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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. [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 (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]

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.

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.

Sweet cherry is a self-incompatible fruit species and shows a gametophytic self-incompatibility (GSI) system, which is genetically determined by a polymorphic locus (S), encoding at least two linked genes that determine the pistil and pollen phenotypes. Therefore, most sweet cherry genotypes are self-incompatible and many are cross-incompatible. The aim of this work was to identify the Sallelic constitutions and incompatibility groups in eight indigenous sweet cherry genotypes collected in orchards of individual growers in the region of Čačak, i.e. villages Jezdina (GТ-3), Prislonica (GТ-17, GТ-18, GТ-19, GТ-20 and GТ21) and Ljubić (GТ-22 and GТ-23). The allelic constitution of S-RNase gene in each genotype was determined by polymerase chain reaction (PCR) with consensus and allele-specific primers. Eight alleles (S1, S2, S3, S4, S5, S6, S9 and S13) that generated seven Sgenotypes [S1S2 (GТ-18), S1S4 (GТ-20 and GТ-22), S3S5 (GТ-3), S3S6 (GТ-21), S3S9 (GТ-17), S4S5 (GТ-23) and S4S13 (GТ-19)] were detected. Among the revealed alleles, S4 was the most frequent (25%), followed by S1 and S3 (18.75% to each allele). Based on the Sallelic constitution, the indigenous sweet cherry genotypes were assigned to the following incompatibility groups: I, V, VI, VII, IX, XVI and XLV. The obtained results represent crucial information on cross-compatibility of these Serbian accessions that is very important for both cherry breeders and growers. [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.)

Gametophytic self-incompatibility, one of the key characters for production of seedless citrus, is known in pummelo [Citrus maxima (Burm.) Merr.], mandarin (C. reticulata Blanco) and their hybrid cultivars. The allelic variation in Citrus was reported for the self-incompatibility gene (S). However, S allele frequencies and S genotypes of self-incompatible and semi-self-incompatible Citrus cultivars were known only in a few S alleles. In the present study, S3 and S11 alleles were examined with homozygous S1 seedlings (S3S3 and S11S11) of self-incompatible Clementine mandarin (C. clementina hort. ex Tanaka; S3S11) a putative hybrid between self-compatible 'Mediterranean' mandarin (C. deliciosa Ten.) and self-compatible sweet orange [C. sinensis (L.) Osbeck]. One-hundred-forty-six Citrus accessions, including 43 pummelo accessions, were pollinated with each of the homozygous S1 seedlings (S3S3 and S11S11). Pollen tube arrest in the styles of their pollinated pistils indicated that 25 accessions, including Clementine, have an S3 allele each and 18 accessions, including Clementine, have an S11 allele each. Frequency of accessions with S3 allele was 17.7% (25 of 141 accessions examined) and S3 allele frequency was 11.8% (25 of 212 alleles excluding Sf self-compatible allele). Frequency of accessions with S11 allele was 13.2% (18 of 136 accessions examined) and S11 allele frequency was 8.8% (18 of 205 alleles excluding Sf allele). Pummelo accessions have S3 and S11 alleles with the frequencies of 12.2% and 33.3% respectively, suggesting that the two alleles originated from pummelo. Clementine shared S3 allele with two navel oranges, ten common oranges and two tangors, but not with three blood oranges. In the three blood oranges, it was suggested that stylar part mutation (S3sm) resulting in cross compatibility with S3 pollen occurred in their common ancestor. Clementine shared S11 allele with two tangors but did not share it with 'Mediterranean' mandarin. The present result for S3 and S11 alleles in Citrus accessions suggests that the two S alleles expanded their distribution in Citrus plants including sweet orange along with stylar part mutation for S3 allele during long history of cultivation. [ABSTRACT FROM AUTHOR]

Self-incompatibility (SI) mechanisms prevent self-fertilization in flowering plants based on specific discrimination between self- and non-self pollen. Since this trait promotes outcrossing and avoids inbreeding it is a widespread mechanism of controlling sexual plant reproduction. Growers and breeders have effectively exploited SI as a tool for manipulating domesticated crops for thousands of years. However, only within the past thirty years have studies begun to elucidate the underlying molecular features of SI. The specific S-determinants and some modifier factors controlling SI have been identified in the sporophytic system exhibited by Brassica species and in the two very distinct gametophytic systems present in Papaveraceae on one side and in Solanaceae, Rosaceae, and Plantaginaceae on the other. Molecular level studies have enabled SI to SC transitions (and vice versa) to be intentionally manipulated using marker assisted breeding and targeted approaches based on transgene integration, silencing, and more recently CRISPR knock-out of SI-related factors. These scientific advances have, in turn, provided a solid basis to implement new crop production and plant breeding practices. Applications of self-(in)compatibility include widely differing objectives such as crop yield and quality improvement, marker-assisted breeding through SI genotyping, and development of hybrids for overcoming intra- and interspecific reproductive barriers. Here, we review scientific progress as well as patented applications of SI, and also highlight future prospects including further elucidation of SI systems, deepening our understanding of SI-environment relationships, and new perspectives on plant self/non-self recognition.

During the last decade, S-genotyping has been extensively investigated in fruit tree crops such as those belonging to the Prunus genus, including plums. In plums, S-allele typing has been largely studied in diploid species but works are scarcer in polyploid species due to the complexity of the polyploid genome. This study was conducted in order to analyze the S-genotypes of 30 diploid P. salicina, 17 of them reported here for the first time, and 29 hexaploid plums (24 of P. domestica and 5 of P. insititia). PCR analysis allowed identifying nine S-alleles in the P. salicina samples allocating the 30 accessions in 16 incompatibility groups, two of them identified here for the first time. In addition, pollen tube growth was studied in self-pollinated flowers of 17 Tunisian P. salicina under the microscope. In 16 samples, including one carrying the Se allele, which has been correlated with self-compatibility, the pollen tubes were arrested in the style. Only in one cultivar (“Bedri”), the pollen tubes reached the base of the style. Twelve S-alleles were identified in the 24 P. domestica and 5 P. insititia accessions, assigning accessions in 16 S-genotypes. S-genotyping results were combined with nine SSR loci to analyze genetic diversity. Results showed a close genetic relationship between P. domestica and P. salicina and between P. domestica and P. insititia corroborating that S-locus genotyping is suitable for molecular fingerprinting in diploid and polyploid Prunus species.

Highlights • S -genotypes of 15 Spanish almond local cultivars determined herein for the first time. • Specific primers for almond S 10 -RNase herein designed are useful for S -genotyping. • A new S-RNase allele numbered as S 53 was identified in Spanish almond cultivars. • A compilation table of almond cross-incompatibility groups is provided. • The information of this table is very useful in almond research and breeding. Abstract Most almond cultivars are self-incompatible, and so to obtain a yield they need to be pollinated with cross-compatible cultivars. For an efficient use of these cultivars in growing and breeding, over the years many have been S -genotyped. This information has been included in consecutive tables of cross-incompatibility groups, which are essentially an update of the previously proposed. However, the information of these tables has not always been reconciled and their inconsistencies have not been corrected. In this work the S -genotypes of 15 Spanish almond local cultivars, included in a research program for the preservation of almond biodiversity, were determined for the first time. For this, PCR with consensus primers for the Prunus S-RNases, PCR using S-RNase allele specific primers designed herein, and also cloning and sequencing were performed. As a result, a new S-RNase allele numbered as S 53 could be identified, and a compilation table of almond cross-incompatibility groups is provided including the information from this and previous studies. In this novel Table, twelve new cross-incompatibility groups have been established, two former groups were omitted, and those cultivars with inconsistent genotypes have been removed. The information of this table will facilitate the use of a very high number of almond cultivars in research and breeding. Determination of the S -genotype might be considered as an important addition to a set of identity markers of almond genetic resources, what is of particular interest in the characterization of the agricultural biodiversity. [ABSTRACT FROM AUTHOR]

The genus Pyrus is characterized by an S-RNase-based gametophytic self-incompatibility (GSI) system, a mechanism that promotes outbreeding and prevents self-fertilization. While the S-genotype of the most widely known pear cultivars was already described, little is known on the S-allele variability within local accessions. The study was conducted on 86 accessions encompassing most of the local Sicilian varieties selected for their traits of agronomic interest and complemented with some accessions of related wild species (P. pyrifolia Nakai, P. amygdaliformis Vill.) and some national and international cultivars used as references. The employment of consensus and specific primers enabled the detection of 24 S-alleles combined in 48 S-genotypes. Results shed light on the distribution of the S-alleles among accessions, with wild species and international cultivars characterized by a high diversity and local accessions showing a more heterogeneous distribution of the S-alleles, likely reflecting a more complex history of hybridization. The S-allele distribution was largely in agreement with the genetic structure of the studied collection. In particular, the “wild” genetic background was often characterized by the same S-alleles detected in P. pyrifolia and P. amygdaliformis. The analysis of the S-allele distribution provided novel insight into the contribution of the wild and international cultivars to the genetic background of the local Sicilian or national accessions. Furthermore, these results provide information that can be readily employed by breeders for the set-up of novel mating schemes.

Key message: Data obtained from Illumina resequencing of 63 apple cultivars were used to obtain full-length S-RNase sequences using a strategy based on both alignment and de novo assembly of reads.Abstract: The reproductive biology of apple is regulated by the S-RNase-based gametophytic self-incompatibility system, that is genetically controlled by the single, multi-genic and multi-allelic S locus. Resequencing of apple cultivars provided a huge amount of genetic data, that can be aligned to the reference genome in order to characterize variation to a genome-wide level. However, this approach is not immediately adaptable to the S-locus, due to some peculiar features such as the high degree of polymorphism, lack of colinearity between haplotypes and extensive presence of repetitive elements. In this study we describe a dedicated procedure aimed at characterizing S-RNase alleles from resequenced cultivars. The S-genotype of 63 apple accessions is reported; the full length coding sequence was determined for the 25 S-RNase alleles present in the 63 resequenced cultivars; these included 10 previously incomplete sequences (S5, S6a, S6b, S8, S11, S23, S39, S46, S50 and S58). Moreover, sequence divergence clearly suggests that alleles S6a and S6b, proposed to be neutral variants of the same alleles, should be instead considered different specificities. The promoter sequences have also been analyzed, highlighting regions of homology conserved among all the alleles. [ABSTRACT FROM AUTHOR]

Italian almond germplasm is characterized by a wide diversity in several growing areas among which Sicily is one of the most important. Analysis with consensus and specific primers and DNA sequencing was performed to investigate S-RNase genetic diversity and to elucidate the homology rate within a genetic pool of 27 Italian accessions. Interestingly, some of the self-compatible cultivars did not show the presence of Sf allele. Amplicons from consensus and allele-specific PCR primers revealed a high level of variability. Sequencing of all the S-RNase amplicons derived from consensus primers allowed the identification of two new S-RNase alleles (S51 and S52). Surprisingly, despite the AA replacement mutation, S51 did not exhibit any change of its S-RNase function. Additionally, several mutations, with no effect on amino acid composition, were detected in the intron and/or in the ORF of four known alleles (Sg, S10, S31 and S35). Genetic variation, regarding point mutations and only detected by sequencing, was revealed among 11 of 27 tested cultivars. The new sources of variability might have an interest for product traceability. [ABSTRACT FROM AUTHOR]

Gametophytic self-incompatibility in Japanese pear (Pyrus pyrifolia Nakai) is controlled by the single, multiallelic S-locus. Information about the S-genotypes is important for breeding and the selection of pollen donors for fruit production. Rapid and reliable S-genotype identification system is necessary for efficient breeding of new cultivars in Japanese pear. We designed S allele-specific PCR primer pairs for ten previously reported S-RNase alleles (S¹-S9 and Sk) as simple and reliable method. Specific nucleotide sequences were chosen to design the primers to amplify fragments of only the corresponding S alleles. The developed primer pairs were evaluated by using homozygous S-genotypes (S¹/S¹-S9/S9 and S4sm/S4sm) and 14 major Japanese pear cultivars, and found that S allele-specific primer pairs can identify S-genotypes effectively. The S allele-specific primer pairs developed in this study will be useful for efficient S-genotyping and for marker-assisted selection in Japanese pear breeding programs. [ABSTRACT FROM AUTHOR]

The Pyrus species exhibit the so-called S-RNase-based gametophytic self-incompatibility system, which is considered to be the most widespread self-incompatibility system among flowering plants. In this study, 57 Iranian pear ( Pyrus communis L.) domestic cultivars and wild genotypes, plus 21 European pear cultivars used as references, were genotyped adopting a PCR-based genotyping assay using consensus and allele-specific primers. The results revealed traces of significant genetic contribution in the Iranian traditional varieties and genotypes from other Pyrus species; the genetic contribution of Japanese pear clearly emerged with the detection of some Pyrus pyrifolia S-RNase alleles. Moreover, our results highlighted the presence of three new S-RNase alleles (named S, S, and S) that were not previously identified in P. communis, possibly introduced in the germplasm of cultivated pear through gene transfer from other cultivated or wild species. [ABSTRACT FROM AUTHOR]

Breeding for higher levels of health-promoting components are among the priorities for many fruit crops. Such an approach requires breeding material displaying great variations in the trait to be improved. Commercial sweet cherry cultivars demonstrate only limited variability in fruit antioxidant parameters and hence this study was carried out to characterize some Ukrainian sweet cherry cultivars for their antioxidant and antiradical capacity and total phenolic and anthocyanin contents. Ukrainian cultivars had significantly higher ferric reducing antioxidant power and total phenolic contents compared with commercial cultivars. Levels of all four parameters varied considerably in the Ukrainian germplasm, with some cultivars having outstanding values. A total of eight different self-incompatibility genotypes were assigned to the 14 accessions tested. Assigning cultivars to the appropriate incompatibility groups allowed the design of parental combinations which may result in new genotypes with enhanced functional properties and other valuable fruit traits. Based on the S-genotypes, parental combinations were proposed to ensure 50 or 100 % self-compatible seedlings in the offspring population. The most perspective Ukrainian cultivars like 'Dagestanka' and 'Kodrinskaya' also had reasonable fruit weight (over 8 g), high flesh to pit ratio (approx. 18) and soluble solid contents (over 16 %). Our results demonstrated that adequate variation is available in Ukrainian germplasm for improving fruit health benefits in sweet cherry through directed hybridizations. [ABSTRACT FROM AUTHOR]

Self-incompatibility of sweet cherry ( Prunus avium L.) is controlled by the multiallelic S-locus. While many cultivars and wild accessions have been S-genotyped, only limited data are available on accessions native to the center of origin of this species. Therefore, this study was carried out to determine the S-genotype of 11 landrace cultivars and 17 local genotypes selected from populations growing wild at the Black Sea coast. Eleven sweet cherries ( S- S, S, and S- S) and some wild cherries ( S- S, S, and S) S- RNase alleles were detected. The results indicate that Turkish cultivars represent a broader gene pool as compared with international cultivars. A new ( S) and a doubtful allele (provisionally labelled as S) as well as the sour cherry S-allele were identified in sweet cherry. These data and others (SSR variants within the S- RNase introns) confirmed that allele pools of sweet and sour cherries in the Black Sea region are overlapping. A new cross-incompatibility group, XLV ( S S), was also proposed. Allele-specific primers were designed for S- S, S, S, and S. A phylogenetic analysis of the cherry S- RNase and its trans-specific sister alleles reliably mirrored the assumed length of the time period after the divergence of species in the subgenera Cerasus and Prunophora. Most variations (insertions/deletions and single-nucleotide polymorphisms) in the S- RNase gene were silent and, hence, have not been exposed to natural selection. The results are discussed from the aspects of S-allele evolution and phylogenetic relationships among cherries and other Prunus species. [ABSTRACT FROM AUTHOR]

Apple exhibits self-incompatibility controlled by the multiallelic S-locus. Twenty-three old apple cultivars were S-genotyped using three different approaches (allele-specific polymerase chain reaction (PCR) + cleaved amplified polymorphic sequences (CAPS), consensus PCR + sequencing and consensus PCR + CAPS) to compare the robustness and reliability of these techniques and characterise genotypes from the Carpathian basin that might be useful in resistance breeding. Best results were obtained using the ASPF3 and ASPR3S consensus primer pair that detected 96% of all alleles carried by the 23 cultivars tested. Flow cytometry analysis was also needed to control the completeness of the genotypes as was seen in case of a tetraploid cultivar with only three assigned S-alleles. The genetic disparity between the old Carpathian basin and modern apple cultivars was indicated by differences in allele frequency data ( S, S and S) as well as single nucleotide polymorphisms in S, S, S S and S and indels in S and S alleles. An alignment of partial genomic sequences indicated trans-specific and trans-generic evolution of S-ribonuclease alleles in the Maloideae subfamily ( S and S) and a possibly recent introgression event ( S) between Malus × domestica and Malus sylvestris. These data suggest that the genome of old cultivars from the Carpathian basin was enriched by several Malus taxa and are free from the consequences of modern breeding. These cultivars may contribute to the widening of the genetic basis of cultivated apple and prevent genetic erosion in future commercial cultivars. [ABSTRACT FROM AUTHOR]

pple ( Malus domestica Borkh.) possesses gametophytic self-incompatibility (GSI) which is controlled by S-RNase in the pistil as well as a pollen S-determinant that has not been well characterized. The identification of S-locus F-box brother ( SFBB) genes, which are good candidates for the pollen S-determinant in apple and pear, indicated the presence of multiple S-allelic polymorphic F-box genes at the S-locus. In apple, two SFBB gene groups have been described, while there are at least three groups in pear. In this report, we identified five MdSLFB ( S- RNase- linked F- box) genes from four different S-genotypes of apple. These genes showed pollen- and S-allele-specific expression with a high polymorphism among S-alleles. The phylogenetic tree suggested that some of them belong to SFBBα or β groups as described previously, while others appear to be different from SFBBs. In particular, the presence of MdSLFB3 and MdSLFB9 suggested that there are more S-allelic polymorphic F-box gene groups in the S-locus besides α and β. Based on the sequence polymorphism of MdSLFBs, we developed an S-genotyping system for apple cultivars. In addition, we isolated twelve MdSLFB- like genes, which showed pollen-specific expression without S-allelic polymorphism. [ABSTRACT FROM AUTHOR]

Abstract: In Israel four European pear cultivars are grown: ‘Spadona’ is the main cultivar and ‘Coscia’, ‘Gentile’ and ‘Spadochina’ are its pollinators. However, molecular S-genotyping revealed that ‘Spadona’ is semi-compatible with its three pollinators. This explains, at least in part, the relatively low pear yield in Israel. The Syrian pear (Pyrus syriaca) grows wild in Israel and blooms intensively, overlapping the blooming of the cultivated European pears. Cross-fertilization between Syrian pear and ‘Spadona’ was shown to be efficient suggesting that Syrian pear might be a potent pollinator for ‘Spadona’. Twenty-six Syrian pear seedlings, from different sites in north-east Israel were S-genotyped identifying 11 that are fully compatible with the four European pear varieties cultivated in Israel. By this screening, 24 different S-RNases were cloned; ten of them are new, whereas the other fourteen had been identified previously. In addition, seedlings of two wild pear species were also S-genotyped. Two seedlings from Pyrus betulifolia and one from Pyrus korshinskii were found to be genetically compatible with the four European pear cultivars. From these seedlings four S-RNases were cloned, two are new, one had been cloned previously and one was identical to an S-RNase allele cloned from Syrian pear in this work. [Copyright &y& Elsevier]

With 3 figures and 2 tables Consensus polymerase chain reaction (PCR) primers amplifying the first and second intron regions of the S-RNase gene and DNA sequencing as well as fruit set analysis after controlled pollinations were used for S-genotyping of Eastern European almond cultivars. Complete S-genotypes have been proposed for 19 accessions, and partial S-genotypes have been proposed for three additional accessions. Based on DNA sequences and fruit set analysis, two novel cross-incompatibility groups have been proposed: group XXI ( S11 S31H) including ‘Szigetcsépi 55’ (3/9) and ‘Eriane’ and group XXII ( S36 S37) for ‘Nikitskyi’ and ‘Óriás kagyló’ (4/4). Four new alleles were identified, S36 ( ), S37 ( ), S38 ( ) and S39 ( ). The combination of consensus PCR primers amplifying the first and second intron regions could reliably discriminate the S-RNase alleles identified in this study from those described previously. Our results supply long-awaited information on almond S-allele diversity from regions between the main cultivation centres and the centre of origin of this species. [ABSTRACT FROM AUTHOR]

Loquat ( Eriobotrya japonica Lindl.) is a fruit tree species of the Pyrinae subtribe of the Rosaceae that behaves as self incompatible. Since self-incompatibility in the Rosaceae is of the gametophytic type where a stylar ribonuclease ( S-RNase) controls the female function of pollen–pistil recognition, consensus primers derived from the alignment of S-RNase sequences from other Pyrinae species were used to search for S-RNases in loquat. As a result, the first four S-RNases were sequenced for this species. The genomic sequences obtained showed the structural features of Pyrinae S-RNases. Moreover, microscopic observations of pollen tube growth in the style confirmed the inter-(in)compatibility relationships predicted from the molecular analyses. Phylogenetic analysis of the deduced amino acid sequences with other Pyrinae S-RNases confirmed that divergence of S-alleles in loquat and the Pyrinae predated speciation. This study reports for the first time the genomic characterization of S-RNases in loquat, providing a sound basis for an appropriate selection of pollinator cultivars and an adequate design of breeding programs. [ABSTRACT FROM AUTHOR]

A series of PCR methods were used to detect S- RNase alleles and SFB alleles and to determine S-genotypes in 25 accessions of myrobalan ( Prunus cerasifera L.). Firstly, primers flanking the polymorphic second intron were used to identify S- RNases in agarose gels. These primers amplified one or two bands per accession in 25 accessions. Then consensus primers were designed for amplifying the polymorphic first intron, unique to Prunus S- RNases, for automated fluorescent detection. Each accession produced one or two peaks. New primers were then developed to amplify the intron in the SFB gene, for detection by fluorescence. Cross-referencing PCR bands and peaks indicated 15 S-alleles were present in the 25 accessions. Cloning, sequencing and comparison with published data indicated that the amplified products were S- RNase alleles. Sequence information was used to design primers specific for each S- RNase. Full and consistent S-genotypes were obtained by cross-comparing PCR data for 23 of the 25 accessions, and two accessions appeared to have a single allele. Pollen-tube microscopy indicated function of some but not all of the S-alleles sequenced. [ABSTRACT FROM AUTHOR]

Abstract: Self-incompatibility of almond [Prunus dulcis (Mill.) D.A. Webb] is controlled by the S-locus with 30 described allelic variants. In this study, PCR amplification, cloning and DNA sequence analysis revealed a new S-RNase allele in a Hungarian cultivar, ‘Tétényi bőtermő’. This new allele was labelled as S 31. Since S 31 is characterized by almost identical intron sizes as S 9, consensus PCR was not successful in discrimination of the alleles, even if fluorescently labelled fragments were sized on an automated sequencer. Therefore, an allele-specific forward primer (PdS31-F) was designed to anneal selectively within the second intron of the S 31-RNase gene and used in combination with the EM-PC3consRD consensus primer. This allowed for the successful discrimination of S 31 from S 9. The PdS31-F primer and allele-specific PCR in general might be useful in the identification of different alleles with matching intron sizes that might occur during screening for S-alleles in a more diverse population, e.g. local cultivars from Central Europe to Asia. [Copyright &y& Elsevier]

Cherry laurel ( Prunus laurocerasus L.) is an extreme polyploid (2 n = 22 x ) species of the Rosaceae family where gametophytic self-incompatibility (GSI) prevents inbreeding. This study was carried out to identify the S -ribonuclease alleles ( S -RNases) of P. laurocerasus using PCR amplification of the first and second intron region of the S-RNase gene, cloning and sequencing. A total of 23 putative S -RNase alleles ( S 1 - S 20 , S 5 m , S 13 m , and S 18 m ) were sequenced from the second (C2) to the fifth conserved region (C5), and they shared significant homology to other Prunus S -RNases. The length of the sequenced amplicons ranged from 505 to 1,544 bp, and similar sizes prevented the proper discrimination of some alleles based on PCR analysis. We have found three putatively non-functional alleles ( S 5 m , S 18 m , and S 9 ) coding for truncated proteins. Although firm conclusions cannot be drawn, our data seem to support that heteroallelic pollen cannot induce self-compatibility in this polyploid Prunus species. The identities in the deduced amino acid sequences between the P. laurocerasus and other Prunus S -RNases ranged between 44 and 100%, without a discontinuity gap separating the identity percentages of trans-specific and more distantly related alleles. The phylogenetic position, the identities in nucleotide sequences of the second intron and in deduced amino acid sequences found one or more trans-specific alleles for all but S 10 , S 14 , S 18 , and S 20 cherry laurel RNases. The analysis of mutational frequencies in trans-specific allele pairs indicated the region RC4-C5 accepts the most amino acid replacements and hence it may contribute to allele-specificity. Our results form the basis of future studies to confirm the existence and function of the GSI system in this extreme polyploid species and the alleles identified will be also useful for phylogenetic studies of Prunus S -RNases as the number of S-RNase sequences was limited in the Racemose group of Prunus (where P. laurocerasus belongs to)., 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., (Copyright © 2021 Halász, Molnár, Ilhan, Ercisli and Hegedűs.)

Self-incompatibility (SI) mechanisms prevent self-fertilization in flowering plants based on specific discrimination between self- and non-self pollen. Since this trait promotes outcrossing and avoids inbreeding it is a widespread mechanism of controlling sexual plant reproduction. Growers and breeders have effectively exploited SI as a tool for manipulating domesticated crops for thousands of years. However, only within the past thirty years have studies begun to elucidate the underlying molecular features of SI. The specific S -determinants and some modifier factors controlling SI have been identified in the sporophytic system exhibited by Brassica species and in the two very distinct gametophytic systems present in Papaveraceae on one side and in Solanaceae, Rosaceae, and Plantaginaceae on the other. Molecular level studies have enabled SI to SC transitions (and vice versa ) to be intentionally manipulated using marker assisted breeding and targeted approaches based on transgene integration, silencing, and more recently CRISPR knock-out of SI-related factors. These scientific advances have, in turn, provided a solid basis to implement new crop production and plant breeding practices. Applications of self-(in)compatibility include widely differing objectives such as crop yield and quality improvement, marker-assisted breeding through SI genotyping, and development of hybrids for overcoming intra- and interspecific reproductive barriers. Here, we review scientific progress as well as patented applications of SI, and also highlight future prospects including further elucidation of SI systems, deepening our understanding of SI-environment relationships, and new perspectives on plant self/non-self recognition., (Copyright © 2020 Muñoz-Sanz, Zuriaga, Cruz-García, McClure and Romero.)

During the last decade, S -genotyping has been extensively investigated in fruit tree crops such as those belonging to the Prunus genus, including plums. In plums, S -allele typing has been largely studied in diploid species but works are scarcer in polyploid species due to the complexity of the polyploid genome. This study was conducted in order to analyze the S -genotypes of 30 diploid P. salicina , 17 of them reported here for the first time, and 29 hexaploid plums (24 of P. domestica and 5 of P. insititia ). PCR analysis allowed identifying nine S -alleles in the P. salicina samples allocating the 30 accessions in 16 incompatibility groups, two of them identified here for the first time. In addition, pollen tube growth was studied in self-pollinated flowers of 17 Tunisian P. salicina under the microscope. In 16 samples, including one carrying the Se allele, which has been correlated with self-compatibility, the pollen tubes were arrested in the style. Only in one cultivar ("Bedri"), the pollen tubes reached the base of the style. Twelve S -alleles were identified in the 24 P. domestica and 5 P. insititia accessions, assigning accessions in 16 S -genotypes. S -genotyping results were combined with nine SSR loci to analyze genetic diversity. Results showed a close genetic relationship between P. domestica and P. salicina and between P. domestica and P. insititia corroborating that S -locus genotyping is suitable for molecular fingerprinting in diploid and polyploid Prunus species.