Mapping quantitative trait loci associated with self-(in)compatibility in goji berries (Lycium barbarum).

BACKGROUND: Goji (Lycium barbarum L.) is a perennial deciduous shrub widely distributed in arid and semiarid regions of Northwest China. It is highly valued for its medicinal and functional properties. Most goji varieties are naturally self-incompatible, posing challenges in breeding and cultivation. Self-incompatibility is a complex genetic trait, with ongoing debates regarding the number of self-incompatible loci. To date, no genetic mappings has been conducted for S loci or other loci related to self-incompatibility in goji. RESULTS: We used genome resequencing to create a high-resolution map for detecting de novo single-nucleotide polymorphisms (SNP) in goji. We focused on 229 F1 individuals from self-compatible '13-19' and self-incompatible 'new 9' varieties. Subsequently, we conducted a quantitative trait locus (QTL) analysis on traits associated with self-compatibility in goji berries. The genetic map consisted of 249,327 SNPs distributed across 12 linkage groups (LGs), spanning a total distance of 1243.74 cM, with an average interval of 0.002 cM. Phenotypic data related to self-incompatibility, such as average fruit weight, fruit rate, compatibility index, and comparable compatibility index after self-pollination and geitonogamy, were collected for the years 2021-2022, as well as for an extra year representing the mean data from 2021 to 2022 (2021/22). A total of 43 significant QTL, corresponding to multiple traits were identified, accounting for more than 11% of the observed phenotypic variation. Notably, a specific QTL on chromosome 2 consistently appeared across different years, irrespective of the relationship between self-pollination and geitonogamy. Within the localization interval, 1180 genes were annotated, including Lba02g01102 (annotated as an S-RNase gene), which showed pistil-specific expression. Cloning of S-RNase genes revealed that the parents had two different S-RNase alleles, namely S1S11 and S2S8. S-genotype identification of the F1 population indicated segregation of the four S-alleles from the parents in the offspring, with the type of S-RNase gene significantly associated with self-compatibility. CONCLUSIONS: In summary, our study provides valuable insights into the genetic mechanism underlying self-compatibility in goji berries. This highlights the importance of further positional cloning investigations and emphasizes the importance of integration of marker-assisted selection in goji breeding programs.

The homomorphic self-incompatibility system in Oleaceae is controlled by a hemizygous genomic region expressing a gibberellin pathway gene.

In flowering plants, outcrossing is commonly ensured by self-incompatibility (SI) systems. These can be homomorphic (typically with many different allelic specificities) or can accompany flower heteromorphism (mostly with just two specificities and corresponding floral types). The SI system of the Oleaceae family is unusual, with the long-term maintenance of only two specificities but often without flower morphology differences. To elucidate the genomic architecture and molecular basis of this SI system, we obtained chromosome-scale genome assemblies of Phillyrea angustifolia individuals and related them to a genetic map. The S-locus region proved to have a segregating 543-kb indel unique to one specificity, suggesting a hemizygous region, as observed in all distylous systems so far studied at the genomic level. Only one of the predicted genes in this indel region is found in the olive tree, Olea europaea, genome, also within a segregating indel. We describe complete association between the presence/absence of this gene and the SI types determined for individuals of seven distantly related Oleaceae species. This gene is predicted to be involved in catabolism of the gibberellic acid (GA) hormone, and experimental manipulation of GA levels in developing buds modified the male and female SI responses of the two specificities in different ways. Our results provide a unique example of a homomorphic SI system, where a single conserved gibberellin-related gene in a hemizygous indel underlies the long-term maintenance of two groups of reproductive compatibility.

A hemizygous supergene controls homomorphic and heteromorphic self-incompatibility systems in Oleaceae.

Self-incompatibility (SI) has evolved independently multiple times and prevents self-fertilization in hermaphrodite angiosperms. Several groups of Oleaceae such as jasmines exhibit distylous flowers, with two compatibility groups each associated with a specific floral morph.1 Other Oleaceae species in the olive tribe have two compatibility groups without associated morphological variation.2,3,4,5 The genetic basis of both homomorphic and dimorphic SI systems in Oleaceae is unknown. By comparing genomic sequences of three olive subspecies (Olea europaea) belonging to the two compatibility groups, we first locate the genetic determinants of SI within a 700-kb hemizygous region present only in one compatibility group. We then demonstrate that the homologous hemizygous region also controls distyly in jasmine. Phylogenetic analyses support a common origin of both systems, following a segmental genomic duplication in a common ancestor. Examination of the gene content of the hemizygous region in different jasmine and olive species suggests that the mechanisms determining compatibility groups and floral phenotypes (whether homomorphic or dimorphic) in Oleaceae rely on the presence/absence of two genes involved in gibberellin and brassinosteroid regulation.

Self-(in)compatibility in Tunisian apple accessions [Malus domestica. Borkh]: S-genotypes identification and pollen tube growth analysis.

MAIN CONCLUSION: Self-incompatibility studies have revealed a potential use of Tunisian apple resources for crop improvement and modern breeding programs and a likely correlation between the pollen tube growth and flowering period. Apples [Malus domestica. Borkh] exhibit an S-RNase-based gametophytic self-incompatibility (GSI) system. Four primer combinations were used to S-genotype eighteen Tunisian local apple accessions and twelve introduced accessions that served as references. Within the Tunisian local accessions, S2, S3, S7, and S28 S-alleles were the most frequent and were assigned to 14 S-genotypes; among them, S7S28, S3S7, S2S5, and S2S3 were the most abundant. PCA plot showed that population structuring was affected by the S-alleles frequencies and revealed a modern origin of the Tunisian varieties rather than being ancient ones. Nonetheless, the results obtained with 17 SSR markers showed a separate grouping of local Tunisian accessions that calls into question the hypothesis discussed. Pollination experiments showed that the pollen started to germinate within 24 h of pollination but 48 h after pollination in the "El Fessi" accession. The first pollen tubes arrived in the styles within 36 h of pollination in two early flowering accessions known as "Arbi" and "Bokri", and after 72 h of pollination in late flowering "El Fessi" and 48 h after pollination in remaining accessions. The first pollen tube arrests were observed in accessions "Arbi" and "Bokri" within 84 h of pollination, within 108 h of pollination in "El Fessi" and within 108 h of pollination in remaining accessions. In the apple accession called "Boutabgaya," the pollen tubes reached the base of the style within 120 h of pollination without being aborted. Nevertheless, the self-compatible nature of "Boutabgaya" needs more studies to be confirmed. However, our results revealed the malfunction of the female component of the GSI in this accession. To conclude, this work paved the path for further studies to enhance the insight (i) into the relation between the flowering period and the pollen tube growth, (ii) self-compatible nature of "Boutabgaya", and (iii) the origin of the Tunisian apple.

Is self-incompatibility a reproductive barrier for hybridization in a sympatric species?

PREMISE: Barriers at different reproductive stages contribute to reproductive isolation. Self-incompatibility (SI) systems that prevent self-pollination could also act to control interspecific pollination and contribute to reproductive isolation, preventing hybridization. Here we evaluated whether SI contributes to reproductive isolation among four co-occurring Opuntia species that flower at similar times and may hybridize with each other. METHODS: We assessed whether Opuntia cantabrigiensis, O. robusta, O. streptacantha, and O. tomentosa, were self-compatible and formed hybrid seeds in five manipulation treatments to achieve self-pollination, intraspecific cross-pollination, open pollination (control), interspecific crosses or apomixis, then recorded flowering phenology and synchrony. RESULTS: All species flowered in the spring with a degree of synchrony, so that two pairs of species were predisposed to interspecific pollination (O. cantabrigiensis with O. robusta, O. streptacantha with O. tomentosa). All species had distinct reproductive systems: Opuntia cantabrigiensis is self-incompatible and did not produce hybrid seeds as an interspecific pollen recipient; O. robusta is a dioecious species, which formed a low proportion of hybrid seeds; O. streptacantha and O. tomentosa are self-compatible and produced hybrid seeds. CONCLUSIONS: Opuntia cantabrigiensis had a strong pollen-pistil barrier, likely due to its self-incompatibility. Opuntia robusta, the dioecious species, is an obligate outcrosser and probably partially lost its ability to prevent interspecific pollen germination. Given that the self-compatible species can set hybrid seeds, we conclude that pollen-pistil interaction and high flowering synchrony represent weak barriers; whether reproductive isolation occurs later in their life cycle (e.g., germination or seedling survival) needs to be determined.

Molecular and evolutionary mechanisms of self-incompatibility in angiosperms.

As an intraspecific outcrossing mechanism, self-incompatibility (SI) widely adopted by hermaphroditic plants is usually controlled by a polymorphic multi-allelic S locus. Typically, six molecular types of SI have been found, including type-I controlled by the pistil S S-RNase and pollen S SLFs commonly spread in Plantaginaceae, Solanaceae, Rosaceae and Rutaceae, type-II by SRK and SCR in Brassicaceae, type-III by PrsS and PrpS in Papaveraceae, type-IV by CYP-GLO2-KFB-CCM-PUM in Primulaceae, type-V by TsSPH1-TsYUC6-TsBAHD in Turneraceae and type-VI by HPS10-S and DUF247I-S in Poaceae, with type-I characterized as a non-self recognition system but types-II, -III and -VI self ones. Furthermore, remarkable progresses have been made in their origin and evolutionary mechanisms recently. Among them, type-I SI possessed a single origin in the most recent common ancestor of eudicots and types II-V dynamically evolved following its losses, while type-VI SI exclusively existed in monocot Poaceae may be regained after the loss of the ancient type-I. Here, we mainly review the molecular and evolutionary mechanisms of angiosperm SI systems, thus providing a helpful reference for their theoretical research and breeding application.

Dominance in self-compatibility between subgenomes of allopolyploid Arabidopsis kamchatica shown by transgenic restoration of self-incompatibility.

The evolutionary transition to self-compatibility facilitates polyploid speciation. In Arabidopsis relatives, the self-incompatibility system is characterized by epigenetic dominance modifiers, among which small RNAs suppress the expression of a recessive SCR/SP11 haplogroup. Although the contribution of dominance to polyploid self-compatibility is speculated, little functional evidence has been reported. Here we employ transgenic techniques to the allotetraploid plant A. kamchatica. We find that when the dominant SCR-B is repaired by removing a transposable element insertion, self-incompatibility is restored. This suggests that SCR was responsible for the evolution of self-compatibility. By contrast, the reconstruction of recessive SCR-D cannot restore self-incompatibility. These data indicate that the insertion in SCR-B conferred dominant self-compatibility to A. kamchatica. Dominant self-compatibility supports the prediction that dominant mutations increasing selfing rate can pass through Haldane's sieve against recessive mutations. The dominance regulation between subgenomes inherited from progenitors contrasts with previous studies on novel epigenetic mutations at polyploidization termed genome shock.

Low copy numbers for mitochondrial DNA moderates the strength of nuclear-cytoplasmic incompatibility in plants.

Plant cells contain only small amounts of mitochondrial DNA (mtDNA), with the genomic information shared among multiple mitochondria. The biological relevance and molecular mechanism underlying this hallmark of plant cells has been unclear. Here, we report that Arabidopsis thaliana plants exhibited significantly reduced growth and mitochondrial dysfunction when the mtDNA copy number was increased to the degree that each mitochondrion possessed DNA. The amounts of mitochondrion-encoded transcripts increased several fold in the presence of elevated mtDNA levels. However, the efficiency of RNA editing decreased with this excess of mitochondrion-encoded transcripts, resulting in impaired assembly of mitochondrial complexes containing mtDNA-encoded subunits, such as respiratory complexes I and IV. These observations indicate the occurrence of nuclear-mitochondrial incompatibility in the cells with increased amounts of mtDNA and provide an initial answer to the fundamental question of why plant cells have much lower mtDNA levels than animal cells. We propose that keeping mtDNA levels low moderates nuclear-mitochondrial incompatibility and that this may be a crucial factor driving plant cells to restrict the copy numbers of mtDNA.

Genome-Wide Analysis of the RNase T2 Family and Identification of Interacting Proteins of Four ClS-RNase Genes in 'XiangShui' Lemon.

S-RNase plays vital roles in the process of self-incompatibility (SI) in Rutaceae plants. Data have shown that the rejection phenomenon during self-pollination is due to the degradation of pollen tube RNA by S-RNase. The cytoskeleton microfilaments of pollen tubes are destroyed, and other components cannot extend downwards from the stigma and, ultimately, cannot reach the ovary to complete fertilisation. In this study, four S-RNase gene sequences were identified from the 'XiangShui' lemon genome and ubiquitome. Sequence analysis revealed that the conserved RNase T2 domains within S-RNases in 'XiangShui' lemon are the same as those within other species. Expression pattern analysis revealed that S3-RNase and S4-RNase are specifically expressed in the pistils, and spatiotemporal expression analysis showed that the S3-RNase expression levels in the stigmas, styles and ovaries were significantly higher after self-pollination than after cross-pollination. Subcellular localisation analysis showed that the S1-RNase, S2-RNase, S3-RNase and S4-RNase were found to be expressed in the nucleus according to laser confocal microscopy. In addition, yeast two-hybrid (Y2H) assays showed that S3-RNase interacted with F-box, Bifunctional fucokinase/fucose pyrophosphorylase (FKGP), aspartic proteinase A1, RRP46, pectinesterase/pectinesterase inhibitor 51 (PME51), phospholipid:diacylglycerol acyltransferase 1 (PDAT1), gibberellin receptor GID1B, GDT1-like protein 4, putative invertase inhibitor, tRNA ligase, PAP15, PAE8, TIM14-2, PGIP1 and p24beta2. Moreover, S3-RNase interacted with TOPP4. Therefore, S3-RNase may play an important role in the SI of 'XiangShui' lemon.

Three types of genes underlying the Gametophyte factor1 locus cause unilateral cross incompatibility in maize.

Unilateral cross incompatibility (UCI) occurs between popcorn and dent corn, and represents a critical step towards speciation. It has been reported that ZmGa1P, encoding a pectin methylesterase (PME), is a male determinant of the Ga1 locus. However, the female determinant and the genetic relationship between male and female determinants at this locus are unclear. Here, we report three different types, a total of seven linked genes underlying the Ga1 locus, which control UCI phenotype by independently affecting pollen tube growth in both antagonistic and synergistic manners. These include five pollen-expressed PME genes (ZmGa1Ps-m), a silk-expressed PME gene (ZmPME3), and another silk-expressed gene (ZmPRP3), encoding a pathogenesis-related (PR) proteins. ZmGa1Ps-m confer pollen compatibility. Presence of ZmPME3 causes silk to reject incompatible pollen. ZmPRP3 promotes incompatibility pollen tube growth and thereby breaks the blocking effect of ZmPME3. In addition, evolutionary genomics analyses suggest that the divergence of the Ga1 locus existed before maize domestication and continued during breeding improvement. The knowledge gained here deepen our understanding of the complex regulation of cross incompatibility.

The proteome reveals the involvement of serine/threonine kinase in the recognition of self- incompatibility in almond.

The self-incompatibility recognition mechanism determines whether the gametophyte is successfully fertilized between pollen tube SCF (SKP1-CUL1-F-box-RBX1) protein and pistil S-RNase protein during fertilization is unclear. In this study, the pistils of two almond cultivars 'Wanfeng' and 'Nonpareil' were used as the experimental materials after self- and nonself/cross-pollination, and pistils from the stamen-removed flowers were used as controls. We used fluorescence microscopy to observe the development of pollen tubes after pollination and 4D-LFQ to detect the protein expression profiles of 'Wanfeng' and 'Nonpareil' pistils and in controls. The results showed that it took 24-36 h for the development of the pollen tube to 1/3 of the pistil, and a total of 7684 differentially accumulated proteins (DAPs) were identified in the pistil after pollinating for 36 h, of which 7022 were quantifiable. Bioinformatics analysis based on the function of DAPs, identified RNA polymerases (4 DAPs), autophagy (3 DAPs), oxidative phosphorylation (3 DAPs), and homologous recombination (2 DAPs) pathways associated with the self-incompatibility process. These results were confirmed by parallel reaction monitoring (PRM), protein interaction and bioinformatics analysis. Taken together, these results provide the involvement of serine/threonine kinase protein in the reaction of pollen tube recognition the nonself- and the self-S-RNase protein. SIGNIFICANCE: Gametophytic self-incompatibility (GSI) is controlled by the highly polymorphic S locus or S haplotype, with two linked self-incompatibility genes, one encoding the S-RNase protein of the pistil S-determinant and the other encoding the F-box/SLF/SFB (S haplotype-specific F-box protein) protein of the pollen S-determinant. The recognition mechanism between pollen tube SCF protein and pistil S-RNase protein is divided into nonself- and self-recognition hypothesis mechanisms. At present, two hypothetical mechanisms cannot explain the recognition between pollen and pistil well, so the mechanism of gametophytic self-incompatibility recognition is still not fully revealed. In this experiment, we investigated the molecular mechanism of pollen-pistil recognition in self-incompatibility using self- and nonself-pollinated pistils of almond cultivars 'Wanfeng' and 'Nonpareil'. Based on our results, we proposed a potential involvement of the MARK2 (serine/threonine kinase) protein in the reaction of pollen tube recognition of the nonself- and the self-S-RNase protein. It provides a new way to reveal how almond pollen tubes recognize the self and nonself S-RNase enzyme protein.

Origin, loss, and regain of self-incompatibility in angiosperms.

The self-incompatibility (SI) system with the broadest taxonomic distribution in angiosperms is based on multiple S-locus F-box genes (SLFs) tightly linked to an S-RNase termed type-1. Multiple SLFs collaborate to detoxify nonself S-RNases while being unable to detoxify self S-RNases. However, it is unclear how such a system evolved, because in an ancestral system with a single SLF, many nonself S-RNases would not be detoxified, giving low cross-fertilization rates. In addition, how the system has been maintained in the face of whole-genome duplications (WGDs) or lost in other lineages remains unclear. Here we show that SLFs from a broad range of species can detoxify S-RNases from Petunia with a high detoxification probability, suggestive of an ancestral feature enabling cross-fertilization and subsequently modified as additional SLFs evolved. We further show, based on its genomic signatures, that type-1 was likely maintained in many lineages, despite WGD, through deletion of duplicate S-loci. In other lineages, SI was lost either through S-locus deletions or by retaining duplications. Two deletion lineages regained SI through type-2 (Brassicaceae) or type-4 (Primulaceae), and one duplication lineage through type-3 (Papaveraceae) mechanisms. Thus, our results reveal a highly dynamic process behind the origin, maintenance, loss, and regain of SI.

Challenges and Perspectives in the Study of Self-Incompatibility in Orchids.

Self-incompatibility affects not only the formation of seeds, but also the evolution of species diversity. A robust understanding of the molecular mechanisms of self-incompatibility is essential for breeding efforts, as well as conservation biology research. In recent years, phenotypic and multiple omics studies have revealed that self-incompatibility in Orchidaceae is mainly concentrated in the subfamily Epidendroideae, and the self-incompatibility phenotypes are diverse, even in the same genus, and hormones (auxin and ethylene), and new male and female determinants might be involved in SI response. This work provides a good foundation for future studies of the evolution and molecular mechanisms of self-incompatibility. We review recent research progress on self-incompatibility in orchids at the morphological, physiological, and molecular levels, provide a general overview of self-incompatibility in orchids, and propose future research directions.

Polyamines Involved in Regulating Self-Incompatibility in Apple.

Apple exhibits typical gametophytic self-incompatibility, in which self-S-RNase can arrest pollen tube growth, leading to failure of fertilization. To date, there have been few studies on how to resist the toxicity of self-S-RNase. In this study, pollen tube polyamines were found to respond to self-S-RNase and help pollen tubes defend against self-S-RNase. In particular, the contents of putrescine, spermidine, and spermine in the pollen tube treated with self-S-RNase were substantially lower than those treated with non-self-S-RNase. Further analysis of gene expression of key enzymes in the synthesis and degradation pathways of polyamines found that the expression of DIAMINE OXIDASE 4 (MdDAO4) as well as several polyamine oxidases such as POLYAMINE OXIDASES 3 (MdPAO3), POLYAMINE OXIDASES 4 (MdPAO4), and POLYAMINE OXIDASES 6 (MdPAO6) were significantly up-regulated under self-S-RNase treatment, resulting in the reduction of polyamines. Silencing MdPAO6 in pollen tubes alleviates the inhibitory effect of self-S-RNase on pollen tube growth. In addition, exogenous polyamines also enhance pollen tube resistance to self-S-RNase. Transcriptome sequencing data found that polyamines may communicate with S-RNase through the calcium signal pathway, thereby regulating the growth of the pollen tubes. To summarize, our results suggested that polyamines responded to the self-incompatibility reaction and could enhance pollen tube tolerance to S-RNase, thus providing a potential way to break self-incompatibility in apple.

Widespread coexistence of self-compatible and self-incompatible phenotypes in a diallelic self-incompatibility system in Ligustrum vulgare (Oleaceae).

The breakdown of self-incompatibility (SI) in angiosperms is one of the most commonly observed evolutionary transitions. While multiple examples of SI breakdown have been documented in natural populations, there is strikingly little evidence of stable within-population polymorphism with both inbreeding (self-compatible) and outcrossing (self-incompatible) individuals. This absence of breeding system polymorphism corroborates theoretical expectations that predict that in/outbreeding polymorphism is possible only under very restricted conditions. However, theory also predicts that a diallelic sporophytic SI system should facilitate the maintenance of such polymorphism. We tested this prediction by studying the breeding system of Ligustrum vulgare L., an insect-pollinated hermaphroditic species of the Oleaceae family. Using stigma tests with controlled pollination and paternity assignment of open-pollinated progenies, we confirmed the existence of two self-incompatibility groups in this species. We also demonstrated the occurrence of self-compatible individuals in different populations of Western Europe arising from a mutation affecting the functioning of the pollen component of SI. Our results show that the observed low frequency of self-compatible individuals in natural populations is compatible with theoretical predictions only if inbreeding depression is very high.

A nonS-locus F-box gene breaks self-incompatibility in diploid potatoes.

Potato is the third most important staple food crop. To address challenges associated with global food security, a hybrid potato breeding system, aimed at converting potato from a tuber-propagated tetraploid crop into a seed-propagated diploid crop through crossing inbred lines, is under development. However, given that most diploid potatoes are self-incompatible, this represents a major obstacle which needs to be addressed in order to develop inbred lines. Here, we report on a self-compatible diploid potato, RH89-039-16 (RH), which can efficiently induce a mating transition from self-incompatibility to self-compatibility, when crossed to self-incompatible lines. We identify the S-locusinhibitor (Sli) gene in RH, capable of interacting with multiple allelic variants of the pistil-specific S-ribonucleases (S-RNases). Further, Sli gene functions like a general S-RNase inhibitor, to impart SC to RH and other self-incompatible potatoes. Discovery of Sli now offers a path forward for the diploid hybrid breeding program.

Neofunctionalisation of the Sli gene leads to self-compatibility and facilitates precision breeding in potato.

Genetic gain in potato is hampered by the heterozygous tetraploid genome of cultivated potato. Converting potato into a diploid inbred-line based F1-hybrid crop provides a promising route towards increased genetic gain. The introduction of a dominant S-locus inhibitor (Sli) gene into diploid potato germplasm allows efficient generation of self-fertilized seeds and thus the development of potato inbred lines. Little is known about the structure and function of the Sli locus. Here we describe the mapping of Sli to a 12.6 kb interval on chromosome 12 using a recombinant screen approach. One of two candidate genes present in this interval shows a unique sequence that is exclusively present in self-compatible lines. We describe an expression vector that converts self-incompatible genotypes into self-compatible and a CRISPR-Cas9 vector that converts SC genotypes into SI. The Sli gene encodes an F-box protein that is specifically expressed in pollen from self-compatible plants. A 533 bp insertion in the promotor of that gene leads to a gain of function mutation, which overcomes self-pollen rejection.

Plant biology: Stigmatic ROS decide whether pollen is accepted or rejected.

A new study reports that self-incompatibility in Brassica triggers the production of stigmatic ROS that are responsible for the rejection of incompatible pollen.

RNase-based self-incompatibility in cacti.

Approximately one-half of all flowering plants express genetically based physiological mechanisms that prevent self-fertilisation. One such mechanism, termed RNase-based self-incompatibility, employs ribonucleases as the pistil component. Although it is widespread, it has only been characterised in a handful of distantly related families, partly due to the difficulties presented by life history traits of many plants, which complicate genetic research. Many species in the cactus family are known to express self-incompatibility but the underlying mechanisms remain unknown. We demonstrate the utility of a candidate-based RNA-seq approach, combined with some unusual features of self-incompatibility-causing genes, which we use to uncover the genetic basis of the underlying mechanisms. Specifically, we assembled transcriptomes from Schlumbergera truncata (crab cactus or false Christmas cactus), and interrogated them for tissue-specific expression of candidate genes, structural characteristics, correlation with expressed phenotype(s), and phylogenetic placement. The results were consistent with operation of the RNase-based self-incompatibility mechanism in Cactaceae. The finding yields additional evidence that the ancestor of nearly all eudicots possessed RNase-based self-incompatibility, as well as a clear path to better conservation practices for one of the most charismatic plant families.