S locus genes and the evolution of self-fertility in Arabidopsis thaliana

Loss of self-incompatibility (SI) in Arabidopsis thaliana was accompanied by inactivation of genes required for SI, including S-LOCUS RECEPTOR KINASE (SRK) and S-LOCUS CYSTEINE-RICH PROTEIN (SCR), coadapted genes that constitute the SI specificity-determining S haplotype. Arabidopsis accessions are polymorphic for PsiSRK and PsiSCR, but it is unknown if the species harbors structurally different S haplotypes, either representing relics of ancestral functional and structurally heteromorphic S haplotypes or resulting from decay concomitant with or subsequent to the switch to self-fertility. We cloned and sequenced the S haplotype from C24, in which self-fertility is due solely to S locus inactivation, and show that this haplotype was produced by interhaplotypic recombination. The highly divergent organization and sequence of the C24 and Columbia-0 (Col-0) S haplotypes demonstrate that the A. thaliana S locus underwent extensive structural remodeling in conjunction with a relaxation of selective pressures that once preserved the integrity and linkage of coadapted SRK and SCR alleles. Additional evidence for this process was obtained by assaying 70 accessions for the presence of C24- or Col-0-specific sequences. Furthermore, analysis of SRK and SCR polymorphisms in these accessions argues against the occurrence of a selective sweep of a particular allele of SCR, as previously proposed.     

Inbreeding depression in self-incompatible North-American Arabidopsis lyrata: disentangling genomic and S-locus-specific genetic load

Newly formed selfing lineages may express recessive genetic load and suffer inbreeding depression. This can have a genome-wide genetic basis, or be due to loci linked to genes under balancing selection. Understanding the genetic architecture of inbreeding depression is important in the context of the maintenance of self-incompatibility and understanding the evolutionary dynamics of S-alleles. We addressed this using North-American subspecies of Arabidopsis lyrata. This species is normally self-incompatible and outcrossing, but some populations have undergone a transition to selfing. The goals of this study were to: (1) quantify the strength of inbreeding depression in North-American populations of A. lyrata; and (2) disentangle the relative contribution of S-linked genetic load compared with overall inbreeding depression. We enforced selfing in self-incompatible plants with known S-locus genotype by treatment with CO₂, and compared the performance of selfed vs outcrossed progeny. We found significant inbreeding depression for germination rate (δ=0.33), survival rate to 4 weeks (δ=0.45) and early growth (δ=0.07), but not for flowering rate. For two out of four S-alleles in our design, we detected significant S-linked load reflected by an under-representation of S-locus homozygotes in selfed progeny. The presence or absence of S-linked load could not be explained by the dominance level of S-alleles. Instead, the random nature of the mutation process may explain differences in the recessive deleterious load among lineages.     

Robust Self-Incompatibility in the Absence of a Functional ARC1 Gene in Arabidopsis thaliana

Self-incompatibility (SI) is the primary determinant of the outbreeding mode of sexual reproduction in the Brassicaceae. All Arabidopsis thaliana accessions analyzed to date carry mutations that disrupt SI functions by inactivating the SI specificity-determining S locus or SI modifier loci. S-locus genes isolated from self-incompatible close relatives of A. thaliana restore robust SI in several accessions that harbor only S-locus mutations and confer transient SI in accessions that additionally harbor mutations at modifier loci. Self-incompatible transgenic A. thaliana plants have proved to be valuable for analysis of the recognition and signaling events that underlie SI in the Brassicaceae. Here, we review results demonstrating that S-locus genes are necessary and sufficient for SI signaling and for restoration of a strong and developmentally stable SI phenotype in several accessions of A. thaliana. The data indicate that introduction of a functional E3 ligase-encoding ARC1 gene, which is deleted in all accessions that have been analyzed to date, is not required for SI signaling leading to inhibition of self pollen or for reversion of A. thaliana to its fully self-incompatible ancestral state.It is well established that specific pollen recognition in the self-incompatibility (SI) response of the Brassicaceae is determined by allele-specific interactions that occur at the stigma surface between two highly polymorphic proteins encoded in the S locus: the S-locus receptor kinase SRK and its ligand, the S-locus cysteine-rich protein SCR. Arabidopsis thaliana lacks a functional SI system and harbors nonfunctional S-locus variants that contain defective alleles of the SRK and/or SCR genes (Kusaba et al., 2001; Sherman-Broyles et al., 2007; Tang et al., 2007; Shimizu et al., 2008; Boggs et al., 2009a; Tsuchimatsu et al., 2010; Dwyer et al., 2013). Despite being highly self-fertile, A. thaliana can be made to express SI upon transformation with functional SRK-SCR gene pairs isolated from its self-incompatible close relatives (Nasrallah et al., 2002, 2004; Boggs et al., 2009a, 2009b). The first transfer of the SI trait into A. thaliana was achieved using the SRKb-SCRb gene pair isolated from the Sb locus of Arabidopsis lyrata (Kusaba et al., 2001; Nasrallah et al., 2002, 2004). Many of the subsequent studies that have been performed in the transgenic A. thaliana SRK-SCR system have used plants transformed with p548, a plasmid that we constructed by inserting the A. lyrata SRKb and SCRb genes with their 5′ and 3′ regulatory sequences into the pBIN+ binary vector (Nasrallah et al., 2004).Indriolo et al. (2014) recently used the p548 plasmid to generate SRKb-SCRb transformants and test the role of the ARM Repeat Containing1 (ARC1) gene in SI. ARC1 was originally identified as a Brassica napus protein that interacts with the SRK kinase domain in yeast (Gu et al., 1998), and it was subsequently inferred to be required for SI because downregulation of the ARC1 gene in B. napus (Stone et al., 1999) and A. lyrata (Indriolo et al., 2012), as well as overexpression of ARC1’s target, Exo70A1, in B. napus (Samuel et al., 2009), caused partial breakdown of the SI response. However, the involvement of the proposed SRK-ARC1-Exo70A1 pathway in SI has been questioned because the ARC1 gene was found to be deleted in all A. thaliana accessions analyzed to date (Kitashiba et al., 2011; Indriolo et al., 2012), including those in which the SRKb-SCRb transgenes confer a strong SI phenotype (Kitashiba et al., 2011). Additionally, overexpression of Exo70A1 did not cause weakening of the SI response in A. thaliana SRKb-SCRb plants (Kitashiba et al., 2011).Indriolo et al. (2014) reported on their characterization of the SI response in plants of the Sha and Columbia-0 (Col-0) accessions, which they either transformed with the p548 plasmid alone or cotransformed with p548 and a plasmid containing an ARC1 gene isolated from A. lyrata or B. napus. They concluded that, along with SRK and SCR, “ARC1 is the third component that is required to return A. thaliana to its ancestral self-incompatibility state.” However, this conclusion is inconsistent with results of previous studies of SI in transgenic A. thaliana SRK-SCR transformants, which have shown that several A. thaliana accessions are rendered fully self-incompatible by transformation with the p548 plasmid without the addition of a functional ARC1 gene. Contrary to Indriolo et al.’s assertion that in previous studies of A. thaliana SRK-SCR transformants, “the self-pollen rejection response was incomplete,” we reported that among 11 A. thaliana accessions tested by transformation with the p548 plasmid, five accessions (C24, Cvi-0, Hodja, Kas-2, and Sha) were converted to full SI by expression of the SRKb and SCRb genes alone (Nasrallah et al., 2004; Boggs et al., 2009a). Importantly, the SI phenotype of these self-incompatible SRKb-SCRb transformants faithfully recapitulates the SI phenotype of naturally self-incompatible Brassicaceae with respect to the four defining features of SI in this family: (1) site of pollen inhibition at the stigma surface, (2) intensity of the response, (3) developmental regulation over the course of stigma maturation, and (4) heritability. These features suggest that the inhibition of self pollen in self-incompatible A. thaliana SRK-SCR transformants is achieved via the same signaling pathway as that utilized by other self-incompatible Brassicaceae species.     

Molecular Characterization and Evolution of Self-Incompatibility Genes in Arabidopsis thaliana: The Case of the Sc Haplotype

The switch from an outcrossing mode of mating enforced by self-incompatibility to self-fertility in the Arabidopsis thaliana lineage was associated with mutations that inactivated one or both of the two genes that comprise the self-incompatibility (SI) specificity-determining S-locus haplotype, the S-locus receptor kinase (SRK) and the S-locus cysteine-rich (SCR) genes, as well as unlinked modifier loci required for SI. All analyzed A. thaliana S-locus haplotypes belong to the SA, SB, or SC haplotypic groups. Of these three, the SC haplotype is the least well characterized. Its SRKC gene can encode a complete open-reading frame, although no functional data are available, while its SCRC sequences have not been isolated. As a result, it is not known what mutations were associated with inactivation of this haplotype. Here, we report on our analysis of the Lz-0 accession and the characterization of its highly rearranged SC haplotype. We describe the isolation of its SCRC gene as well as the subsequent isolation of SCRC sequences from other SC-containing accessions and from the A. lyrata S36 haplotype, which is the functional equivalent of the A. thaliana SC haplotype. By performing transformation experiments using chimeric SRK and SCR genes constructed with SC- and S36-derived sequences, we show that the SRKC and SCRC genes of Lz-0 and at least a few other SC-containing accessions are nonfunctional, despite SCRC encoding a functional full-length protein. We identify the probable mutations that caused the inactivation of these genes and discuss our results in the context of mechanisms of S-locus inactivation in A. thaliana.     

Single gene control of postzygotic self-incompatibility in poke milkweed, Asclepias exaltata L

Most individuals of Asclepias exaltata are self-sterile, but all plants lack prezygotic barriers to self-fertilization. To determine whether postzygotic rejection of self-fertilized ovules is due to late-acting self-incompatibility or to extreme, early acting inbreeding depression, we performed three diallel crosses among self-sterile plants related as full-sibs. The full-sibs segregated into four compatibility classes, suggesting that late acting self-incompatibility is controlled by a single gene (S-locus). Crosses between plants sharing one or both alleles at the S-locus are incompatible. An additional diallel cross was done among full-sib progeny from a cross of a self-sterile and a self-fertile plant. These progeny grouped into two compatibility classes, and plants within classes displayed varying levels of self-fertility. This suggests that the occasional self-fertility documented in natural pollinations is caused by pseudo-self-fertility alleles that alter the functioning of the S-locus.     

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.     

Diverse phosphate and auxin transport loci distinguish phosphate tolerant from sensitive Arabidopsis accessions

Phosphorus (P) is an essential element for plant growth often limiting agroecosystems. To identify genetic determinants of performance under variable phosphate (Pi) supply, we conducted genome-wide association studies on five highly predictive Pi starvation response traits in 200 Arabidopsis (Arabidopsis thaliana) accessions. Pi concentration in Pi-limited organs had the strongest, and primary root length had the weakest genetic component. Of 70 trait-associated candidate genes, 17 responded to Pi withdrawal. The PHOSPHATE TRANSPORTER1 gene cluster on chromosome 5 comprises PHT1;1, PHT1;2, and PHT1;3 with known impact on P status. A second locus featured uncharacterized endomembrane-associated auxin efflux carrier encoding PIN-LIKES7 (PILS7) which was more strongly suppressed in Pi-limited roots of Pi-starvation sensitive accessions. In the Col-0 background, Pi uptake and organ growth were impaired in both Pi-limited pht1;1 and two pils7 T-DNA insertion mutants, while Pi -limited pht1;2 had higher biomass and pht1;3 was indistinguishable from wild-type. Copy number variation at the PHT1 locus with loss of the PHT1;3 gene and smaller scale deletions in PHT1;1 and PHT1;2 predicted to alter both protein structure and function suggest diversification of PHT1 is a key driver for adaptation to P limitation. Haplogroup analysis revealed a phosphorylation site in the protein encoded by the PILS7 allele from stress-sensitive accessions as well as additional auxin-responsive elements in the promoter of the “stress tolerant” allele. The former allele’s inability to complement the pils7-1 mutant in the Col-0 background implies the presence of a kinase signaling loop controlling PILS7 activity in accessions from P-rich environments, while survival in P-poor environments requires fine-tuning of stress-responsive root auxin signaling.

A series of insertion/deletion nucleotide polymorphisms at PHOSPHATE TRANSPORTER1 and PIN-LIKES7 loci confer natural variation in low phosphate tolerance in 200 Arabidopsis accessions.     

Arabidopsis sos1 mutant in a salt-tolerant accession revealed an importance of salt acclimation ability in plant salt tolerance

An analysis of the salinity tolerance of 354 Arabidopsis thaliana accessions showed that some accessions were more tolerant to salt shock than the reference accession, Col-0, when transferred from 0 to 225 mM NaCl. In addition, several accessions, including Zu-0, showed marked acquired salt tolerance after exposure to moderate salt stress. It is likely therefore that Arabidopsis plants have at least two types of tolerance, salt shock tolerance and acquired salt tolerance. To evaluate a role of well-known salt shock tolerant gene SOS1 in acquired salt tolerance, we isolated a sos1 mutant from ion-beam-mutagenized Zu-0 seedlings. The mutant showed severe growth inhibition under salt shock stress owing to a single base deletion in the SOS1 gene and was even more salt sensitive than Col-0. Nevertheless, it was able to survive after acclimation on 100 mM NaCl for 7 d followed by 750 mM sorbitol for 20 d, whereas Col-0 became chlorotic under the same conditions. We propose that genes for salt acclimation ability are different from genes for salt shock tolerance and play an important role in the acquisition of salt or osmotic tolerance.     

Genetic Architecture of Natural Variation of Telomere Length in Arabidopsis thaliana

Telomeres represent the repetitive sequences that cap chromosome ends and are essential for their protection. Telomere length is known to be highly heritable and is derived from a homeostatic balance between telomeric lengthening and shortening activities. Specific loci that form the genetic framework underlying telomere length homeostasis, however, are not well understood. To investigate the extent of natural variation of telomere length in Arabidopsis thaliana, we examined 229 worldwide accessions by terminal restriction fragment analysis. The results showed a wide range of telomere lengths that are specific to individual accessions. To identify loci that are responsible for this variation, we adopted a quantitative trait loci (QTL) mapping approach with multiple recombinant inbred line (RIL) populations. A doubled haploid RIL population was first produced using centromere-mediated genome elimination between accessions with long (Pro-0) and intermediate (Col-0) telomere lengths. Composite interval mapping analysis of this population along with two established RIL populations (Ler-2/Cvi-0 and Est-1/Col-0) revealed a number of shared and unique QTL. QTL detected in the Ler-2/Cvi-0 population were examined using near isogenic lines that confirmed causative regions on chromosomes 1 and 2. In conclusion, this work describes the extent of natural variation of telomere length in A. thaliana, identifies a network of QTL that influence telomere length homeostasis, examines telomere length dynamics in plants with hybrid backgrounds, and shows the effects of two identified regions on telomere length regulation.     

On the Evolution of Genetic Incompatibility Systems. VI. a Three-Locus Modifier Model for the Origin of Gametophytic Self-Incompatibility

Recent genetic analyses have demonstrated that self-incompatibility in flowering plants derives from the coordinated expression of a system of loci. To address the selective mechanisms through which a genetic system of this kind evolves, I present a three-locus model for the origin of gametophytic self-incompatibility. Conventional models assume that a single locus encodes all physiological effects associated with self-incompatibility and that the viability of offspring depends only on whether they were derived by selfing or outcrossing. My model explicitly represents the genetic determination of offspring viability by a locus subject to symmetrically overdominant selection. Initially, the level of expression of the proto-S locus is insufficient to induce self-incompatibility. Weak gametophytic self-incompatibility arises upon the introduction of a rare allele at an unlinked modifier locus which enhances the expression of the proto-S locus. While conventional models predict that the origin of self-incompatibility requires at least two- to threefold levels of inbreeding depression, I find that the comparatively low levels of inbreeding depression generated by a single overdominant locus can ensure the invasion of an enhancer of self-incompatibility under sufficiently high rates of receipt of self-pollen. Associations among components of the incompatibility system promote the origin of self-incompatibility. Enhancement of heterozygosity at the initially neutral proto-S locus improves offspring viability through associative overdominance. Further, the modifier that enhances the expression of self-incompatibility develops a direct association with heterozygosity at the overdominant viability locus. These results suggest that the evolutionary processes by which incompatibility systems originate may differ significantly from those associated with their breakdown. The genetic mechanism explored here may apply to the evolution of other systems that restrict reproduction, including maternal-fetal incompatibility in mammals.     

On the origin of the widespread self-compatible allotetraploid Capsella bursa-pastoris (Brassicaceae)

Polyploidy, or whole-genome duplication, is a common speciation mechanism in plants. An important barrier to polyploid establishment is a lack of compatible mates. Because self-compatibility alleviates this problem, it has long been hypothesized that there should be an association between polyploidy and self-compatibility (SC), but empirical support for this prediction is mixed. Here, we investigate whether the molecular makeup of the Brassicaceae self-incompatibility (SI) system, and specifically dominance relationships among S-haplotypes mediated by small RNAs, could facilitate loss of SI in allopolyploid crucifers. We focus on the allotetraploid species Capsella bursa-pastoris, which formed ~300 kya by hybridization and whole-genome duplication involving progenitors from the lineages of Capsella orientalis and Capsella grandiflora. We conduct targeted long-read sequencing to assemble and analyze eight full-length S-locus haplotypes, representing both homeologous subgenomes of C. bursa-pastoris. We further analyze small RNA (sRNA) sequencing data from flower buds to identify candidate dominance modifiers. We find that C. orientalis-derived S-haplotypes of C. bursa-pastoris harbor truncated versions of the male SI specificity gene SCR and express a conserved sRNA-based candidate dominance modifier with a target in the C. grandiflora-derived S-haplotype. These results suggest that pollen-level dominance may have facilitated loss of SI in C. bursa-pastoris. Finally, we demonstrate that spontaneous somatic tetraploidization after a wide cross between C. orientalis and C. grandiflora can result in production of self-compatible tetraploid offspring. We discuss the implications of this finding on the mode of formation of this widespread weed.Subject terms: Evolution, Polyploidy in plants, Plant evolution, Haplotypes     

S-RNase-mediated self-incompatibility

The Solanaceae, Rosaceae, and Scrophulariaceae families all possess an RNase-mediated self-incompatibility mechanism through which their pistils can recognize and reject self-pollen to prevent inbreeding. The highly polymorphic S-locus controls the self-incompatibility interaction, and the S-locus of the Solanaceae has been shown to be a multi-gene complex in excess of 1.3 Mb. To date, the function of only one of the S-locus genes, the S-RNase gene, has been determined. This article reviews the current status of the search for the pollen S-gene and the current models for how S-haplotype specific inhibition of pollen tubes can be accomplished by S-RNases.     

The ARC1 E3 Ligase Promotes Two Different Self-Pollen Avoidance Traits in Arabidopsis

Flowering plants have evolved various strategies for avoiding self-pollen to drive genetic diversity. These strategies include spatially separated sexual organs (herkogamy), timing differences between male pollen release and female pistil receptivity (dichogamy), and self-pollen rejection. Within the Brassicaceae, these outcrossing systems are the evolutionary default state, and many species display these traits, including Arabidopsis lyrata. In contrast to A. lyrata, closely related Arabidopsis thaliana has lost these self-pollen traits and thus represents an excellent system to test genes for reconstructing these evolutionary traits. We previously demonstrated that the ARC1 E3 ligase is required for self-incompatibility in two diverse Brassicaceae species, Brassica napus and A. lyrata, and is frequently deleted in self-compatible species, including A. thaliana. In this study, we examined ARC1’s requirement for reconstituting self-incompatibility in A. thaliana and uncovered an important role for ARC1 in promoting a strong and stable pollen rejection response when expressed with two other A. lyrata self-incompatibility factors. Furthermore, we discovered that ARC1 promoted an approach herkogamous phenotype in A. thaliana flowers. Thus, ARC1’s expression resulted in two different A. lyrata traits for self-pollen avoidance and highlights the key role that ARC1 plays in the evolution and retention of outcrossing systems.     

The ARC1 E3 Ligase Gene Is Frequently Deleted in Self-Compatible Brassicaceae Species and Has a Conserved Role in Arabidopsis lyrata Self-Pollen Rejection

Self-pollen rejection is an important reproductive regulator in flowering plants, and several different intercellular signaling systems have evolved to elicit this response. In the Brassicaceae, the self-incompatibility system is mediated by the pollen S-locus Cys-Rich/S-locus Protein11 (SCR/SP11) ligand and the pistil S Receptor Kinase (SRK). While the SCR/SP11-SRK recognition system has been identified in several species across the Brassicaceae, less is known about the conservation of the SRK-activated cellular responses in the stigma, following self-pollen contact. The ARM Repeat Containing1 (ARC1) E3 ubiquitin ligase functions downstream of SRK for the self-incompatibility response in Brassica, but it has been suggested that ARC1 is not required in Arabidopsis species. Here, we surveyed the presence of ARC1 orthologs in several recently sequenced genomes from Brassicaceae species that had diversified ∼20 to 40 million years ago. Surprisingly, the ARC1 gene was deleted in several species that had lost the self-incompatibility trait, suggesting that ARC1 may lose functionality in the transition to self-mating. To test the requirement of ARC1 in a self-incompatible Arabidopsis species, transgenic ARC1 RNA interference Arabidopsis lyrata plants were generated, and they exhibited reduced self-incompatibility responses resulting in successful fertilization. Thus, this study demonstrates a conserved role for ARC1 in the self-pollen rejection response within the Brassicaceae.     

Perturbation of Parentally Biased Gene Expression during Interspecific Hybridization

Interspecific hybridization often induces epigenetic remodeling that leads to transposon activation, gene expression changes, and loss of imprinting. These genomic changes can be deleterious and contribute to postzygotic hybrid incompatibility. In Arabidopsis, loss of genomic imprinting of PHERES1 and presumed failure of Polycomb Repressive Complex contributes to seed inviability observed in A. thaliana X A. arenosa interspecific hybrids. We used this species pair to further analyze the relationship between parentally biased gene expression and postzygotic hybrid incompatibility using two A. thaliana accessions, Col-0 and C24, with differential seed survival. We found that parentally biased expression was perturbed to a similar degree in both A. thaliana hybrids for PHERES1, HDG3, and six other normally paternally expressed genes. We propose that early genome remodeling and loss of imprinting of seed development genes induces lethality in both compatible and incompatible hybrids.     

Expression of Distinct Self-Incompatibility Specificities in Arabidopsis thaliana

The interplay of balancing selection within a species and rapid gene evolution between species can confound our ability to determine the functional equivalence of interspecific and intergeneric pairs of alleles underlying reproduction. In crucifer plants, mating specificity in the barrier to self-fertilization called self-incompatibility (SI) is controlled by allele-specific interactions between two highly polymorphic and co-evolving proteins, the S-locus receptor kinase (SRK) and its S-locus cysteine rich (SCR) ligand. These proteins have diversified both within and between species such that it is often difficult to determine from sequence information alone if they encode the same or different SI specificity. The self-fertile Arabidopsis thaliana was derived from an obligate outbreeding ancestor by loss of self-incompatibility, often in conjunction with inactivation of SRK or SCR. Nevertheless, some accessions of A. thaliana can express self-incompatibility upon transformation with an SRK–SCR gene pair isolated from its self-incompatible close relative A. lyrata. Here we show that several additional and highly diverged SRK/SCR genes from A. lyrata and another crucifer plant, Capsella grandiflora, confer self-incompatibility in A. thaliana, either as intact genes isolated from genomic libraries or after manipulation to generate chimeric fusions. We describe how the use of this newly developed chimeric protein strategy has allowed us to test the functional equivalence of SRK/SCR gene pairs from different taxa and to assay the functionality of endogenous A. thaliana SRK and SCR sequences.MATING reactions in plants, fungi, and animals are strongly influenced by molecular recognition machineries that act as gauges of genetic relatedness (Brown and Casselton 2001; Nasrallah 2005; Yamazaki and Beauchamp 2007). Many plants with hermaphroditic flowers have evolved inbreeding avoidance mechanisms, known as self-incompatibility (SI) systems. These systems are based on the ability of the female reproductive apparatus (the pistil) to discriminate among genetically distinct pollen grains, resulting in the failure of self-pollination despite functional female and male reproductive structures. In the Brassicaceae (crucifers), specific recognition of pollen by the epidermal cells of the stigma (a structure located at the tip of the pistil) is controlled by haplotypes of the S locus, and activation of the SI response leading to inhibition of pollen tube growth occurs if pollen and stigma are derived from plants that express the same S-locus haplotype (S haplotype). Within self-incompatible crucifer species, the number of S haplotypes and corresponding SI specificities is usually high, with >50 reported in some species (Watanabe et al. 2000), and SI dictates that self-incompatible plants are typically heterozygous and carry two S haplotypes. Each S haplotype is composed of two highly polymorphic genes that are the determinants of SI specificity in stigma and pollen (Stein et al. 1991; Schopfer et al. 1999). The S-locus receptor kinase (SRK) gene encodes a single-pass transmembrane serine/threonine kinase localized on the surface of stigma epidermal cells, and the S-locus cysteine-rich protein (SCR) gene encodes a small peptide localized in the pollen coat. SCR is the ligand for SRK and will bind to the extracellular domain of SRK (hereafter eSRK) only if both proteins are encoded by the same S-locus haplotype (Kachroo et al. 2001; Takayama et al. 2001; Chookajorn et al. 2004). The binding of SCR to its cognate eSRK triggers an intracellular phosphorylation cascade that results in pollen rejection by a poorly understood mechanism.A mechanistic understanding of the recognition phase of SI requires detailed structure–function analyses of SRK and SCR aimed at identifying the amino acid residues that determine their allele-specific interaction and explaining the puzzling dominance/recessive interactions exhibited by different SRK alleles in the heterozygous stigmas of self-incompatible plants (Hatakeyama et al. 2001; Mable et al. 2003; Prigoda et al. 2005). Such structure–function studies require an experimental system that allows efficient in vivo functional analysis of large numbers of SRK and SCR sequence variants generated in vitro by site-directed mutagenesis or domain swapping between proteins that determine different SI specificities. The recent transfer of the SI trait into Arabidopsis thaliana has established this species as a model organism for mechanistic and evolutionary studies of mating systems in crucifers (Nasrallah et al. 2002, 2004). However, to date, only one SI specificity, that which is determined by the Sb haplotype of A. lyrata, has been successfully introduced into A. thaliana and shown to alter the plant''s mating reaction from strict autogamy to full SI. To exploit fully the A. thaliana transgenic SI model, additional S haplotypes must be introduced into this species. In addition to facilitating mechanistic studies of the SRK–SCR interaction and dominance relationships, the expression of multiple SI specificities in A. thaliana promises to shed light on processes underlying the diversification of SRK and SCR genes. For example, expression in A. thaliana of SI specificities derived from different crucifer species will allow direct assays of the functional equivalence or nonequivalence of the corresponding S haplotypes, an issue that is difficult to resolve on the basis of sequence information alone.Although conceptually simple, expressing different SI specificities by transformation with different SRK/SCR gene pairs is not a straightforward proposition. Difficulties stem largely from the availability of appropriate cloned SRK/SCR variants for use in transformation experiments. A large number of SRK/SCR gene pairs are available from Brassica species as a result of extensive and long-standing studies of SI. However, attempts to restore SI in transgenic A. thaliana using Brassica S-locus genes had met with failure (Bi et al. 2000; J. B. Nasrallah, unpublished data), possibly because of the inability of Brassica SRKs to interact productively with A. thaliana components of the SI signal transduction pathway. In the past few years, studies of SI were initiated in self-incompatible species more closely related to A. thaliana, such as A. lyrata, A. halleri, and Capsella grandiflora. However, with a few exceptions, these studies produced only partial SRK and SCR sequences amplified from genomic DNA (Schierup et al. 2001; Prigoda et al. 2005; Bechsgaard et al. 2006; Paetsch et al. 2006). The challenging task of cloning the very highly polymorphic SCR sequences and complete SRK and SCR genes, which requires genomic library construction and in many cases chromosome walking, has only been accomplished for two S haplotypes of A. lyrata, Sb (hereafter AlSb, which was used in previous transformation studies (Nasrallah et al. 2002, 2004), and Sa (AlSa; Kusaba et al. 2001), and for the S7 haplotype of C. grandiflora (CgS7; Nasrallah et al. 2007).In this article, we report the isolation of two new SRK/SCR gene pairs from genomic libraries of A. lyrata and expression of the corresponding SI specificities in A. thaliana. We also describe a novel strategy for rapid and efficient transfer of several distinct SI specificities into A. thaliana, which only requires knowledge of the eSRK sequence and SCR second-exon sequences that encode the mature SCR protein.