-locus. In subdivided populations, however, population subdivision has complicated effects on both the number of SI alleles and the level of inbreeding depression, rendering the maintenance of SI difficult to predict. Here, we explore the conditions for the invasion of a self-compatible mutant in a structured population. We find that the maintenance of SI is strongly compromised when a population becomes subdivided. We show that this effect is mainly caused by the decrease in the local diversity of SI alleles rather than by a change in the dynamics of inbreeding depression. Strikingly, we also find that the diversity of SI alleles at the whole population level is a poor predictor of the maintenance of SI. We discuss the implications of our results for the interpretation of empirical data on the loss of SI in natural populations.     

Functional gametophytic self-incompatibility in a peripheral population of Solanum peruvianum (Solanaceae)

The transition from self-incompatibility to self-compatibility is a common transition in angiosperms often reported in populations at the edge of species range limits. Geographically distinct populations of wild tomato species (Solanum section Lycopersicon (Solanaceae)) have been described as polymorphic for mating system with both self-incompatible and self-compatible populations. Using controlled pollinations and sequencing of the S-RNase mating system gene, we test the compatibility status of a population of S. peruvianum located near its southern range limit. Pollinations among plants of known genotypes revealed strong self-incompatibility; fruit set following compatible pollinations was significantly higher than following incompatible pollinations for all tested individuals. Sequencing of the S-RNase gene in parents and progeny arrays was also as predicted under self-incompatibility. Molecular variation at the S-RNase locus revealed a diverse set of alleles, and heterozygosity in over 500 genotyped individuals. We used controlled crosses to test the specificity of sequences recovered in this study; in all cases, results were consistent with a unique allelic specificity for each tested sequence, including two alleles sharing 92% amino-acid similarity. Site-specific patterns of selection at the S-RNase gene indicate positive selection in regions of the gene associated with allelic specificity determination and purifying selection in previously characterized conserved regions. Further, there is broad convergence between the present and previous studies in specific amino-acid positions inferred to be evolving under positive selection.     

MATE AVAILABILITY AND FECUNDITY SELECTION IN MULTI-ALLELIC SELF-INCOMPATIBILITY SYSTEMS IN PLANTS

We investigate mate availability in different models of multiallelic self-incompatibility systems in mutation-selection-drift balance in finite populations. Substantial differences among self-incompatibility systems occur in average mate availability, and in variances of mate availability among individual plants. These differences are most pronounced in small populations in which low mate availability may reduce seed set in some types of sporophytic self-incompatibility. In cases where the pollination system causes a restriction in the number of pollen genotypes available to an individual plant, the fecundity of that plant depends on the availability of compatible pollen, which is determined by its genotype at the incompatibility locus. This leads to an additional component of selection acting on self-incompatibility systems, which we term “fecundity selection.” Fecundity selection increases the number of alleles maintained in finite populations and increases mate availability in small populations. The strength of fecundity selection is dependent on the type of self-incompatibility. In some cases, fecundity selection markedly alters the equilibrium dynamics of self-incompatibility alleles. We discuss the population genetic consequences of mate availability and fecundity selection in the contexts of conservation management of self-incompatible plant species and experimental investigations on self-incompatibility in natural populations.