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As self-incompatibility is a major issue in pummelo breeding and production, its mechanism in citrus was analyzed to improve breeding efficiency and reduce production costs. Rutaceae belongs to S-RNase type of gametophytic self-incompatibility. While the function of S-RNase/SLF and the mechanism of self-incompatibility have been studied extensively, the transcriptional regulation of S-RNase has been less studied. We performed transcriptome sequencing with the styles of ‘Shatian’ pummelo on the day of anthesis and 1–5 days before anthesis, and found that the transcript level of S-RNase gradually decreased with flower development. By analyzing differentially expressed genes and correlation with the expression trend of S-RNase, we identified a candidate gene, CgHSFB1, and utilized biochemical experiments such as yeast one-hybrid assay, electrophoretic mobility shift assay and dual-luciferase assay, as well as transient transformation of citrus calli and Citrus microcarpa and demonstrated that CgHSFB1 could directly bind to the S1-RNase promoter and repress the expression of S1-RNase, which is involved in the pummelo self-incompatibility response. In contrast, CgHSFB1 did not bind to the promoter of S2-RNase, and there was specificity in the regulation of S-RNase.Key message: Transcription factor CgHSFB1 regulates the expression of S -RNase and is thus involved in self-incompatibility in citrus. [ABSTRACT FROM AUTHOR]

Summary: Self‐incompatibility (SI) is a widespread prezygotic mechanism for flowering plants to avoid inbreeding depression and promote genetic diversity. Citrus has an S‐RNase‐based SI system, which was frequently lost during evolution. We previously identified a single nucleotide mutation in Sm‐RNase, which is responsible for the loss of SI in mandarin and its hybrids. However, little is known about other mechanisms responsible for conversion of SI to self‐compatibility (SC) and we identify a completely different mechanism widely utilized by citrus. Here, we found a 786‐bp miniature inverted‐repeat transposable element (MITE) insertion in the promoter region of the FhiS2‐RNase in Fortunella hindsii Swingle (a model plant for citrus gene function), which does not contain the Sm‐RNase allele but are still SC. We demonstrate that this MITE plays a pivotal role in the loss of SI in citrus, providing evidence that this MITE insertion prevents expression of the S‐RNase; moreover, transgenic experiments show that deletion of this 786‐bp MITE insertion recovers the expression of FhiS2‐RNase and restores SI. This study identifies the first evidence for a role for MITEs at the S‐locus affecting the SI phenotype. A family‐wide survey of the S‐locus revealed that MITE insertions occur frequently adjacent to S‐RNase alleles in different citrus genera, but only certain MITEs appear to be responsible for the loss of SI. Our study provides evidence that insertion of MITEs into a promoter region can alter a breeding strategy and suggests that this phenomenon may be broadly responsible for SC in species with the S‐RNase system. [ABSTRACT FROM AUTHOR]

Self‐incompatibility (SI) is an intraspecific reproductive barrier widely present in angiosperms. The SI system with the broadest occurrence in angiosperms is based on an S‐RNase linked to a cluster of multiple S‐locus F‐box (SLF) genes found in the Solanaceae, Plantaginaceae, Rosaceae, and Rutaceae. Recent studies reveal that non‐self S‐RNase is degraded by the Skip Cullin F‐box (SCF)SLF‐mediated ubiquitin–proteasome system in a collaborative manner in Petunia, but how self‐RNase functions largely remains mysterious. Here, we show that S‐RNases form S‐RNase condensates (SRCs) in the self‐pollen tube cytoplasm through phase separation and the disruption of SRC formation breaks SI in self‐incompatible Petunia hybrida. We further find that the pistil SI factors of a small asparagine‐rich protein HT‐B and thioredoxin h together with a reduced state of the pollen tube all promote the expansion of SRCs, which then sequester several actin‐binding proteins, including the actin polymerization factor PhABRACL, the actin polymerization activity of which is reduced by S‐RNase in vitro. Meanwhile, we find that S‐RNase variants lacking condensation ability fail to recruit PhABRACL and are unable to induce actin foci formation required for pollen tube growth inhibition. Taken together, our results demonstrate that phase separation of S‐RNase promotes SI response in P. hybrida, revealing a new mode of S‐RNase action. [ABSTRACT FROM AUTHOR]

The S-RNase gene plays an essential role in the gametophytic self-incompatibility (GSI) system of Pyrus. It codes for the stylar-expressed S-RNase protein which inhibits the growth of incompatible pollen tubes through cytotoxicity and the induction of programmed cell death in the pollen tube. While research on the Pyrus GSI system has primarily focused on the S-RNase gene, there is still a lack of insight into its spatiotemporal expression profile and the factors that regulate it. Previous studies have suggested that S-RNase expression in the style is influenced by pollination and is dependent on the compatibility type. We here continue on this basic hypothesis by analyzing the spatiotemporal expression of the S-RNase alleles in Pyrus communis "Conference" styles in response to different types of pollination; namely, upon full- and semi-compatible pollination and upon incompatible selfing. The results revealed that temporal dynamics of S-RNase expression are influenced by the pollen's compatibility type, indicating the presence of a signaling mechanism between pollen and style to control S-RNase production during pollen tube growth. In our experiment, S-RNase expression continuously decreased after cross-pollination and in the unpollinated control. However, after a fully incompatible pollination, S-RNase expression remained constant. Finally, semi-compatible pollination showed a initially constant S-RNase expression for both alleles followed by a strong decrease in expression. Based on these results and previous findings, we propose a regulatory mechanism to explain the effect of pollination and the associated compatibility type on S-RNase expression in the style. This proposed mechanism could be used as a starting point for future research. [ABSTRACT FROM AUTHOR]

Sour cherry is a tetraploid species, and gametophytic self-incompatibility (SI) operates in this species in the same way as in other stone fruit trees. However, while self-compatibility is most common in sour cherry and self-compatibility (SC) genotypes are rarely found, both SI and self-compatible (SC) types are selected in sour cherry. In this work, S-alleles have been identified for 70 sour cherry accessions and cultivars from the Shabestar regions of Iran, with S-genotypes of 68 cultivars identified for the first time. To identify the S-alleles, PCR-based methods were used. The amplification of the different alleles using combinations of the four forward primers (PaConsI-F, PruC2, PaConsII-F, EM-PC2consFD) and the five reverse primers (PruC4R, PCE-R, PaConsI-Rnew, PaConsII-R, EM-PC5consRD) revealed that they were the most useful for the identification of the sour cherry alleles. Nine known S-haplotypes (S6, S4, S9, S6m, S6m2, S24, S26, S35, S36a) were identified. In our study, alleles S6, S9, and S6m2 had a high frequency. It was shown that the consensus primers can be used to detect incompatibility alleles in sour cherry accessions. Our study has proved that the diversity of S alleles between the studied accessions was low, indicating low genetic diversity, which could also be due to the selection of superior genotypes by farmers.

Self-incompatibility (SI) is a genetic mechanism common in flowering plants to prevent self-fertilization. Among citrus species, several pummelo, mandarin, and mandarin-like accessions show SI behavior. In these species, SI is coupled with a variable degree of parthenocarpy ensuring the production of seedless fruits, a trait that is highly appreciated by consumers. In Citrus, recent evidences have shown the presence of a gametophytic SI system based on S-ribonucleases (SRNases) ability to impair self-pollen tube growth in the upper/middle part of the style. In the present study, we combined PCR analysis and next-generation sequencing technologies, to define the presence of S7- and S11-Rnases in the Sgenotype of the Citrus clementina (Hort. ex Tan.), the self-incompatible ‘Comune’ clementine and its self-compatible natural mutant ‘Monreal’. The reference genome of ‘Monreal’ clementine is presented for the first time, providing more robust results on the genetic sequence of the newly discovered S7-RNase. SNP discovery analysis coupled with the annotation of the variants detected enabled the identification of 7,781 SNPs effecting 5,661 genes in ‘Monreal’ compared to the reference genome of C. clementina. Transcriptome analysis of unpollinated pistils at the mature stage from both clementine genotypes revealed the lack of expression of S7-RNase in ‘Monreal’ suggesting its involvement in the loss of the SI response. RNA-seq analysis followed by gene ontology studies enabled the identification of 2,680 differentially expressed genes (DEGs), a significant number of those is involved in oxidoreductase and transmembrane transport activity. Merging of DNA sequencing and RNA data led to the identification of 164 DEGs characterized by the presence of at least one SNP predicted to induce mutations with a high effect on their amino acid sequence. Among them, four candidate genes referring to two Agamous-like MADS-box proteins, to MYB111 and to MLO-like protein 12 were validated. Moreover, the transcription factor MYB111 appeared to contain a binding site for the 2.0-kb upstream sequences of the S7- and S11- RNase genes. These results provide useful information about the genetic bases of SI indicating that SNPs present in their sequence could be responsible for the differential expression and the regulation of S7-RNase and consequently of the SI mechanism. [ABSTRACT FROM AUTHOR]

The S-RNase gene plays an essential role in the gametophytic self-incompatibility (GSI) system of Pyrus. It codes for the stylar-expressed S-RNase protein which inhibits the growth of incompatible pollen tubes through cytotoxicity and the induction of programmed cell death in the pollen tube. While research on the Pyrus GSI system has primarily focused on the S-RNase gene, there is still a lack of insight into its spatiotemporal expression profile and the factors that regulate it. Previous studies have suggested that S-RNase expression in the style is influenced by pollination and is dependent on the compatibility type. We here continue on this basic hypothesis by analyzing the spatiotemporal expression of the S-RNase alleles in Pyrus communis “Conference” styles in response to different types of pollination; namely, upon full- and semi-compatible pollination and upon incompatible selfing. The results revealed that temporal dynamics of S-RNase expression are influenced by the pollen’s compatibility type, indicating the presence of a signaling mechanism between pollen and style to control S-RNase production during pollen tube growth. In our experiment, S-RNase expression continuously decreased after cross-pollination and in the unpollinated control. However, after a fully incompatible pollination, S-RNase expression remained constant. Finally, semi-compatible pollination showed a initially constant S-RNase expression for both alleles followed by a strong decrease in expression. Based on these results and previous findings, we propose a regulatory mechanism to explain the effect of pollination and the associated compatibility type on S-RNase expression in the style. This proposed mechanism could be used as a starting point for future research.

Abstract Due to self-incompatibility (SI) prevents self-fertilization, natural or artificial cross-pollination has been conducted in many orchards to stabilize fruit yield. However, it is still puzzled which routes of self S-RNase arresting pollen tube growth. Herein, 17 COBRA genes were isolated from pear genome. Of these genes, the pollen-specifically expressed PbCOB.A.1 and PbCOB.A.2 positively mediates pollen tube growth. The promoters of PbCOB.A.1 and/or PbCOB.A.2 were bound and activated by PbABF.E.2 (an ABRE-binding factor) and PbC2H2.K16.2 (a C2H2-type zinc finger protein). Notably, the expressions of PbCOB.A.1, PbCOB.A.2, and PbC2H2.K16.2 were repressed by self S-RNase, suggesting that self S-RNase reduces the expression of PbCOB.A.1 and PbCOB.A.2 by decreasing the expression of their upstream factors, such as PbC2H2.K16.2, to arrest pollen tube growth. PbCOB.A.1 or PbCOB.A.2 accelerates the growth of pollen tubes treated by self S-RNase, but can hardly affect level of reactive oxygen species and deploymerization of actin cytoskeleton in pollen tubes and cannot physically interact with any reported proteins involved in SI. These results indicate that PbCOB.A.1 and PbCOB.A.2 may not relieve S-RNase toxicity in incompatible pollen tube. The information provides a new route to elucidate the arresting pollen tube growth during SI reaction. Graphical Abstract

Tomato (Solanum lycopersicum L.) domestication and later introduction into Europe resulted in a genetic bottleneck that reduced genetic variation. Crosses with other wild tomato species from the Lycopersicon clade can be used to increase genetic diversity and improve important agronomic traits such as stress tolerance. However, many species in the Lycopersicon clade have intraspecific and interspecific incompatibility, such as gametophytic self-incompatibility and unilateral incompatibility. In this review, we provide an overview of the known incompatibility barriers in Lycopersicon. We begin by addressing the general mechanisms self-incompatibility, as well as more specific mechanisms in the Rosaceae, Papaveraceae, and Solanaceae. Incompatibility in the Lycopersicon clade is discussed, including loss of self-incompatibility, species exhibiting only self-incompatibility and species presenting both self-compatibility and selfincompatibility. We summarize unilateral incompatibility in general and specifically in Lycopersicon, with details on the ’self-compatible x selfincompatible’ rule, implications of self-incompatibility in unilateral incompatibility and self-incompatibility-independent pathways of unilateral incompatibility. Finally, we discuss advances in the understanding of compatibility barriers and their implications for tomato breeding. [ABSTRACT FROM AUTHOR]

Due to self-incompatibility (SI) prevents self-fertilization, natural or artificial cross-pollination has been conducted in many orchards to stabilize fruit yield. However, it is still puzzled which routes of self S-RNase arresting pollen tube growth. Herein, 17 COBRA genes were isolated from pear genome. Of these genes, the pollen-specifically expressed PbCOB.A.1 and PbCOB.A.2 positively mediates pollen tube growth. The promoters of PbCOB.A.1 and/or PbCOB.A.2 were bound and activated by PbABF.E.2 (an ABRE-binding factor) and PbC2H2.K16.2 (a C2H2-type zinc finger protein). Notably, the expressions of PbCOB.A.1, PbCOB.A.2, and PbC2H2.K16.2 were repressed by self S-RNase, suggesting that self S-RNase reduces the expression of PbCOB.A.1 and PbCOB.A.2 by decreasing the expression of their upstream factors, such as PbC2H2.K16.2, to arrest pollen tube growth. PbCOB.A.1 or PbCOB.A.2 accelerates the growth of pollen tubes treated by self S-RNase, but can hardly affect level of reactive oxygen species and deploymerization of actin cytoskeleton in pollen tubes and cannot physically interact with any reported proteins involved in SI. These results indicate that PbCOB.A.1 and PbCOB.A.2 may not relieve S-RNase toxicity in incompatible pollen tube. The information provides a new route to elucidate the arresting pollen tube growth during SI reaction. [ABSTRACT FROM AUTHOR]

Self-incompatibility (SI) is a genetic mechanism common in flowering plants to prevent self-fertilization. Among citrus species, several pummelo, mandarin, and mandarin-like accessions show SI behavior. In these species, SI is coupled with a variable degree of parthenocarpy ensuring the production of seedless fruits, a trait that is highly appreciated by consumers. In Citrus, recent evidences have shown the presence of a gametophytic SI system based on S-ribonucleases (S-RNases) ability to impair self-pollen tube growth in the upper/middle part of the style. In the present study, we combined PCR analysis and next-generation sequencing technologies, to define the presence of S7- and S11-Rnases in the S-genotype of the Citrus clementina (Hort. ex Tan.), the self-incompatible ‘Comune’ clementine and its self-compatible natural mutant ‘Monreal’. The reference genome of ‘Monreal’ clementine is presented for the first time, providing more robust results on the genetic sequence of the newly discovered S7-RNase. SNP discovery analysis coupled with the annotation of the variants detected enabled the identification of 7,781 SNPs effecting 5,661 genes in ‘Monreal’ compared to the reference genome of C. clementina. Transcriptome analysis of unpollinated pistils at the mature stage from both clementine genotypes revealed the lack of expression of S7-RNase in ‘Monreal’ suggesting its involvement in the loss of the SI response. RNA-seq analysis followed by gene ontology studies enabled the identification of 2,680 differentially expressed genes (DEGs), a significant number of those is involved in oxidoreductase and transmembrane transport activity. Merging of DNA sequencing and RNA data led to the identification of 164 DEGs characterized by the presence of at least one SNP predicted to induce mutations with a high effect on their amino acid sequence. Among them, four candidate genes referring to two Agamous-like MADS-box proteins, to MYB111 and to MLO-like protein 12 were validated. Moreover, the transcription factor MYB111 appeared to contain a binding site for the 2.0-kb upstream sequences of the S7- and S11-RNase genes. These results provide useful information about the genetic bases of SI indicating that SNPs present in their sequence could be responsible for the differential expression and the regulation of S7-RNase and consequently of the SI mechanism.

Tomato (Solanum lycopersicum L.) domestication and later introduction into Europe resulted in a genetic bottleneck that reduced genetic variation. Crosses with other wild tomato species from the Lycopersicon clade can be used to increase genetic diversity and improve important agronomic traits such as stress tolerance. However, many species in the Lycopersicon clade have intraspecific and interspecific incompatibility, such as gametophytic self-incompatibility and unilateral incompatibility. In this review, we provide an overview of the known incompatibility barriers in Lycopersicon. We begin by addressing the general mechanisms self-incompatibility, as well as more specific mechanisms in the Rosaceae, Papaveraceae, and Solanaceae. Incompatibility in the Lycopersicon clade is discussed, including loss of self-incompatibility, species exhibiting only self-incompatibility and species presenting both self-compatibility and self-incompatibility. We summarize unilateral incompatibility in general and specifically in Lycopersicon, with details on the ’self-compatible x self-incompatible’ rule, implications of self-incompatibility in unilateral incompatibility and self-incompatibility-independent pathways of unilateral incompatibility. Finally, we discuss advances in the understanding of compatibility barriers and their implications for tomato breeding.

Self-incompatible pitaya varieties have low fruit-setting rates under natural conditions, leading to higher production costs and hindering industrial prosperity. Through transcriptome sequencing, we obtained the 36,900 longest transcripts (including 9167 new transcripts) from 60 samples of flowers. Samples were collected pre- and post-pollination (at 0 h, 0.5 h, 2 h, 4 h, and 12 h) from two varieties of pitaya (self-compatible Jindu No. 1 and self-incompatible Cu Sha). Using the RNA-Seq data and comparison of reference genomes, we annotated 28,817 genes in various databases, and 1740 genes were optimized in their structure for annotation. There were significant differences in the expression of differentially expressed genes (DEGs) in the pitaya stigmas under different pollination types, especially at the late post-pollination stage, where the expression of protease genes increasedal significantly under cross-pollination. We identified DEGs involved in the ribosomal, ubiquitination-mediated, and phyto-signaling pathways that may be involved in pitaya SI regulation. Based on the available transcriptome data and bioinformatics analysis, we tentatively identified HuS-RNase2 as a candidate gynogenetic S gene in the pitaya GSI system. [ABSTRACT FROM AUTHOR]

The T2/RNase gene family is widespread in eukaryotes, and particular members of this family play critical roles in the gametophytic self-incompatibility (GSI) system in plants. Wild diploid strawberry (Fragaria) species have diversified their sexual systems via self-incompatible and self-compatible traits, yet how these traits evolved in Fragaria remains elusive. By integrating the published and de novo assembled genomes and the newly generated RNA-seq data, members of the RNase T2 gene family were systematically identified in six Fragaria species, including three self-incompatible species (Fragaria nipponica, Fragaria nubicola, and Fragaria viridis) and three self-compatible species (Fragaria nilgerrensis, Fragaria vesca, and Fragaria iinumae). In total, 115 RNase T2 genes were identified in the six Fragaria genomes and can be classified into three classes (I–III) according to phylogenetic analysis. The identified RNase T2 genes could be divided into 22 homologous gene sets according to amino acid sequence similarity and phylogenetic and syntenic relationships. We found that extensive gene loss and pseudogenization coupled with small-scale duplications mainly accounted for variations in the RNase T2 gene numbers in Fragaria. Multiple copies of homologous genes were mainly generated from tandem and segmental duplication events. Furthermore, we newly identified five S-RNase genes in three self-incompatible Fragaria genomes, including two in F. nipponica, two in F. viridis, and one in F. nubicola, which fit for typical features of a pistil determinant, including highly pistil-specific expression, highly polymorphic proteins and alkaline isoelectric point (pI), while no S-RNase genes were found in all three self-compatible Fragaria species. Surprisingly, these T2/S-RNase genes contain at least one large intron (>10 kb). This study revealed that the rapid evolution of T2/S-RNase genes within the Fragaria genus could be associated with its sexual mode, and repeated evolution of the self-compatible traits in Fragaria was convergent via losses of S-RNase.

The Gametophytic Self-Incompatibility (GSI) system in diploid potato (Solanum tuberosum L.) poses a substantial barrier in diploid potato breeding by hindering the generation of inbred lines. One solution is gene editing to generate selfcompatible diploid potatoes which will allow for the generation of elite inbred lines with fixed favorable alleles and heterotic potential. The S-RNase and HT genes have been shown previously to contribute to GSI in the Solanaceae family and self-compatible S. tuberosum lines have been generated by knocking out SRNase gene with CRISPR-Cas9 gene editing. This study employed CRISPR-Cas9 to knockout HT-B either individually or in concert with S-RNase in the diploid self-incompatible S. tuberosum clone DRH-195. Using mature seed formation from self-pollinated fruit as the defining characteristic of self-compatibility, HTB-only knockouts produced little or no seed. In contrast, double knockout lines of HT-B and S-RNase displayed levels of seed production that were up to three times higher than observed in the S-RNase-only knockout, indicating a synergistic effect between HT-B and S-RNase in self-compatibility in diploid potato. This contrasts with compatible cross-pollinations, where S-RNase and HT-B did not have a significant effect on seed set. Contradictory to the traditional GSI model, self-incompatible lines displayed pollen tube growth reaching the ovary, yet ovules failed to develop into seeds indicating a potential late-acting self-incompatibility in DRH-195. Germplasm generated from this study will serve as a valuable resource for diploid potato breeding. [ABSTRACT FROM AUTHOR]

The relationships of interspecific compatibility and incompatibility in Solanum section Petota are complex. Inquiry into these relationships in tomato and its wild relatives has elucidated the pleiotropic and redundant function of S-RNase and HT which tandemly and independently mediate both interspecific and intraspecific pollen rejection. Our findings presented here are consistent with previous work conducted in Solanum section Lycopersicon showing that S-RNase plays a central role in interspecific pollen rejection. Statistical analyses also demonstrated that HT-B alone is not a significant factor in these pollinations; demonstrating the overlap in gene function between HT-A and HT-B, as HT-A, was present and functional in all genotypes used. We were not able to replicate the general absence of prezygotic stylar barriers observable in S. verrucosum, which has been attributed to the lack of S-RNase, indicating that other non-S-RNase factors play a significant role. We also demonstrated that Sli played no significant role in these interspecific pollinations, directly conflicting with previous research. It is possible that S. chacoense as a pollen donor is better able to bypass stylar barriers in 1EBN species such as S. pinnatisectum. Consequently, S. chacoense may be a valuable resource in accessing these 1EBN species regardless of Sli status. [ABSTRACT FROM AUTHOR]

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The Chinese pear (Pyrus spp.) exhibits typical gametophytic self-incompatibility (GSI), which inhibits self-crossing and promotes out-crossing, similar to other fruit species in the Rosaceae family. Thus, S-compatible cultivars are required in pear orchards to ensure successful pollination and stable yields. In this study, 84 native Chinese pear accessions were genotyped by allele-specific PCR using one pair allele consensus primers and 29 pairs of S-allele-specific primers that were designed in this study. After cloning and sequencing the PCR products, the S-genotypes of all 84 pear accessions, including wild and cultivated accessions, were determined. The reported 34 S-alleles and a novel S-allele were isolated from these pear accessions. These S-alleles were expressed specifically in the style. Sequence analysis identified that six pear cultivars originated in China shared the same S-RNases with P. communis (Pc). These findings supported the hypothesis that oriental and occidental Pyrus spp. may share the same pool of alleles at the S-locus. A novel S-RNase was isolated in ‘Putiandouli’, ‘Daguoshanli’, ‘Yunhongli 1′, and ‘Dianli’ and deposited as S67-RNase under accession number MT773568. Furthermore, the deduced amino acid sequences exhibited high similarity (99.56%) to S32-RNase in Malus. The high similarity between S-RNase in Pyrus and Malus indicated that the existence of S-RNase could have predated speciation between Pyrus and Malus. Furthermore, S-allele information was rearranged in Asian and European pears to provide information for selecting the best pollinator for widely cultivated pear cultivars in China. This information is useful for pear production, cross-breeding, and understanding the mechanism of the self-incompatibility reaction.

SUMMARY: Gametophytic self‐incompatibility (GSI) has been widely studied in flowering plants, but studies of the mechanisms underlying pollen tube growth arrest by self S‐RNase in GSI species are limited. In the present study, two leucine‐rich repeat extensin genes in pear (Pyrus bretschneideri), PbLRXA2.1 and PbLRXA2.2, were identified based on transcriptome and quantitative real‐time PCR analyses. The expression levels of these two LRX genes were significantly higher in the pollen grains and pollen tubes of the self‐compatible cultivar 'Jinzhui' (harboring a spontaneous bud mutation) than in those of the self‐incompatible cultivar 'Yali'. Both PbLRXA2.1 and PbLRXA2.2 stimulated pollen tube growth and attenuated the inhibitory effects of self S‐RNase on pollen tube growth by stabilizing the actin cytoskeleton and enhancing cell wall integrity. These results indicate that abnormal expression of PbLRXA2.1 and PbLRXA2.2 is involved in the loss of self‐incompatibility in 'Jinzhui'. The PbLRXA2.1 and PbLRXA2.2 promoters were directly bound by the ABRE‐binding factor PbABF.D.2. Knockdown of PbABF.D.2 decreased PbLRXA2.1 and PbLRXA2.2 expression and inhibited pollen tube growth. Notably, the expression of PbLRXA2.1, PbLRXA2.2, and PbABF.D.2 was repressed by self S‐RNase, suggesting that self S‐RNase can arrest pollen tube growth by restricting the PbABF.D.2–PbLRXA2.1/PbLRXA2.2 signal cascade. These results provide novel insight into pollen tube growth arrest by self S‐RNase. Significance Statement: PbLRXA2.1 and PbLRX.A2.2 mediate pollen tube growth and attenuate the inhibitory effects of self S‐RNase on pollen tube growth by stabilizing the actin cytoskeleton and enhancing cell wall integrity. Self S‐RNase can arrest pollen tube growth by restricting the PbABF.D.2–PbLRXA2.1/PbLRXA2.2 signal cascade. [ABSTRACT FROM AUTHOR]

The Gametophytic Self-Incompatibility (GSI) system in diploid potato (Solanum tuberosum L.) poses a substantial barrier in diploid potato breeding by hindering the generation of inbred lines. One solution is gene editing to generate self-compatible diploid potatoes which will allow for the generation of elite inbred lines with fixed favorable alleles and heterotic potential. The S-RNase and HT genes have been shown previously to contribute to GSI in the Solanaceae family and self-compatible S. tuberosum lines have been generated by knocking out S-RNase gene with CRISPR-Cas9 gene editing. This study employed CRISPR-Cas9 to knockout HT-B either individually or in concert with S-RNase in the diploid self-incompatible S. tuberosum clone DRH-195. Using mature seed formation from self-pollinated fruit as the defining characteristic of self-compatibility, HT-B-only knockouts produced little or no seed. In contrast, double knockout lines of HT-B and S-RNase displayed levels of seed production that were up to three times higher than observed in the S-RNase-only knockout, indicating a synergistic effect between HT-B and S-RNase in self-compatibility in diploid potato. This contrasts with compatible cross-pollinations, where S-RNase and HT-B did not have a significant effect on seed set. Contradictory to the traditional GSI model, self-incompatible lines displayed pollen tube growth reaching the ovary, yet ovules failed to develop into seeds indicating a potential late-acting self-incompatibility in DRH-195. Germplasm generated from this study will serve as a valuable resource for diploid potato breeding.

سیب (Malus spp.) یکی از مهم­ترین درختان میوه مناطق معتدله است. رنگ گوشت میوه سیب، از سفید تا قرمز متغیر است و تجمع مقدار بالایی از آنتوسیانین باعث قرمز بودن گوشت میوه آن می‌باشد، چنین افزایشی در مقدار آنتوسیانین در بسیاری از موارد به دلیل بیان بالای ژن MdMYB10 در بسیاری از بافت­ها از جمله گوشت میوه است، با این وجود،‌ در مواردی،‌ یک مکان ژنی پیوسته با آلل S3 مکان ژنی S-RNase، به عنوان مسئول قرمزی گوشت میوه در بعضی از ژنوتیپ­ها پیشنهاد شده است. برای بررسی سازوکار قرمز بودن رنگ گوشت میوه در برخی ژنوتیپ­های محلی، استخراج DNA ژنومی از نمونه­های برگی بدست آمده از 9 ژنوتیپ سیب با گوشت قرمز و گوشت سفید انجام شد و آللوتیپ آنها از نظر ناحیه پیشبری ژن MdMYB10 و نیز وجود آلل S3 در مکان ژنی S-RNase با استفاده از تکنیک واکنش زنجیره‌ای پلی‌مراز برای تکثیر با آغازگرهای اختصاصی هر ژن و الکتروفورز ژل آگارز قطعات تکثیر شده، مورد ارزیابی قرار گرفت. نتایج این پژوهش نشان داد که ژنوتیپ‌های گوشت سفید در ناحیه پیشبری ژن MdMYB10 فاقد آلل R6 بوده و همگی هموزیگوت R1R1 تشخیص داده شدند. در حالی‌که، ارقام سیب گوشت قرمز دارای حداقل یک آلل R6 در ناحیه پیشبری بوده و از نظر ژنوتیپ مکان ژنی خودناسازگاری S-RNase، آلل S3 را دارا بودند. بنابراین، می­توان نمونه­های مذکور را هیبریدی از واریته ‘Niedzwetzkyana’ دانست. ممکن است گوشت قرمزی در یکی از ژنوتیپ­های مورد بررسی که برای ناحیه پیشبری R1R1 بود، مربوط به مکان ژنی پیوسته با آلل S3 مکان ژنی S-RNase باشد. استفاده از این ژنوتیپ‌های گوشت قرمز برای اصلاح میوه­هایی با آنتوسیانین بالا توصیه می‌گردد.

Self-incompatible pitaya varieties have low fruit-setting rates under natural conditions, leading to higher production costs and hindering industrial prosperity. Through transcriptome sequencing, we obtained the 36,900 longest transcripts (including 9167 new transcripts) from 60 samples of flowers. Samples were collected pre- and post-pollination (at 0 h, 0.5 h, 2 h, 4 h, and 12 h) from two varieties of pitaya (self-compatible Jindu No. 1 and self-incompatible Cu Sha). Using the RNA-Seq data and comparison of reference genomes, we annotated 28,817 genes in various databases, and 1740 genes were optimized in their structure for annotation. There were significant differences in the expression of differentially expressed genes (DEGs) in the pitaya stigmas under different pollination types, especially at the late post-pollination stage, where the expression of protease genes increasedal significantly under cross-pollination. We identified DEGs involved in the ribosomal, ubiquitination-mediated, and phyto-signaling pathways that may be involved in pitaya SI regulation. Based on the available transcriptome data and bioinformatics analysis, we tentatively identified HuS-RNase2 as a candidate gynogenetic S gene in the pitaya GSI system.

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. [ABSTRACT FROM AUTHOR]

Pummelo (Citrus grandis L. Osbeck) exhibits S-RNase-based self-incompatibility (SI), during which S-RNase cytotoxicity inhibits pollen tubes in an S-haplotype specific manner. The entry of S-RNase into self-pollen tubes triggers a series of reactions. However, these reactions are still poorly understood in pummelo. In the present study, we used S-RNases as baits to screen a pummelo pollen cDNA library and characterized a myo-inositol oxygenase (CgMIOX3) that physically interacts with S-RNases. CgMIOX3 is highly expressed in pummelo pollen tubes and its down-regulation leads to a reduction in pollen tube growth. Upon entering pollen tubes, S-RNases increase the expression of CgMIOX3 and enhance its activity by directly binding to it in an S-haplotype-independent manner. CgMIOX3 improves pollen tube growth under oxidative stress through ascorbic acid accumulation and increases the length of self-pollen tubes. Furthermore, over-expression of CgMIOX3 increases the relative length of self-pollen tubes growing in the style of petunia (Petunia hybrida). This study provides intriguing insights into the pumelo SI system, revealing a regulatory mechanism mediated by CgMIOX3 that plays an important role in the resistance of pollen tubes to S-RNase cytotoxicity., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The relationships of interspecific compatibility and incompatibility in Solanum section Petota are complex. Inquiry into these relationships in tomato and its wild relatives has elucidated the pleiotropic and redundant function of S-RNase and HT which tandemly and independently mediate both interspecific and intraspecific pollen rejection. Our findings presented here are consistent with previous work conducted in Solanum section Lycopersicon showing that S-RNase plays a central role in interspecific pollen rejection. Statistical analyses also demonstrated that HT-B alone is not a significant factor in these pollinations; demonstrating the overlap in gene function between HT-A and HT-B, as HT-A, was present and functional in all genotypes used. We were not able to replicate the general absence of prezygotic stylar barriers observable in S. verrucosum, which has been attributed to the lack of S-RNase, indicating that other non-S-RNase factors play a significant role. We also demonstrated that Sli played no significant role in these interspecific pollinations, directly conflicting with previous research. It is possible that S. chacoense as a pollen donor is better able to bypass stylar barriers in 1EBN species such as S. pinnatisectum. Consequently, S. chacoense may be a valuable resource in accessing these 1EBN species regardless of Sli status.

The pollination of self-incompatible diploid sweet cherry is determined by the S-locus alleles. We resolved the S-alleles of 50 sweet cherry cultivars grown in Estonia and determined their incompatibility groups, which were previously unknown for most of the tested cultivars. We used consensus primers SI-19/20, SI-31/32, PaConsI, and PaConsII followed by allele-specific primers and sequencing to identify sweet cherry S-genotypes. Surprisingly, 48% (24/50) of the tested cultivars, including 17 Estonian cultivars, carry the rare S-allele S17, which had initially been described in wild sweet cherries in Belgium and Germany. The S17-allele in Estonian cultivars could originate from 'Leningradskaya tchernaya' (S6|S17), which has been extensively used in Estonian sweet cherry breeding. Four studied cultivars carrying S17 are partly self-compatible, whereas the other 20 cultivars with S17 have not been reported to be self-compatible. The recommended pollinator of seven self-incompatible sweet cherries is of the same S-genotype, including four with S17-allele, suggesting heritable reduced effectiveness of self-infertility. We classified the newly genotyped sweet cherry cultivars into 15 known incompatibility groups, and we proposed four new incompatibility groups, 64–67, for S-locus genotypes S3|S17, S4|S17, S5|S17, and S6|S17, respectively, which makes them excellent pollinators all across Europe. Alternatively, the frequency of S17 might be underestimated in Eastern European populations and some currently unidentified sweet cherry S-alleles might potentially be S17. [ABSTRACT FROM AUTHOR]

There has been an increased interest in true potato seeds (TPS) as planting material because of their advantages over seed tubers. TPS produced from a tetraploid heterozygous bi-parental population produces non-uniform segregating progenies, which have had limited uniformity in yield and quality in commercial cultivation, and, thus, limited success. Inbreeding depression and self-incompatibility hamper the development of inbred lines in both tetraploid and diploid potatoes, impeding hybrid development efforts. Diploid potatoes have gametophytic self-incompatibility (SI) controlled by S-locus, harboring the male-dependent S-locus F-box (SLF/SFB) and female-dependent Stylar-RNase (S-RNase). Manipulation of these genes using biotechnological tools may lead to loss of self-incompatibility. Self-compatibility can also be achieved by the introgression of S-locus inhibitor (Sli) found in the self-compatible (SC) natural mutants of Solanum chacoense. The introgression of Sli through conventional breeding methods has gained much success. Recently, the Sli gene has been cloned from diverse SC diploid potato lines. It is expressed gametophytically and can overcome the SI in different diploid potato genotypes through conventional breeding or transgenic approaches. Interestingly, it has a 533 bp insertion in its promoter elements, a MITE transposon, making it a SC allele. Sli gene encodes an F-box protein PP2-B10, which consists of an F-box domain linked to a lectin domain. Interaction studies have revealed that the C-terminal region of Sli interacts with most of the StS-RNases, except StS-RNase 3, 9, 10, and 13, while full-length Sli cannot interact with StS-RNase 3, 9, 11, 13, and 14. Thus, Sli may play an essential role in mediating the interactions between pollen and stigma and function like SLFs to interact with and detoxify the S-RNases during pollen tube elongation to confer SC to SI lines. These advancements have opened new avenues in the diploid potato hybrid. [ABSTRACT FROM AUTHOR]

Knowledge of the genotypes for the self-incompatibility locus (S-locus) in apple varieties and in genotypes being used as parents is critical for breeding and commercial production. We present a high-throughput set of molecular markers for the identification of 13 common S-RNase alleles (S1, S2, S3, S5, S7, S8, S9, S10, S20, S23, S24, S25 and S28). This set is composed of seven allele-specific quantitative PCR-based High-Resolution Melting assays and four multi-allelic SSR markers. Validation of these markers was performed using 86 apple accessions, including cultivars with known S-genotypes and recent commercial varieties arising from the Plant & Food Research (PFR) cultivar breeding programme. We also characterized the S-genotypes of 183 genotypes representing some of the most valuable parents within PFR’s cultivar breeding programme. The results of this work demonstrate the practical usefulness of this marker set to provide accurate cross-compatibility information to optimise choice of pollenisers in commercial apple orchard design, and to identify compatible parents and guide parental selection when executing apple breeding programmes, to optimise fruit crop yield and quality.

[Display omitted] • Apis mellifera showed a positive effect on the fruit-set ratio of Japanese pear. • Apis mellifera and Andrena spp. deposited pollen grains equal to or greater than Panurginus crawfordi. • Solitary bees were abundant in the late flowering period. • Flies were observed stably irrespective of temperatures during the survey period. • The three pollinator groups transferred pollen grains derived from pollinizer trees. Previously we suggested the significance of some of the hymenopteran (Andrenidae, Apidae, and Halictidae) and dipteran (Syrphidae) families in Japanese pear pollination. However, they might contribute differentially to the pollination reflecting their pollination efficiencies. For this study, we investigated the pollination efficiency of three insect pollinator groups at an insect-pollinated Japanese pear orchard: Apis mellifera (Hymenoptera: Apidae), solitary bees (Hymenoptera), and flies (Diptera). First, we examined the contribution of the insect pollinator groups to the fruit-set ratio of inflorescences. Results indicated that the floral visits of A. mellifera had a positive effect on the fruit-set ratio. Then, for two consecutive years, the numbers of Rosaceae pollen grains deposited on the stigma after a single floral visit by insects were examined. The obtained results were not consistent, but data in one year showed that A. mellifera and Andrena spp. (Hymenoptera: Andrenidae) deposited more pollen grains than Panurginus crawfordi (Hymenoptera: Andrenidae) did. Third, we examined the presence of pollen grains derived from the pollinizer variety among those removed from the body surfaces of the flower-visiting insects. Results indicated the involvement of the three pollinator groups in pollen grain transfer of the pollinizer variety. Finally, we recorded the abundance of flower-visiting insects by walking around the study site. Results demonstrated that flies were observed stably irrespective of temperatures during the survey period, unlike A. mellifera and solitary bees, which exhibited little flower-visiting activity under low temperatures. Results also demonstrated that solitary bees were abundant at the study site in the late flowering period. These results suggest that hymenopterans and dipterans contribute differentially but mutually complementarily to open insect pollination in the Japanese pear orchard. [ABSTRACT FROM AUTHOR]

Oil palm (Elaeis guineensis Jacq.) is a well-known vegetable oil-yielding crop. Seedlessness is one of the most prominent traits in oil palm due to its low processing costs and high oil content. Nevertheless, an extensive study on molecular mechanisms regulating seedless phenotype formation in oil palm is very limited so far. In this study, stigma, style, and ovary from seedless and seeded (Tenera and Pisifera) oil palm trees were used to investigate the possible mechanism. Results showed that non-pollination resulted in no fruits, and self- and cross-pollinations resulted in seedless fruits, while boron treatment had no effect on seedless phenotype formation, implying that seedless trees have incomplete self and outcrossing incompatibility. Furthermore, the transcriptome data analysis highlighted eight programmed cell death (PCD) genes and three groups of PCD-related genes: 4-coumarate-CoA ligase (4CL), S-RNase, and MADS-box. The majority of these genes were significantly up-regulated in the stigma and style of Seedless palm trees compared to Tenera and Pisifera. In addition, the co-expression network analysis confirmed the significant correlation among these genes. Moreover, two simple sequence repeats (SSR) markers (S41 and S44) were developed to identify the seedless phenotype. The up-regulation of 4CL and MADS-box TFs activated the expression of PCD genes; on the other hand, S-RNase resulted in pollen tube RNA degradation and triggered PCD. While the link between PCD and seedless phenotype formation in oil palm has not been extensively studied to date, these findings suggest a role of PCD in pollen tube lethality, leading to double fertilization failure and the seedless phenotype. [ABSTRACT FROM AUTHOR]

Oil palm (Elaeis guineensis Jacq.) is a well-known vegetable oil-yielding crop. Seedlessness is one of the most prominent traits in oil palm due to its low processing costs and high oil content. Nevertheless, an extensive study on molecular mechanisms regulating seedless phenotype formation in oil palm is very limited so far. In this study, stigma, style, and ovary from seedless and seeded (Tenera and Pisifera) oil palm trees were used to investigate the possible mechanism. Results showed that non-pollination resulted in no fruits, and self- and cross-pollinations resulted in seedless fruits, while boron treatment had no effect on seedless phenotype formation, implying that seedless trees have incomplete self and outcrossing incompatibility. Furthermore, the transcriptome data analysis highlighted eight programmed cell death (PCD) genes and three groups of PCD-related genes: 4-coumarate-CoA ligase (4CL), S-RNase, and MADS-box. The majority of these genes were significantly up-regulated in the stigma and style of Seedless palm trees compared to Tenera and Pisifera. In addition, the co-expression network analysis confirmed the significant correlation among these genes. Moreover, two simple sequence repeats (SSR) markers (S41 and S44) were developed to identify the seedless phenotype. The up-regulation of 4CL and MADS-box TFs activated the expression of PCD genes; on the other hand, S-RNase resulted in pollen tube RNA degradation and triggered PCD. While the link between PCD and seedless phenotype formation in oil palm has not been extensively studied to date, these findings suggest a role of PCD in pollen tube lethality, leading to double fertilization failure and the seedless phenotype.

The self-incompatibility (SI) system in the Solanaceae is comprised of cytotoxic pistil S-RNases which are countered by S-locus F-box (SLF) resistance factors found in pollen. Under this barrier-resistance architecture, mating system transitions from SI to self-compatibility (SC) typically result from loss-of-function mutations in genes encoding pistil SI factors such as S-RNase. However, the nature of these mutations is often not well characterized. Here we use a combination of S-RNase sequence analysis, transcript profiling, protein expression and reproductive phenotyping to better understand different mechanisms that result in loss of S-RNase function. Our analysis focuses on 12 S-RNase alleles identified in SC species and populations across the tomato clade. In six cases, the reason for gene dysfunction due to mutations is evident. The six other alleles potentially encode functional S-RNase proteins but are typically transcriptionally silenced. We identified three S-RNase alleles which are transcriptionally silenced under some conditions but actively expressed in others. In one case, expression of the S-RNase is associated with SI. In another case, S-RNase expression does not lead to SI, but instead confers a reproductive barrier against pollen tubes from other tomato species. In the third case, expression of S-RNase does not affect self, interspecific or inter-population reproductive barriers. Our results indicate that S-RNase expression is more dynamic than previously thought, and that changes in expression can impact different reproductive barriers within or between natural populations. [ABSTRACT FROM AUTHOR]

Loquat [Eriobotrya japonica (E. japonica)], a small genus of the subtribe Malinae that consists of ≈30 species, is an evergreen rosaceous fruit tree that is native to southeastern China, and some wild species that possess novel, favorable traits have excellent breeding potential. For example, Eriobotrya bengalensis blooms in late spring and ripens in early autumn in Guizhou Province, China, which prevents cold injury in winter by breeding spring-flowering cultivars using the special characters. Therefore, in the present study, the pollination treatments of cut-style pollination were evaluated that may promote successful distant hybridization in Eriobotrya japonica ‘Dawuxing’ × Eriobotrya deflexa and E. japonica ‘Dawuxing’ × E. bengalensis. The results indicated that the impairment of the pollen tube growth in the upper third of the style after pollen germination is an important factor leading to the failure of distant hybridization between the species tested in E. japonica, and that cut-style pollination can effectively overcome prefertilization barriers of the distant hybridization combination. Furthermore, the results of allele-specific polymerase chain reaction (AS-PCR) showed that S-genotypes, in accordance with the S-RNase heredity to separate the rule completely in offspring, should be both parents' S-RNase, and that the random 50 seedlings of Eb-2 and Ed-2 are true hybrids.

The self-incompatibility (SI) system in the Solanaceae is comprised of cytotoxic pistil S-RNases which are countered by S-locus F-box (SLF) resistance factors found in pollen. Under this barrier-resistance architecture, mating system transitions from SI to self-compatibility (SC) typically result from loss-of-function mutations in genes encoding pistil SI factors such as S-RNase. However, the nature of these mutations is often not well characterized. Here we use a combination of S-RNase sequence analysis, transcript profiling, protein expression and reproductive phenotyping to better understand different mechanisms that result in loss of S-RNase function. Our analysis focuses on 12 S-RNase alleles identified in SC species and populations across the tomato clade. In six cases, the reason for gene dysfunction due to mutations is evident. The six other alleles potentially encode functional S-RNase proteins but are typically transcriptionally silenced. We identified three S-RNase alleles which are transcriptionally silenced under some conditions but actively expressed in others. In one case, expression of the S-RNase is associated with SI. In another case, S-RNase expression does not lead to SI, but instead confers a reproductive barrier against pollen tubes from other tomato species. In the third case, expression of S-RNase does not affect self, interspecific or inter-population reproductive barriers. Our results indicate that S-RNase expression is more dynamic than previously thought, and that changes in expression can impact different reproductive barriers within or between natural populations.

Summary: In self‐incompatible Petunia species, the pistil S‐RNase acts as cytotoxin to inhibit self‐pollination but is polyubiquitinated by the pollen‐specific nonself S‐locus F‐box (SLF) proteins and subsequently degraded by the ubiquitin‐proteasome system (UPS), allowing cross‐pollination. However, it remains unclear how S‐RNase is restricted by the UPS.Using biochemical analyses, we first show that Petunia hybrida S3‐RNase is largely ubiquitinated by K48‐linked polyubiquitin chains at three regions, R I, R II and R III. R I is ubiquitinated in unpollinated, self‐pollinated and cross‐pollinated pistils, indicating its occurrence before PhS3‐RNase uptake into pollen tubes, whereas R II and R III are exclusively ubiquitinated in cross‐pollinated pistils.Transgenic analyses showed that removal of R II ubiquitination resulted in significantly reduced seed sets from cross‐pollination and that of R I and R III to a lesser extent, indicating their increased cytotoxicity. Consistent with this, the mutated R II of PhS3‐RNase resulted in a marked reduction of its degradation, whereas that of R I and R III resulted in less reduction.Taken together, we demonstrate that PhS3‐RNase R II functions as a major ubiquitination region for its destruction and R I and R III as minor ones, revealing that its cytotoxicity is primarily restricted by a stepwise UPS mechanism for cross‐pollination in P. hybrida. [ABSTRACT FROM AUTHOR]

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.

Molecular tools were largely used to characterise native almond (Prunus dulcis Mill.) cultivars, to elucidate their diversification and to establish their membership to Mediterranean germplasm pools. Hereby, the efficiency of the application of PCR techniques to study the genetic diversity of S alleles in almond was tested to identify self-incompatible cultivar combinations and groups. Hence, an analysis with consensus AS1II and AmyC5R and with allele-specific primers was performed for 25 almond accessions including 17 local Tunisian cultivars, 7 advanced selections, and 1 extra group from Italy in order to identify their S-genotype. Interestingly, all genotypes were assigned to their specific self-incompatible groups and a new group (LI) has been established. The two cultivars 'Dillou' and 'Porto' both considered as self-compatible have been demonstrated to be self-incompatible. Results reveal also a discrepancy for the S-genotype assigned to 'Zahaaf' preserved in the International almond collection. Thus, S genes genotyping of commercially used or newly released cultivars is very useful as we demonstrated that it may show that common knowledge may be partly inaccurate. For important varieties, this immediately has a consequence for cultivation practices, also for smallholder orchards as changing an incorrect practice may in time increase yield and also guarantee yield stability. [ABSTRACT FROM AUTHOR]

Premise: Long‐distance dispersal has been important in explaining the present distributions of many plant species. Despite being infrequent, such dispersal events have considerable evolutionary consequences, because bottlenecks during colonization can result in reduced genetic diversity. We examined the phylogeographic history of Lycium carolinianum, a widespread taxon that ranges from southeastern North America to several Pacific islands, with intraspecific diversity in sexual and mating systems. Methods: We used Bayesian, likelihood, and coalescent approaches with nuclear and plastid sequence data and genome‐wide single nucleotide polymorphisms to reconstruct the dispersal history of this species. We also compared patterns of genetic variation in mainland and island populations using single nucleotide polymorphisms and allelic diversity at the S‐RNase mating system gene. Results: Lycium carolinianum is monophyletic and dispersed once from the North American mainland, colonizing the Pacific islands ca. 40,100 years ago. This dispersal was accompanied by a loss of genetic diversity in SNPs and the S‐RNase locus due to a colonization bottleneck and the loss of self‐incompatibility. Additionally, we documented at least two independent transitions to gynodioecy: once following the colonization of the Hawaiian Islands and loss of self‐incompatibility, and a second time associated with polyploidy in the Yucatán region of Mexico. Conclusions: Long‐distance dispersal via fleshy, bird dispersed fruits best explains the unusually widespread distribution of L. carolinianum. The collapse of diversity at the S‐RNase locus in island populations suggests that self‐fertilization may have facilitated the subsequent colonization of Pacific islands following a single dispersal from mainland North America. [ABSTRACT FROM AUTHOR]

There has been an increased interest in true potato seeds (TPS) as planting material because of their advantages over seed tubers. TPS produced from a tetraploid heterozygous bi-parental population produces non-uniform segregating progenies, which have had limited uniformity in yield and quality in commercial cultivation, and, thus, limited success. Inbreeding depression and self-incompatibility hamper the development of inbred lines in both tetraploid and diploid potatoes, impeding hybrid development efforts. Diploid potatoes have gametophytic self-incompatibility (SI) controlled by S-locus, harboring the male-dependent S-locus F-box (SLF/SFB) and female-dependent Stylar-RNase (S-RNase). Manipulation of these genes using biotechnological tools may lead to loss of self-incompatibility. Self-compatibility can also be achieved by the introgression of S-locus inhibitor (Sli) found in the self-compatible (SC) natural mutants of Solanum chacoense. The introgression of Sli through conventional breeding methods has gained much success. Recently, the Sli gene has been cloned from diverse SC diploid potato lines. It is expressed gametophytically and can overcome the SI in different diploid potato genotypes through conventional breeding or transgenic approaches. Interestingly, it has a 533 bp insertion in its promoter elements, a MITE transposon, making it a SC allele. Sli gene encodes an F-box protein PP2-B10, which consists of an F-box domain linked to a lectin domain. Interaction studies have revealed that the C-terminal region of Sli interacts with most of the StS-RNases, except StS-RNase 3, 9, 10, and 13, while full-length Sli cannot interact with StS-RNase 3, 9, 11, 13, and 14. Thus, Sli may play an essential role in mediating the interactions between pollen and stigma and function like SLFs to interact with and detoxify the S-RNases during pollen tube elongation to confer SC to SI lines. These advancements have opened new avenues in the diploid potato hybrid.

Abstract The objective of this study was to characterize the parents and respective populations of apple trees regarding S-alleles to confirm their genealogy and to evaluate the efficiency of the molecular markers used. Sixteen specific sets of primers were used for identification of apple S-alleles by PCR. Two segregating populations of the Epagri Apple Breeding Program resulting from crosses between ‘Fred Hough’ × ‘Monalisa’ and ‘M-11/00’ × ‘M-13/91’ were evaluated. The expected segregations are 1:1:1:1 for full compatibility and 1:1 for semi-compatibility, which can be confirmed by the X2 test. The ‘Fred Hough’ (S5S19) × ‘Monalisa’ (S2S10) cross proved to be fully compatible; and two triploids were identified among the hybrids as well. The ‘M-11/00’ (S3S19) × ‘M-13/91’ (S3S5) cross was characterized as semi-compatible based on DNA markers, and the segregation of the S-alleles in the hybrids was 1:1, as expected. The segregation of the DNA markers occurred together with their respective S-alleles: S2, S3, S5, S10, and S19. Thus, characterization of the S-alleles not only allowed identification of compatibility between parents but also identified contaminations in segregating populations.

Several citrus varieties show gametophytic self-incompatibility (GSI), which can contribute to seedless fruit production in several cultivars. This study investigated the genes regulating this trait through RNA-seq performed using styles collected from the flowers of Japanese citrus cultivars ‘Hyuganatsu,' ‘Tosabuntan,' ‘Hassaku,' ‘Banpeiyu,' and ‘Sweet Spring'. We screened the transcripts of putative T2 RNases, i.e., the protein family including all S-RNases from S-RNase-based GSI plants, and constructed a phylogenetic tree using the screened T2 RNases and S-RNases retrieved from citrus genome databases and a public database. Three major clusters (class I–III) were formed, among which, the class III cluster contained family specific subclusters formed by S-RNase and a citrus-specific cluster monophyletic to the S-RNase clusters. From the citrus class III cluster, six transcripts were consistent with the S haplotypes previously determined in Japanese citrus accessions, sharing characteristics such as isoelectric point, extracellular localization, molecular weight, intron number and position, and tissue-specific expression with S-RNases. One T2 RNase gene in self-incompatible Hyuganatsu was significantly down-regulated in the styles of a self-compatible mutant of Hyuganatsu in RNA-seq and qPCR analyses. In addition, the inheritance pattern of some T2 RNase genes was consistent with the pattern of the S haplotype in the progeny population of Hyuganatsu and Tosabuntan. As all results supported citrus self-incompatibility being based on S-RNase, we believe that six T2 RNase genes were S-RNases. The homology comparison between the six T2 RNases and S-RNases recently reported in Chinese citrus revealed that three out of six T2 RNases were identical to S-RNases from Chinese citrus. Thus, the other three T2 RNases were finally concluded to be novel citrus S-RNases involved in self-incompatibility.

Several citrus varieties show gametophytic self-incompatibility (GSI), which can contribute to seedless fruit production in several cultivars. This study investigated the genes regulating this trait through RNA-seq performed using styles collected from the flowers of Japanese citrus cultivars 'Hyuganatsu,' 'Tosabuntan,' 'Hassaku,' 'Banpeiyu,' and 'Sweet Spring'. We screened the transcripts of putative T2 RNases, i.e., the protein family including all S-RNases from S-RNase-based GSI plants, and constructed a phylogenetic tree using the screened T2 RNases and S-RNases retrieved from citrus genome databases and a public database. Three major clusters (class I–III) were formed, among which, the class III cluster contained family specific subclusters formed by S-RNase and a citrus-specific cluster monophyletic to the S-RNase clusters. From the citrus class III cluster, six transcripts were consistent with the S haplotypes previously determined in Japanese citrus accessions, sharing characteristics such as isoelectric point, extracellular localization, molecular weight, intron number and position, and tissue-specific expression with S-RNases. One T2 RNase gene in self-incompatible Hyuganatsu was significantly down-regulated in the styles of a self-compatible mutant of Hyuganatsu in RNA-seq and qPCR analyses. In addition, the inheritance pattern of some T2 RNase genes was consistent with the pattern of the S haplotype in the progeny population of Hyuganatsu and Tosabuntan. As all results supported citrus self-incompatibility being based on S-RNase, we believe that six T2 RNase genes were S-RNases. The homology comparison between the six T2 RNases and S-RNases recently reported in Chinese citrus revealed that three out of six T2 RNases were identical to S-RNases from Chinese citrus. Thus, the other three T2 RNases were finally concluded to be novel citrus S-RNases involved in self-incompatibility. [ABSTRACT FROM AUTHOR]

Self-incompatibility (SI) is a genetic mechanism common in flowering plants to prevent self-fertilization. Among citrus species, several pummelo, mandarin, and mandarin-like accessions show SI behavior. In these species, SI is coupled with a variable degree of parthenocarpy ensuring the production of seedless fruits, a trait that is highly appreciated by consumers. In Citrus , recent evidences have shown the presence of a gametophytic SI system based on S-ribonucleases ( S-RNases ) ability to impair self-pollen tube growth in the upper/middle part of the style. In the present study, we combined PCR analysis and next-generation sequencing technologies, to define the presence of S 7 - and S 11 -Rnases in the S -genotype of the Citrus clementina (Hort. ex Tan.), the self-incompatible 'Comune' clementine and its self-compatible natural mutant 'Monreal'. The reference genome of 'Monreal' clementine is presented for the first time, providing more robust results on the genetic sequence of the newly discovered S 7 -RNase. SNP discovery analysis coupled with the annotation of the variants detected enabled the identification of 7,781 SNPs effecting 5,661 genes in 'Monreal' compared to the reference genome of C. clementina . Transcriptome analysis of unpollinated pistils at the mature stage from both clementine genotypes revealed the lack of expression of S 7 -RNase in 'Monreal' suggesting its involvement in the loss of the SI response. RNA-seq analysis followed by gene ontology studies enabled the identification of 2,680 differentially expressed genes (DEGs), a significant number of those is involved in oxidoreductase and transmembrane transport activity. Merging of DNA sequencing and RNA data led to the identification of 164 DEGs characterized by the presence of at least one SNP predicted to induce mutations with a high effect on their amino acid sequence. Among them, four candidate genes referring to two Agamous -like MADS-box proteins, to MYB111 and to MLO -like protein 12 were validated. Moreover, the transcription factor MYB111 appeared to contain a binding site for the 2.0-kb upstream sequences of the S 7 - and S 11 -RNase genes. These results provide useful information about the genetic bases of SI indicating that SNPs present in their sequence could be responsible for the differential expression and the regulation of S 7 -RNase and consequently of the SI mechanism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Bennici, Poles, Di Guardo, Percival-Alwyn, Caccamo, Licciardello, Gentile, Distefano and La Malfa.)

Plants have evolved diverse self-incompatibility (SI) systems for outcrossing. Since Darwin's time, considerable progress has been made toward elucidating this unrivaled reproductive innovation. Recent advances in interdisciplinary studies and applications of biotechnology have given rise to major breakthroughs in understanding the molecular pathways that lead to SI, particularly the strikingly different SI mechanisms that operate in Solanaceae, Papaveraceae, Brassicaceae, and Primulaceae. These best-understood SI systems, together with discoveries in other "nonmodel" SI taxa such as Poaceae, suggest a complex evolutionary trajectory of SI, with multiple independent origins and frequent and irreversible losses. Extensive exploration of self-/nonself-discrimination signaling cascades has revealed a comprehensive catalog of male and female identity genes and modifier factors that control SI. These findings also enable the characterization, validation, and manipulation of SI-related factors for crop improvement, helping to address the challenges associated with development of inbred lines. Here, we review current knowledge about the evolution of SI systems, summarize key achievements in the molecular basis of pollen‒pistil interactions, discuss potential prospects for breeding of SI crops, and raise several unresolved questions that require further investigation., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

SUMMARY: Self‐incompatibility in Petunia is controlled by the polymorphic S‐locus, which contains S‐RNase encoding the pistil determinant and 16–20 S‐locus F‐box (SLF) genes collectively encoding the pollen determinant. Here we sequenced and assembled approximately 3.1 Mb of the S2‐haplotype of the S‐locus in Petunia inflata using bacterial artificial chromosome clones collectively containing all 17 SLF genes, SLFLike1, and S‐RNase. Two SLF pseudogenes and 28 potential protein‐coding genes were identified, 20 of which were also found at the S‐loci of both the S6a‐haplotype of P. inflata and the SN‐haplotype of self‐compatible Petunia axillaris, but not in the S‐locus remnants of self‐compatible potato (Solanum tuberosum) and tomato (Solanum lycopersicum). Comparative analyses of S‐locus sequences of these three S‐haplotypes revealed potential genetic exchange in the flanking regions of SLF genes, resulting in highly similar flanking regions between different types of SLF and between alleles of the same type of SLF of different S‐haplotypes. The high degree of sequence similarity in the flanking regions could often be explained by the presence of similar long terminal repeat retroelements, which were enriched at the S‐loci of all three S‐haplotypes and in the flanking regions of all S‐locus genes examined. We also found evidence of the association of transposable elements with SLF pseudogenes. Based on the hypothesis that SLF genes were derived by retrotransposition, we identified 10 F‐box genes as putative SLF parent genes. Our results shed light on the importance of non‐coding sequences in the evolution of the S‐locus, and on possible evolutionary mechanisms of generation, proliferation, and deletion of SLF genes. Significance Statement: Sequencing the Petunia S‐locus region of the S2‐haplotype containing 17 S‐locus F‐box (SLF) genes, SLFLike1, and the S‐RNase gene allows comparative analyses of the approximately 3.1 Mb assembled sequence with the S‐locus sequences of other S‐haplotypes of Petunia and S‐locus remnants of several self‐compatible solanaceous species. The results from comparison in both the coding and non‐coding flanking sequences of SLF genes shed light on the genomic organization and evolution of the S‐locus in Petunia. [ABSTRACT FROM AUTHOR]

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.

Summary: In self‐incompatible Solanaceae, the pistil protein S‐RNase contributes to S‐specific pollen rejection in conspecific crosses, as well as to rejecting pollen from foreign species or whole clades. However, S‐RNase alone is not sufficient for either type of pollen rejection. We describe a thioredoxin (Trx) type h from Nicotiana alata, NaTrxh, which interacts with and reduces S‐RNase in vitro. Here, we show that expressing a redox‐inactive mutant, NaTrxhSS, suppresses both S‐specific pollen rejection and rejection of pollen from Nicotiana plumbaginifolia. Biochemical experiments provide evidence that NaTrxh specifically reduces the Cys155‐Cys185 disulphide bond of SC10‐Rnase, resulting in a significant increase of its ribonuclease activity. This reduction and increase in S‐RNase activity by NaTrxh helps to explain why S‐RNase alone could be insufficient for pollen rejection. Significance Statement: S‐RNase is very well known for its role in S‐RNase‐based self‐incompatibility and in some types of interspecific pollen rejection. Through a dominant‐negative approach, we show that a thioredoxin (Trx) type h from Nicotiana alata, NaTrxh, is required for both types of pollen rejection. This is a significant advance, although what sets our new contribution apart is that we show that NaTrxh reduces an S‐RNase disulphide and increases its activity, providing a remarkable level of insight into the biochemical mechanism of S‐RNase‐dependent pollen rejection. [ABSTRACT FROM AUTHOR]

Summary: Apple exhibits S‐RNase‐mediated self‐incompatibility. Although the cytotoxic effect of S‐RNase inside the self‐pollen tube has been studied extensively, the underlying defence mechanism in pollen tube in Rosaceae remains unclear. On exposure to stylar S‐RNase, plant defence responses are activated in the pollen tube; however, how these are regulated is currently poorly understood. Here, we show that entry of both self and non‐self S‐RNase into pollen tubes of apple (Malus domestica) stimulates jasmonic acid (JA) production, in turn inducing the accumulation of MdMYC2 transcripts, a transcription factor in the JA signalling pathway widely considered to be involved in plant defence processes. MdMYC2 acts as a positive regulator in the pollen tube activating expression of MdD1, a gene encoding a defence protein. Importantly, MdD1 was shown to bind to the RNase activity sites of S‐RNase leading to inhibition of enzymatic activity. This work provides intriguing insights into an ancient defence mechanism present in apple pollen tubes where MdD1 likely acts as a primary line of defence to inhibit S‐RNase cytotoxicity prior to self/non‐self recognition. [ABSTRACT FROM AUTHOR]