Molecular cloning of two Solanum chacoense S-alleles and a hypothesis concerning their evolution

The polymerase chain reaction (PCR) has been used to clone two S-alleles (S13 and S14) from Solanum chacoense. The two alleles do not cross-hybridize on genomic Southern blots or on northern blots using stylar RNA. Although the S14 message was not detected in a stylar cDNA library prepared from mature flowers, a full-length copy of the S13 coding sequence was isolated by screening with the PCR fragment. We have analysed the sequences of the S13 cDNA and the S14 PCR fragment (60% of the mature protein coding sequence) in the context of S-RNase evolution, and propose that random point mutations may be sufficient to generate new S-alleles. Based on a phylogenetic tree composed of RNase sequences containing the conserved RNase motifs HGLWP and KHGXC, we suggest that gametophytic self-incompatibility genes are RNase genes that have acquired a new function in the gametophytic self-incompatibility system early in the evolution of flowering plants.     

Re-examination of the self-incompatibility genotype of apple cultivars containing putative ’new’ S-alleles

Recently, the self-incompatibility (S-) genotypes of 56 apple cultivars were examined by protein analysis, which led to the identification by Boskovic and Tobutt of 14 putative ’new’ S-alleles, S12 to S25. This paper reports a re-examination of the S-genotypes of some of these cultivars through S-allele ’specific’ PCR and sequence analysis. The results obtained by this analysis indicated that the number of S-alleles that are present in apple is probably smaller than the number proposed by Boskovic and Tobutt. The existence of three ’new’ S-alleles (S20, S22 and S24) was confirmed. The existence of two other putative ’new’ S-alleles (S23 and S25) was, however, contradicted. The coding sequences of the S-alleles that correspond to the S10 and the S25 ribonuclease bands as well as those corresponding to the S22 and the S23 ribonuclease bands were shown to be identical in sequence. Interestingly, the S-allele corresponding to the S22 and the S23 ribonuclease bands shared a high sequence identity (99% identity) with S27, which was previously cloned and sequenced from Baskatong, but which was not included in the analysis conducted by Boskovic and Tobutt. Both S-alleles only differ in point mutations, which are not translated into differences in amino-acid sequence. To our knowledge, this is the first report of two S-alleles that differ at the nucleotide level but still encode for identical S-RNases. The implications of these observations for determining the S-genotypes of plants by PCR analysis or protein analysis are discussed. Received: 10 January 2001 / Accepted: 19 January 2001     

Phylogeny of Leavenworthia S-alleles suggests unidirectional mating system evolution and enhanced positive selection following an ancient population bottleneck

The adoption of self-fertilization from an ancestral outcrossing state is one of the most common evolutionary transitions in the flowering plants. In the mustard family, outcrossing is typically enforced by sporophytic self-incompatibility (SI), but there are also many self-compatible species. The genus Leavenworthia contains taxa that either possess or lack SI. Here, we present data showing that SI is associated with strict outcrossing and that there is widespread trans-specific sequence polymorphism at the locus involved in the recognition of self-pollen (the S-locus). This ancestral polymorphism is consistent with the presence of an outcrossing mating system in the common ancestor of Leavenworthia species, and suggests that there have been several independent losses of SI in the group. When compared with other mustard species, the bulk of Leavenworthia S-allele sequences are highly diverged from those found in other Brassicaceae and show relatively low levels of nucleotide diversity, a pattern that suggests the common ancestor of the genus likely underwent a strong population bottleneck. The hypothesis of postbottleneck S-locus rediversification is supported by tests showing stronger positive selection acting on S-alleles from Leavenworthia than those found in other Brassicaceae.     

A molecular approach to the identification of S-alleles in Brassica oleracea

A molecular technique for the identification of S-alleles involved in self-incompatibility has been used to analyse the S-allele reference collection of Brassica oleracea. The reference collection contains nearly 50 different lines each with a different S-allele present in the homozygous state. The technique consists of amplifying by the polymerase chain reaction (PCR) sequences belonging to the S multigene sequence family using a single pair of conserved primers. PCR products are then analysed further by digestion with six restriction enzymes followed by gel electrophoresis of the digestion products. A simple method of estimating the band sizes of the digestion products is described. The S-locus-related sequences can be distinguished from S-locus glycoprotein and S-receptor kinase genes by the restriction patterns. Furthermore, with any one restriction enzyme, several alleles showed the same restriction pattern. Alleles could therefore be grouped together. With two exceptions, each member of the S-allele reference collection showed a unique set of restriction patterns. Investigation of the exceptions using pollen tube growth tests showed that these accessions represented duplications within the collection. This technique therefore provides a simple and useful method for identifying different S-alleles.     

New findings in apple S-genotype analysis resolve previous confusion and request the re-numbering of some S-alleles

Apple trees display gametophytic self-incompatibility which is controlled by a series of polymorphic S-alleles. To resolve the discrepancies in S-allele assignment that appeared in the literature, we have re-examined the identity of S-alleles known from domestic apple cultivars. Upon an alignment of S-allele nucleotide sequences, we designed allele-specific primer pairs to selectively amplify a single S-allele per reaction. Alternatively, highly similar S-alleles that were co-amplified with the same primer pair were discriminated through their distinct restriction digestion pattern. This is an extension of our previously developed allele-specific PCR amplification approach to reveal the S-genotypes in apple cultivars. Amplification parameters were optimised for the unique detection of the 15 apple S-alleles of which the nucleotide sequences are known. Both the old cultivars with a known S-genotype and a number of more common cultivars were assayed with this method. In most cases, our data coincided with those obtained through phenotypic and S-RNase analysis. However, three S-alleles were shown to relate to RNases that were previously proposed as being encoded by distinct S-alleles. For another S-allele the corresponding gene product has not been discriminated. Consequently, we propose the re-numbering of these four S-alleles. Furthermore, two alleles that were previously identified as S(27a) and S(27b) now received a distinct number, despite their identical S-specificity. To ease widespread future analysis of S-genotypes, we identified common cultivars that may function as a witness for bearing a particular S-allele. We discuss the assignment of new S-alleles which should help to avoid further confusion.     

PD1, an S-like RNase gene from a self-incompatible cultivar of almond

 Many flowering plants contain stylar S-RNases that are involved in self-incompatibility and S-like RNases of which the biological function is uncertain. This paper reports the deduced amino acid sequence of an S-like RNase gene (PD1) from the self-incompatible plant Prunus dulcis (almond). The amino acid sequence of PD1, which was derived from cDNA and genomic DNA clones, showed 34–86% identity to acidic plant S-like RNases reported so far, with the highest degree of similarity being to an S-like RNase from Japanese pear (Pyrus pyrifolia). Based on RNA hybridisation experiments it appears that, like for many other S-like RNases, the expression of PD1 is not pistil-specific. Analysis of the genomic structure revealed the presence of three introns, of which one is similar in location to that of the related S-RNase gene from Solanaceae and Rosaceae. At least four bands hybridising to PD1 were found upon Southern hybridisation, suggesting the presence of a multigene family of S-like RNase genes in almond. The putative biological function of PD1 is discussed. Received: 22 November 1999 / Revision received: 18 February 2000 · Accepted: 13 March 2000     

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.     

Evolutionary genetics of self-incompatibility in the Solanaceae

The self-incompatibility (S) gene in flowering plants has long been appreciated as an example of extreme allelic polymorphism maintained by frequency-dependent selection. Recent studies of population samples of S-allele sequences obtained by RT-PCR from five species of Solanaceae now reveal a picture of conspicuous inter-specific variation in both S-allele number and age. Explanations for this variation are examined with reference to current theory. We propose that changes in species' effective population size, particularly those associated with the evolution of different life histories, best account for interspecific differences in both the number and average age of S alleles.     

Self-incompatibility (S) alleles of the rosaceae encode members of a distinct class of the T2/S ribonuclease superfamily

Stylar riboncleases (RNases) are associated with gametophytic self-incompatibility in two plant families, the Solanaceae and the Rosaceae. The self-incompatibility-associated RNases (S-RNases) of both the Solanaceae and the Rosaceae were recently reported to belong to the T2 RNase gene family, based on the presence of two well-conserved sequence motifs. Here, the cloning and characterization of S-RNase genes from two species of Rosaceae, apple (Malus × domestica) and Japanese pear (Pyrus serotina) is described and these sequences are compared with those of other T2-type RNases. The S-RNases of apple specifically accumulated in styles following maturation of the flower bud. Two cDNA clones for S-RNases from apple, and PCR clones encoding a further two apple S-RNases as well as two Japanese pear S-RNases were isolated and sequenced. The deduced amino acid sequences of the rosaceous S-RNases contained two conserved regions characteristic of the T2/S-type RNases. The sequences showed a high degree of diversity, with similarities ranging from 60.4% to 69.2%. Interestingly, some interspecific sequence similarities were higher than those within a species, possibly indicating that diversification of S-RNase alleles predated speciation in the Rosaceae. A phylogenetic tree of members of the T2/S-RNase superfamily in plants was obtained. The rosaceous S-RNases formed a new lineage in the tree that was distinct from those of the solanaceous S-RNases and the S-like RNases. The findings suggested that self-incompatibility mechanisms in Rosaceae and Solanaceae are similar but arose independently in the course of evolution.     

Self-incompatibility (S) alleles of the rosaceae encode members of a distinct class of the T2/S ribonuclease superfamily

Stylar riboncleases (RNases) are associated with gametophytic self-incompatibility in two plant families, the Solanaceae and the Rosaceae. The self-incompatibility-associated RNases (S-RNases) of both the Solanaceae and the Rosaceae were recently reported to belong to the T2 RNase gene family, based on the presence of two well-conserved sequence motifs. Here, the cloning and characterization of S-RNase genes from two species of Rosaceae, apple (Malus × domestica) and Japanese pear (Pyrus serotina) is described and these sequences are compared with those of other T2-type RNases. The S-RNases of apple specifically accumulated in styles following maturation of the flower bud. Two cDNA clones for S-RNases from apple, and PCR clones encoding a further two apple S-RNases as well as two Japanese pear S-RNases were isolated and sequenced. The deduced amino acid sequences of the rosaceous S-RNases contained two conserved regions characteristic of the T2/S-type RNases. The sequences showed a high degree of diversity, with similarities ranging from 60.4% to 69.2%. Interestingly, some interspecific sequence similarities were higher than those within a species, possibly indicating that diversification of S-RNase alleles predated speciation in the Rosaceae. A phylogenetic tree of members of the T2/S-RNase superfamily in plants was obtained. The rosaceous S-RNases formed a new lineage in the tree that was distinct from those of the solanaceous S-RNases and the S-like RNases. The findings suggested that self-incompatibility mechanisms in Rosaceae and Solanaceae are similar but arose independently in the course of evolution.     

Transient SI and the dynamics of self-incompatibility alleles: a simulation model and empirical test

A stochastic computer simulation model was created to compare the combined effects of selection and genetic drift on the dynamics of S-alleles under full sporophytic self-incompatibility (SI) versus transient SI, a form of partial SI in which flowers become self-compatible as they age. S-alleles were lost more rapidly with transient than with full SI, as is expected with weakened frequency-dependent selection. Based on these results, equilibrium S-allele diversity is expected to be lower with partial SI for populations of comparable size and migration rates. Consistent with model results, a comparison of the proportion of incompatible crosses in full diallel experiments for a fully SI and a transiently SI species in the annual genus Leptosiphon suggests that S-allele diversity is lower in the partially SI species. Results of the simulation model indicate that the transmission advantage of self-fertilization can have complex effects on S-allele dynamics in partial SI systems.     

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.     

Allelic diversity of S-RNase at the self-incompatibility locus in natural flowering cherry populations (Prunus lannesiana var. speciosa)

In the Rosaceae family, which includes Prunus, gametophytic self-incompatibility (GSI) is controlled by a single multiallelic locus (S-locus), and the S-locus product expressed in the pistils is a glycoprotein with ribonuclease activity (S-RNase). Two populations of flowering cherry (Prunus lannesiana var. speciosa), located on Hachijo Island in Japan's Izu Islands, were sampled, and S-allele diversity was surveyed based on the sequence polymorphism of S-RNase. A total of seven S-alleles were cloned and sequenced. The S-RNases of flowering cherry showed high homology to those of Prunus cultivars (P. avium and P. dulcis). In the phylogenetic tree, the S-RNases of flowering cherry and other Prunus cultivars formed a distinct group, but they did not form species-specific subgroups. The nucleotide substitution pattern in S-RNases of flowering cherry showed no excess of nonsynonymous substitutions relative to synonymous substitutions. However, the S-RNases of flowering cherry had a higher Ka/Ks ratio than those of other Prunus cultivars, and a subtle heterogeneity in the nucleotide substitution rates was observed among the Prunus species. The S-genotype of each individual was determined by Southern blotting of restriction enzyme-digested genomic DNA, using cDNA for S-RNase as a probe. A total of 22 S-alleles were identified. All individuals examined were heterozygous, as expected under GSI. The allele frequencies were, contrary to the expectation under GSI, significantly unequal. The two populations studied showed a high degree of overlap, with 18 shared alleles. However, the allele frequencies differed considerably between the two populations.     

RNase T2 genes from rice and the evolution of secretory ribonucleases in plants

The plant RNase T2 family is divided into two different subfamilies. S-RNases are involved in rejection of self-pollen during the establishment of self-incompatibility in three plant families. S-like RNases, on the other hand, are not involved in self-incompatibility, and although gene expression studies point to a role in plant defense and phosphate recycling, their biological roles are less well understood. Although S-RNases have been subjects of many phylogenetic studies, few have included an extensive analysis of S-like RNases, and genome-wide analyses to determine the number of S-like RNases in fully sequenced plant genomes are missing. We characterized the eight RNase T2 genes present in the Oryza sativa genome; and we also identified the full complement of RNase T2 genes present in other fully sequenced plant genomes. Phylogenetics and gene expression analyses identified two classes among the S-like RNase subfamily. Class I genes show tissue specificity and stress regulation. Inactivation of RNase activity has occurred repeatedly throughout evolution. On the other hand, Class II seems to have conserved more ancestral characteristics; and, unlike other S-like RNases, genes in this class are conserved in all plant species analyzed and most are constitutively expressed. Our results suggest that gene duplication resulted in high diversification of Class I genes. Many of these genes are differentially expressed in response to stress, and we propose that protein characteristics, such as the increase in basic residues can have a defense role independent of RNase activity. On the other hand, constitutive expression and phylogenetic conservation suggest that Class II S-like RNases may have a housekeeping role.     

Repeated adaptive introgression at a gene under multiallelic balancing selection

Recently diverged species typically have incomplete reproductive barriers, allowing introgression of genetic material from one species into the genomic background of the other. The role of natural selection in preventing or promoting introgression remains contentious. Because of genomic co-adaptation, some chromosomal fragments are expected to be selected against in the new background and resist introgression. In contrast, natural selection should favor introgression for alleles at genes evolving under multi-allelic balancing selection, such as the MHC in vertebrates, disease resistance, or self-incompatibility genes in plants. Here, we test the prediction that negative, frequency-dependent selection on alleles at the multi-allelic gene controlling pistil self-incompatibility specificity in two closely related species, Arabidopsis halleri and A. lyrata, caused introgression at this locus at a higher rate than the genomic background. Polymorphism at this gene is largely shared, and we have identified 18 pairs of S-alleles that are only slightly divergent between the two species. For these pairs of S-alleles, divergence at four-fold degenerate sites (K = 0.0193) is about four times lower than the genomic background (K = 0.0743). We demonstrate that this difference cannot be explained by differences in effective population size between the two types of loci. Rather, our data are most consistent with a five-fold increase of introgression rates for S-alleles as compared to the genomic background, making this study the first documented example of adaptive introgression facilitated by balancing selection. We suggest that this process plays an important role in the maintenance of high allelic diversity and divergence at the S-locus in flowering plant families. Because genes under balancing selection are expected to be among the last to stop introgressing, their comparison in closely related species provides a lower-bound estimate of the time since the species stopped forming fertile hybrids, thereby complementing the average portrait of divergence between species provided by genomic data.     

Sequence of an S-protein of Lycopersicon peruvianum and comparison with other solanaceous S-proteins

Summary cDNA clones for an S-allele, designated S₅, of the self-incompatibility locus (S-locus) of Lycopersicon peruvianum have been isolated by probing a pistil cDNA library with cDNAs for S-alleles of Petunia inflata and Solanum chacoense. The longest S₅-cDNA is 869 bp and contains an open reading frame of 217 amino acids. An alignment of the deduced amino acid sequence of S₅-protein with that of the 18 S-proteins from five other solanaceous species is presented. Sequence comparison further refines the primary structural features of the S-proteins previously revealed from comparison of subsets of these sequences. Based on this comparison and evidence presented elsewhere, it is proposed that accumulation of point mutations, and not intragenic recombination, is responsible for the generation of new allelic specificities.     

S16, a novel S-RNase allele in the diploid species Solanum chacoense.

Wild potato species have a gametophytic self-incompatibility system controlled by a single multiallelic S locus. In the style, the S-RNase gene codes for an allele-specific ribonuclease that is involved in the rejection of pollen that carries the same S haplotype. This gene has 5 conserved regions (C1-C5) and highly variable regions outside of these areas that play a role in S-RNase allele specificity. In this work, PCR-mediated amplification of genomic DNA from 2 Solanum chacoense accessions was performed using primers designed on the basis of the C1 and C4 conserved regions. By sequencing the PCR products, a new S-RNase allele (S16) was identified in 1 plant of the QBCM argentinian accession. Comparison of the partial sequence (from C2 to C3) of S16 RNase with those of 11 S-RNase genes of other Solanaceae species showed the highest and the lowest similarity scores within the same plant species (respectively, 71% with the S11 and S13 RNase and 35% with the S2 RNase). Differences at the nucleotide level between S16 and S11 RNase alleles are discussed.     

S-allele diversity in Sorbus aucuparia and Crataegus monogyna (Rosaceae: Maloideae)

RT-PCR was used to obtain the first estimates from natural populations of allelic diversity at the RNase-based gametophytic self-incompatibility locus in the Rosaceae. A total of 20 alleles were retrieved from 20 Sorbus aucuparia individuals, whereas 17 alleles were found in 13 Crataegus monogyna samples. Estimates of population-level allele numbers fall within the range observed in the Solanaceae, the only other family with RNase-based incompatibility for which estimates are available. The nucleotide diversity of S-allele sequences was found to be much lower in the two Rosaceae species as compared with the Solanaceae. This was not due to a lower sequence divergence among most closely related alleles. Rather, it is the depth of the entire genealogy that differs markedly in the two families, with Rosaceae S-alleles exhibiting more recent apparent coalescence. We also investigated patterns of selection at the molecular level by comparing nucleotide diversity at synonymous and nonsynonymous sites. Stabilizing selection was inferred for the 5' region of the molecule, while evidence of diversifying selection was present elsewhere.