S-locus Protein Research Articles

Genetic Components of Self-Incompatibility in Brassica Vegetables

Brassica vegetables are very important to human beings. Self-incompatibility (SI) is a common phenomenon in Brassica. Breeding by SI lines is an important way to utilize heterosis of Brassica vegetables. It is believed that the SI inheritance in Brassica species is controlled by three linkage genes on the S-locus, including SRK (S-locus receptor kinase), SCR (S-locus cystine-rich protein)/SP11 (S-locus protein 11), and SLG (S-locus glycoprotein). SRK is the female determinant and SCR/SP11 is the pollen S gene. The expression of SLG is necessary for SRK, and it enhances the SRK-mediated SI reaction. In addition to these three S-locus genes, some other functional molecules also have significant regulatory effects on SI, such as ARC1 (arm repeat containing 1), MLPK (M-locus protein kinase), Exo70A1 (exocyst compounds), THLl/THL2 (thioredoxin H-like), MOD (aquaporin), SLR (S-locus-related glycoprotein), BPCI (pollen calcium-binding protein I), etc. SI is also associated with the dominant/recessive relationship between S alleles. Here, the genetic elements and molecular mechanisms of SI, mainly in Brassica vegetables, are reviewed.

Comprehensive computational analysis of the SRK–SP11 molecular interaction underlying self-incompatibility in Brassicaceae using improved structure prediction for cysteine-rich proteins

Plants employ self-incompatibility (SI) to promote cross-fertilization. In Brassicaceae, this process is regulated by the formation of a complex between the pistil determinant S receptor kinase (SRK) and the pollen determinant S-locus protein 11 (SP11, also known as S-locus cysteine-rich protein, SCR). In our previous study, we used the crystal structures of two eSRK–SP11 complexes in Brassica rapa S8 and S9 haplotypes and nine computationally predicted complex models to demonstrate that only the SRK ectodomain (eSRK) and SP11 pairs derived from the same S haplotype exhibit high binding free energy. However, predicting the eSRK–SP11 complex structures for the other 100 + S haplotypes and genera remains difficult because of SP11 polymorphism in sequence and structure. Although protein structure prediction using AlphaFold2 exhibits considerably high accuracy for most protein monomers and complexes, 46% of the predicted SP11 structures that we tested showed < 75 mean per-residue confidence score (pLDDT). Here, we demonstrate that the use of curated multiple sequence alignment (MSA) for cysteine-rich proteins significantly improved model accuracy for SP11 and eSRK–SP11 complexes. Additionally, we calculated the binding free energies of the predicted eSRK–SP11 complexes using molecular dynamics (MD) simulations and observed that some Arabidopsis haplotypes formed a binding mode that was critically different from that of B. rapa S8 and S9. Thus, our computational results provide insights into the haplotype-specific eSRK–SP11 binding modes in Brassicaceae at the residue level. The predicted models are freely available at Zenodo, https://doi.org/10.5281/zenodo.8047768.

Autophagy is required for self-incompatible pollen rejection in two transgenic Arabidopsis thaliana accessions.

Successful reproduction in the Brassicaceae is mediated by a complex series of interactions between the pollen and the pistil, and some species have an additional layer of regulation with the self-incompatibility trait. While the initial activation of the self-incompatibility pathway by the pollen S-locus protein 11/S locus cysteine-rich protein and the stigma S Receptor Kinase is well characterized, the downstream mechanisms causing self-pollen rejection are still not fully understood. In previous studies, we detected the presence of autophagic bodies with self-incompatible (SI) pollinations in Arabidopsis lyrata and transgenic Arabidopsis thaliana lines, but whether autophagy was essential for self-pollen rejection was unknown. Here, we investigated the requirement of autophagy in this response by crossing mutations in the essential AUTOPHAGY7 (ATG7) and ATG5 genes into two different transgenic SI A. thaliana lines in the Col-0 and C24 accessions. By using these previously characterized transgenic lines that express A. lyrata and Arabidopsis halleri self-incompatibility genes, we demonstrated that disrupting autophagy weakened their SI responses in the stigma. When the atg7 or atg5 mutations were present, an increased number of SI pollen was found to hydrate and form pollen tubes that successfully fertilized the SI pistils. Additionally, we confirmed the presence of GFP-ATG8a-labeled autophagosomes in the stigmatic papillae following SI pollinations. Together, these findings support the requirement of autophagy in the self-incompatibility response and add to the growing understanding of the intracellular mechanisms employed in the transgenic A. thaliana stigmas to reject self-pollen.

FERONIA receptor kinase-regulated reactive oxygen species mediate self-incompatibility in Brassica rapa

Most plants in the Brassicaceae evolve self-incompatibility (SI) to avoid inbreeding and generate hybrid vigor. Self-pollen is recognized by the S-haplotype-specific interaction of the pollen ligand S-locus protein 11 (SP11) (also known as S-locus cysteine-rich protein [SCR]) and its stigma-specific S-locus receptor kinase (SRK). However, mechanistically much remains unknown about the signaling events that culminate in self-pollen rejection. Here, we show that self-pollen triggers high levels of reactive oxygen species (ROS) in stigma papilla cells to mediate SI in heading Chinese cabbage (Brassica rapa L. ssp. pekinensis). We found that stigmatic ROS increased after self-pollination but decreased after compatible(CP)- pollination. Reducing stigmatic ROS by scavengers or suppressing the expression of respiratory burst oxidase homologs (Rbohs), which encode plant NADPH oxidases that produce ROS, both broke down SI. On the other hand, increasing the level of ROS inhibited the germination and penetration of compatible pollen on the stigma, mimicking an incompatible response. Furthermore, suppressing a B.rapa FERONIA (FER) receptor kinase homolog or Rac/Rop guanosine triphosphatase (GTPase) signaling effectively reduced stigmatic ROS and interfered with SI. Our results suggest that FER-Rac/Rop signaling-regulated, NADPH oxidase-produced ROS is an essential SI response leading to self-pollen rejection.

Mechanism of self/nonself-discrimination in Brassica self-incompatibility

Self-incompatibility (SI) is a breeding system that promotes cross-fertilization. In Brassica, pollen rejection is induced by a haplotype-specific interaction between pistil determinant SRK (S receptor kinase) and pollen determinant SP11 (S-locus Protein 11, also named SCR) from the S-locus. Although the structure of the B. rapa S9-SRK ectodomain (eSRK) and S9-SP11 complex has been determined, it remains unclear how SRK discriminates self- and nonself-SP11. Here, we uncover the detailed mechanism of self/nonself-discrimination in Brassica SI by determining the S8-eSRK–S8-SP11 crystal structure and performing molecular dynamics (MD) simulations. Comprehensive binding analysis of eSRK and SP11 structures reveals that the binding free energies are most stable for cognate eSRK–SP11 combinations. Residue-based contribution analysis suggests that the modes of eSRK–SP11 interactions differ between intra- and inter-subgroup (a group of phylogenetically neighboring haplotypes) combinations. Our data establish a model of self/nonself-discrimination in Brassica SI.

Intraspecific Genetic Variation Underlying Postmating Reproductive Barriers between Species in the Wild Tomato Clade (Solanum sect. Lycopersicon)

A goal of speciation genetics is to understand how the genetic components underlying interspecific reproductive barriers originate within species. Unilateral incompatibility (UI) is a postmating prezygotic barrier in which pollen rejection in the female reproductive tract (style) occurs in only one direction of an interspecific cross. Natural variation in the strength of UI has been observed among populations within species in the wild tomato clade. In some cases, molecular loci underlying self-incompatibility (SI) are associated with this variation in UI, but the mechanistic connection between these intra- and inter-specific pollen rejection behaviors is poorly understood in most instances. We generated an F2 population between SI and SC genotypes of a single species, Solanum pennellii, to examine the genetic basis of intraspecific variation in UI against other species, and to determine whether loci underlying SI are genetically associated with this variation. We found that F2 individuals vary in the rate at which UI rejection occurs. One large effect QTL detected for this trait co-localized with the SI-determining S-locus. Moreover, individuals that expressed S-RNase-the S-locus protein involved in SI pollen rejection-in their styles had much more rapid UI responses compared with those without S-RNase protein. Our analysis shows that intraspecific variation at mate choice loci-in this case at loci that prevent self-fertilization-can contribute to variation in the expression of interspecific isolation, including postmating prezygotic barriers. Understanding the nature of such intraspecific variation can provide insight into the accumulation of these barriers between diverging lineages.

Genetic and tissue-specific RNA-sequencing analysis of self-compatible mutant TSC28 in Brassica rapa L. toward identification of a novel self-incompatibility factor.

Self-incompatibility (SI) is a sophisticated system for pollen selectivity to prevent pollination by genetically identical pollen. In Brassica, it is genetically controlled by a single, highly polymorphic S-locus, and the male and female S-determinant factors have been identified as S-locus protein 11 (SP11)/S-locus cysteine-rich protein (SCR) and S-locus receptor kinase (SRK), respectively. However, the overall molecular system and identity of factors in the downstream cascade of the SI reaction remain unclear. Previously, we identified a self-compatible B. rapa mutant line, TSC28, which has a disruption in an unidentified novel factor of the SI signaling cascade. Here, in a genetic analysis of TSC28, using an F2 population from a cross with the reference B. rapa SI line Chiifu-401, the causal gene was mapped to a genetic region of DNA containing markers BrSA64 and ACMP297 in B. rapa chromosome A1. By fine mapping using an F2 population of 1,034 plants, it was narrowed down to a genetic region between DNA markers ACMP297 and BrgMS4028, with physical length approximately 1.01 Mbp. In this genomic region, 113 genes are known to be located and, among these, we identified 55 genes that were expressed in the papilla cells. These are candidates for the gene responsible for the disruption of SI in TSC28. This list of candidate genes will contribute to the discovery of a novel downstream factor in the SP11-SRK signaling cascade in the Brassica SI system.

The Molecular and Cellular Regulation of Brassicaceae Self-Incompatibility and Self-Pollen Rejection.

In flowering plants, sexual reproduction is actively regulated by cell-cell communication between the male pollen and female pistil, and many species possess self-incompatibilitysystems for the selective rejection of self-pollen to maintain genetic diversity. The Brassicaceae self-incompatibility pathway acts early on when pollen grains have landed on the stigmatic papillae at the top of the pistil. Extensive studies have revealed that self-pollen rejection in the Brassicaceae is initiated by an S-haplotype-specific interaction between two polymorphic proteins: the pollen S-locus protein 11/S cysteine-rich (SP11/SCR) ligand and the stigma S receptor kinase (SRK). While the different S-haplotypes are typically codominant, there are several examples of dominant-recessive interactions, and a small RNA-based regulation of SP11/SCR expression has been uncovered as a mechanism behind these genetic interactions. Recent research has also added to our understanding of various cellular components in the pathway leading from the SP11/SCR-SRK interaction, including two signaling proteins, the M-locus protein kinase (MLPK) and the ARM-repeat containing 1 (ARC1) E3 ligase, as well as calcium fluxes and induction of autophagy in the stigmatic papillae. Finally, a better understanding of the compatible pollen responses that are targeted by the self-incompatibility pathway is starting to emerge, and this will allow us to more fully understand how the Brassicaceae self-incompatibility pathway causes self-pollen rejection. Here, we provide an overview of the field, highlighting recent contributions to our understanding of Brassicaceae self-incompatibility, and draw comparisons to a recently discovered unilateral incompatibility system.

Structural basis for specific self-incompatibility response in Brassica.

Self-incompatibility (SI) is a widespread mechanism in flowering plants which prevents self-fertilization and inbreeding. In Brassica, recognition of the highly polymorphic S-locus cysteine-rich protein (SCR; or S-locus protein 11) by the similarly polymorphic S-locus receptor kinase (SRK) dictates the SI specificity. Here, we report the crystal structure of the extracellular domain of SRK9 (eSRK9) in complex with SCR9 from Brassica rapa. SCR9 binding induces eSRK9 homodimerization, forming a 2:2 eSRK:SCR heterotetramer with a shape like the letter "A". Specific recognition of SCR9 is mediated through three hyper-variable (hv) regions of eSRK9. Each SCR9 simultaneously interacts with hvI and one-half of hvII from one eSRK9 monomer and the other half of hvII from the second eSRK9 monomer, playing a major role in mediating SRK9 homodimerization without involving interaction between the two SCR9 molecules. Single mutations of residues critical for the eSRK9-SCR9 interaction disrupt their binding in vitro. Our study rationalizes a body of data on specific recognition of SCR by SRK and provides a structural template for understanding the co-evolution between SRK and SCR.

Efficient expression of SRK intracellular domain by a modeling-based protein engineering

S-locus protein kinase (SRK) is a receptor kinase that plays a critical role in self-recognition in the Brassicaceae self-incompatibility (SI) response. SRK is activated by binding of its ligand S-locus protein 11 (SP11) and subsequently induced phosphorylation of the intracellular kinase domain. However, a detailed activation mechanism of SRK is still largely unknown because of the difficulty in stably expressing SRK recombinant proteins. Here, we performed modeling-based protein engineering of the SRK kinase domain for stable expression in Escherichia coli. The engineered SRK intracellular domain was expressed about 54-fold higher production than wild type SRK, without loss of the kinase activity, suggesting it could be useful for further biochemical and structural studies.

Calcium signalling mediates self-incompatibility response in the Brassicaceae.

Self-incompatibility in the Brassicaceae is controlled by multiple haplotypes encoding the pollen ligand (S-locus protein 11, SP11, also known as S-locus cysteine-rich protein, SCR) and its stigmatic receptor (S-receptor kinase, SRK). A haplotype-specific interaction between SP11/SCR and SRK triggers the self-incompatibility response that leads to self-pollen rejection, but the signalling pathway remains largely unknown. Here we show that Ca(2+) influx into stigma papilla cells mediates self-incompatibility signalling. Using self-incompatible Arabidopsis thaliana expressing SP11/SCR and SRK, we found that self-pollination specifically induced an increase in cytoplasmic Ca(2+) ([Ca(2+)]cyt) in papilla cells. Direct application of SP11/SCR to the papilla cell protoplasts induced Ca(2+) increase, which was inhibited by D-(-)-2-amino-5-phosphonopentanoic acid (AP-5), a glutamate receptor channel blocker. An artificial increase in [Ca(2+)]cyt in papilla cells arrested wild-type (WT) pollen hydration. Treatment of papilla cells with AP-5 interfered with self-incompatibility, and Ca(2+) increase on the self-incompatibility response was reduced in the glutamate receptor-like channel (GLR) gene mutants. These results suggest that Ca(2+) influx mediated by GLR is the essential self-incompatibility response leading to self-pollen rejection.

Analysis of S-locus and expression of S-alleles of self-compatible rapid-cycling Brassica oleracea 'TO1000DH3'.

Brassica oleracea is a strictly self-incompatible (SI) plant, but rapid-cycling B. oleracea 'TO1000DH3' is self-compatible (SC). Self-incompatibility in Brassicaceae is controlled by multiple alleles of the S-locus. Three S-locus genes, S-locus glycoprotein (SLG), S-locus receptor kinase (SRK) and S-locus protein 11 or S-locus cysteine-rich (SP11/SCR), have been reported to date, all of which are classified into class I and II. In this study, we investigated the molecular mechanism behind alterations of SI to SC in rapid-cycling B. olerace 'TO1000DH3'. Class I SRK were identified by genomic DNA PCR and PCR-RFLP analysis using SRK specific markers and found to be homozygous. Cloning and sequencing of class I SRK revealed a normal kinase domain without any S-domain/transmembrane domain. Moreover, S-locus sequencing analysis revealed only an SLG sequence, but no SP11/SCR. Expression analysis showed no SRK expression in the stigma, although other genes involved in the SI recognition reaction (SLG, MLPK, ARC1, THL) were found to have normal expression in the stigma. Taken together, the above results suggest that structural aberrations such as deletion of the SI recognition genes may be responsible for the breakdown of SI in rapid-cycling B. oleracea 'TO1000DH3'.

Characterization of self-incompatibility genes in the intergeneric hybrid xBrassicoraphanus

Brassica rapa and Raphanus sativus are strictly self-incompatible (SI) plants; however, xBrassicoraphanus (baemoochae) is an intergeneric hybrid synthesized following hybridization of B. rapa and R. sativus that is self-compatible (SC). Self-incompatibility in Brassicaceae is controlled by multiple alleles of the S-locus. Two S-locus genes, SRK (S-locus receptor kinase) and SP11/SCR (S-locus protein 11 or S-locus cysteine-rich), have been reported to date, both of which are classified into class I and II. In this study, we investigate if there is an alteration in the structure or the expression in SRK or SP11 genes behind the alteration of SI to SC in baemoochae. Class I and II SRK were identified by PCR of the genomic DNA of baemoochae using SRK-specific universal primers. Cloning and sequencing of both classes of SRK was conducted and compared with SRK genes of parental species. Comparison analysis showed no genomic alterations and both of them showed expression in the stigma. Similarly, SP11 genes also showed no genomic alterations and normally are expressed in the anther. Other SI-related genes (MLPK, ARC1, THL) also showed normal expression in the stigma and anther. Taken together, these results revealed that no structural/gene expression change in these genes was responsible for the breakdown of SI in baemoochae. Rather, the transformation from SI parents to SC descendants could be responding to epigenetic mechanisms.

Identification of S-haplotypes in DH-lines of broccoli (Brassica oleracea L. var. italica)

SummarySelf-incompatibility (SI) in broccoli (Brassica oleracea L. var. italica) is common, and controlled by the S-locus which contains at least three genes: SLG (the S-locus glycoprotein), SRK (the S-locus receptor kinase), and SCR (the S-locus cysteine-rich protein) also named SP11 (the S-locus protein 11). As the S-locus is complex, the classical term ‘S-allele’ was changed to ‘S-haplotype’. Information on S-alleles in breeding lines is important for breeders to ensure cross-compatibility between lines. In this work, the S-haplotypes of 18 dihaploid (DH) broccoli lines were examined. For each line, fragments of the SRK and SCR/SP11 genes were amplified by PCR and their sequences were determined. The results showed that: (i) out of the 18 DH broccoli lines, 15 belonged to Class II S-haplotypes, while the other three lines were Class I, based on the amplification patterns of SRK and SCR/SP11; (ii) the sequences of Class II S-haplotype SRK and SCR/SP11 fragments of 15 of the DH lines were identical; (iii) the Class I S-haplotypes of three DH lines were different from each other, based on the DNA sequences of their SRK genes; and (iv) one Class II and three Class I S-haplotypes were present in the broccoli DH lines.

Molecular Genetic Aspects of Self-incompatibility in Brassicaceae

Molecular genetic studies of self-incompatibility (SI) are the most accentuating part in the way of advancement of reproductive mechanisms in flowering plants. In the Brassicaceae plants, self-incompatibility has been mapped genetically to a single chromosomal location where several closely linked genes have been identified. Recently, various studies have provided a novel insight into the basis of specificity in the S-receptor kinase (SRK) and S-locus protein 11 or S-locus Cysteine-rich (SP11/SCR) interaction, the nature of the signaling cascade that culminates in the inhibition of ‘self’ pollen, and the physiological and morphological changes that are associated with transitions between the outbreeding and inbreeding modes of mating in the Brassicaceae. In this review, we discuss the current view of the molecular genetic aspects of the self-incompatibility in Brassicaceae.

Development of self-compatible B. rapa by RNAi-mediated S locus gene silencing.

The self-incompatibility (SI) system is genetically controlled by a single polymorphic locus known as the S-locus in the Brassicaceae. Pollen rejection occurs when the stigma and pollen share the same S-haplotype. Recognition of S-haplotype specificity has recently been shown to involve at least two S-locus genes, S-receptor kinase (SRK) and S-locus protein 11 or S locus Cysteine-rich (SP11/SCR) protein. Here, we examined the function of S60, one SP11/SCR allele of B. rapa cv. Osome, using a RNAi-mediated gene silencing approach. The transgenic RNAi lines were highly self-compatible, and this trait was stable in subsequent generations, even after crossing with other commercial lines. These findings also suggested that the resultant self-compatibility could be transferred to commercial cultivars with the desired performances in B. rapa.

Progress on characterization of self-incompatibility in Brassica napus L.

Self-incompatibility (SI) is a widespread mechanism in flowering plants that promotes outbreeding and thereby increases genetic diversity. Recognition specificity in Brassica is achieved by the interaction of the female determinant S-receptor kinase (SRK) and its ligand, the male determinant S-locus protein 11 (SP11). The interaction between SP11 and SRK triggers the signaling cascade in an S-haplotype-specific manner and results in the rejection of self-pollen, but the signal components involved are still not well characterized. S haplotypes are widespread in self-compatible amphidiploid B. napus, and the interaction of heterozygous S haplotypes causes the loss of SI. This review highlights the recent advances made towards understanding the genetic analysis, distribution, and evolution of S haplotypes, the signal factors, and the potential of SI in B. napus hybrid breeding program.

Phylogenetic Analysis of S-Locus Genes Reveals the Complicated Evolution Relationship of S Haplotypes in Brassica

Self-incompatibility (SI) is reported to play a key role in the evolution of species as it promotes their outcrossing through the recognition and rejection of self-pollen grains. In Brassica, two S-locus genes expressed in the stigma, S-locus glycoprotein (SLG) gene and S-locus receptor kinase (SRK) gene, and one expressed in the pollen, S-locus protein 11 (SP11) gene, were linked as an S haplotype. In order to analyze the evolutionary relationships of S haplotypes in Brassica, a total of 39 SRK, 37 SLG, and 58 SP11 sequences of Brassica oleracea, Brassica rapa and Brassica napus were aligned. Two phylogenetic trees with similar pattern were constructed based on the nucleotide sequences of SRK/SLG and SP11, respectively. Class I and class II alleles were clustered into two distinct groups, and alleles from different species, including all the interspecific pairs of S haplotypes, were closely related to each other. The S-locus genes identified in B. napus were intermingled in phylogenetic trees. All these observations showed that class I and class II S haplotypes diverged ahead of the species differentiation in Brassica. The evolution and the genetic diversity of S haplotypes in Brassica were discussed. Moreover, the relationships between S haplotypes and SI phenotypes in Brassica, especially in B. napus, were also discussed.

Trans-acting small RNA determines dominance relationships in Brassica self-incompatibility

A diploid organism has two copies of each gene, one inherited from each parent. The expression of two inherited alleles is sometimes biased by the effects known as dominant/recessive relationships, which determine the final phenotype of the organism. To explore the mechanisms underlying these relationships, we have examined the monoallelic expression of S-locus protein 11 genes (SP11), which encode the male determinants of self-incompatibility in Brassica. We previously reported that SP11 expression was monoallelic in some S heterozygotes, and that the promoter regions of recessive SP11 alleles were specifically methylated in the anther tapetum. Here we show that this methylation is controlled by trans-acting small non-coding RNA (sRNA). We identified inverted genomic sequences that were similar to the recessive SP11 promoters in the flanking regions of dominant SP11 alleles. These sequences were specifically expressed in the anther tapetum and processed into 24-nucleotide sRNA, named SP11 methylation inducer (Smi). Introduction of the Smi genomic region into the recessive S homozygotes triggered the methylation of the promoter of recessive SP11 alleles and repressed their transcription. This is an example showing sRNA encoded in the flanking region of a dominant allele acts in trans to induce transcriptional silencing of the recessive allele. Our finding may provide new insights into the widespread monoallelic gene expression systems.

Evolution of self-compatibility in Arabidopsis by a mutation in the male specificity gene

Ever since Darwin's pioneering research, the evolution of self-fertilisation (selfing) has been regarded as one of the most prevalent evolutionary transitions in flowering plants. A major mechanism to prevent selfing is the self-incompatibility (SI) recognition system, which consists of male and female specificity genes at the S-locus and SI modifier genes. Under conditions that favour selfing, mutations disabling the male recognition component are predicted to enjoy a relative advantage over those disabling the female component, because male mutations would increase through both pollen and seeds whereas female mutations would increase only through seeds. Despite many studies on the genetic basis of loss of SI in the predominantly selfing plant Arabidopsis thaliana, it remains unknown whether selfing arose through mutations in the female specificity gene (S-receptor kinase, SRK), male specificity gene (S-locus cysteine-rich protein, SCR; also known as S-locus protein 11, SP11) or modifier genes, and whether any of them rose to high frequency across large geographic regions. Here we report that a disruptive 213-base-pair (bp) inversion in the SCR gene (or its derivative haplotypes with deletions encompassing the entire SCR-A and a large portion of SRK-A) is found in 95% of European accessions, which contrasts with the genome-wide pattern of polymorphism in European A. thaliana. Importantly, interspecific crossings using Arabidopsis halleri as a pollen donor reveal that some A. thaliana accessions, including Wei-1, retain the female SI reaction, suggesting that all female components including SRK are still functional. Moreover, when the 213-bp inversion in SCR was inverted and expressed in transgenic Wei-1 plants, the functional SCR restored the SI reaction. The inversion within SCR is the first mutation disrupting SI shown to be nearly fixed in geographically wide samples, and its prevalence is consistent with theoretical predictions regarding the evolutionary advantage of mutations in male components.