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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 multigenic and multiallelic S-locus in plants is responsible for the gametophytic self-incompatibility system, which is important to prevent the detrimental effects of self-fertilization and inbreeding depression. Several studies have discussed the importance of punctual mutations, recombination, and natural selection in the generation of allelic diversity in the S-locus. However, there has been no wide-ranging study correlating the molecular evolution and structural aspects of the corresponding proteins in Solanum. Therefore, we evaluated the molecular evolution of one gene in this locus and generated a statistically well-supported phylogenetic tree, as well as evidence of positive selection, helping us to understand the diversification of S alleles in Solanum. The three-dimensional structures of some of the proteins corresponding to the major clusters of the phylogenetic tree were constructed and subsequently submitted to molecular dynamics to stabilize the folding and obtain the native structure. The positively selected amino acid residues were predominantly located in the hyper variable regions and on the surface of the protein, which appears to be fundamental for allele specificity. One of the positively selected residues was identified adjacent to a conserved strand that is crucial for enzymatic catalysis. Additionally, we have shown significant differences in the electrostatic potential among the predicted molecular surfaces in S-RNases. The structural results indicate that local changes in the three-dimensional structure are present in some regions of the molecule, although the general structure seems to be conserved. No previous study has described such structural variations in S-RNases. [ABSTRACT FROM AUTHOR]

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Seedlessness is a desirable character in lemons and other citrus species. Seedless fruit can be induced in many ways, including through self-incompatibility (SI). SI is widely used as an intraspecific reproductive barrier that prevents self-fertilization in flowering plants. Although there have been many studies on SI, its mechanism remains unclear. The 'Xiangshui' lemon is an important seedless cultivar whose seedlessness has been caused by SI. It is essential to identify genes involved in SI in 'Xiangshui' lemon to clarify its molecular mechanism. In this study, candidate genes associated with SI were identified using high-throughput Illumina RNA sequencing (RNA-seq). A total of 61,224 unigenes were obtained (average, 948 bp; N50 of 1,457 bp), among which 47,260 unigenes were annotated by comparison to six public databases (Nr, Nt, Swiss-Prot, KEGG, COG, and GO). Differentially expressed genes were identified by comparing the transcriptomes of no-, self-, and cross-pollinated stigmas with styles of the 'Xiangshui' lemon. Several differentially expressed genes that might be associated with SI were identified, such as those involved in pollen tube growth, programmed cell death, signal transduction, and transcription. NADPH oxidase genes associated with apoptosis were highly upregulated in the self-pollinated transcriptome. The expression pattern of 12 genes was analyzed by quantitative real-time polymerase chain reaction. A putative S- RNase gene was identified that had not been previously associated with self-pollen rejection in lemon or citrus. This study provided a transcriptome dataset for further studies of SI and seedless lemon breeding. [ABSTRACT FROM AUTHOR]

S-RNase-based self-incompatibility in Petunia is a self/non-self recognition system that allows the pistil to reject self-pollen to prevent inbreeding and to accept non-self pollen for outcrossing. Cloning of S-RNase in 1986 marked the beginning of nearly three decades of intensive research into the mechanism of this complex system. S-RNase was shown to be the sole female determinant in 1994, and the first male determinant, S-locus F-box protein1 (SLF1), was identified in 2004. It was discovered in 2010 that additional SLF proteins are involved in pollen specificity, and recently two S-haplotypes of Petunia inflata were found to possess 17 SLF genes based on pollen transcriptome analysis, further increasing the complexity of the system. Here, we first summarize the current understanding of how the interplay between SLF proteins and S-RNase in the pollen tube allows cross-compatible pollination, but results in self-incompatible pollination. We then discuss some of the aspects that are not yet elucidated, including uptake of S-RNase into the pollen tube, nature, and assembly of SLF-containing complexes, the biochemical basis for differential interactions between SLF proteins and S-RNase, and fate of non-self S-RNases in the pollen tube. [ABSTRACT FROM AUTHOR]

Self-compatibility in almond ( Prunus dulcis) is attributed to the presence of the S haplotype, allelic to and dominant over the series of S-alleles controlling self-incompatibility. Some forms of the S haplotype, however, are phenotypically self-incompatible even though their nucleotide sequences are identical. DNA from leaves and styles from genetically diverse almond samples was cloned and sequenced and then analyzed for changes affecting S-RNase variants. Epigenetic changes in several cytosine residues were detected in a fragment of 4,700 bp of the 5′ upstream region of all self-compatible samples of the S-RNases, differentiating them from all self-incompatible samples of S-RNases analyzed. This is the first report of DNA methylation in a Rosaceae species and appears to be strongly associated with inactivation of the S allele. Results facilitate an understanding of the evolution of self-compatibility/self-incompatibility in almond and other Prunus species, and suggest novel approaches for future crop improvement. [ABSTRACT FROM AUTHOR]

The flowering plant Solanum chacoense uses an S-RNase-based self-incompatibility system in order to reject pollen that shares the same genes at the S-locus (S-haplotype) with the style (an incompatible reaction). Two different models have been advanced to explain how compatible pollen tubes are protected from the cytotoxic effects of the S-RNase, sequestration of the S-RNase in a vacuolar compartment or degradation of the S-RNase in the cytoplasm. Here, we examine the subcellular distribution of an S11-RNase 18 and 24 h post pollination (hpp) in compatible and incompatible crosses by immunogold labeling and transmission electron microscopy. We find that the S-RNase is present in the cytoplasm of both compatible and incompatible crosses by 18 hpp, but that almost all the cytoplasmic S-RNase is degraded by 24 hpp in compatible crosses. These results provide compelling evidence that S-RNases are degraded in compatible but not in incompatible pollen tubes. [ABSTRACT FROM AUTHOR]

Self-incompatibility ( SI) is a reproductive isolation mechanism in flowering plants. Plants in the Solanaceae, Rosaceae and Plantaginaceae belong to the gametophytic self-incompatibility type. S- RNase, which is encoded by a female-specific gene located at the S locus, degrades RNA in the pollen tube and causes SI. Recent studies have provided evidence that S- RNase is transported non-selectively into the pollen tube, but have not specified how this transportation is accomplished. We show here that the apple ( Malus domestica) MdABCF protein, which belongs to group F of the ABC transporter family, assists in transportation of S- RNase into the pollen tube. MdABCF is located in the pollen tube membrane and interacts with S- RNase. S- RNase was unable to enter the pollen tube when MdABCF was silenced by antisense oligonucleotide transfection. Our results show that MdABCF assists in transportation of either self or non-self S- RNase into the pollen tube. Moreover, MdABCF coordinates with the cytoskeleton to transport S- RNase. Blockage of S- RNase transport disrupts self-incompatibility in this system. [ABSTRACT FROM AUTHOR]

ROP/Rac GTPase is a conserved class of proteins which play diverse signalling roles in plants. They regulate many fundamental cellular processes such as F-actin dynamics, cell polarity and polar growth. Using apple genomic database analyses, nine ROP family members were cloned for the first time in a fruit tree (apple). Phylogenetic analyses indicated that the nine MdROPs were distributed into two groups, as previously described in the literature for model plants. Expression analyses show all MdROPs were highly expressed in pollen, in particular MdROP1, 3, 4 and 8. Yeast two hybrid and bimolecular fluorescence complementation analyses indicated MdROP8 interacts with S-RNase, a pistil determinate factor in gametophyte self-incompatibility. The pollen tube microtubule is shown to depolymerize in response to S-RNase treatment, during which the expression of MdROP8 rapidly decreased. These results indicate MdROP8 is related to S-RNase mediated self-incompatibility, and gives some useful evidence in modeling the relationship between cytoskeleton depolymerization and pollen tube growth inhibition during the apple SI reaction. [ABSTRACT FROM AUTHOR]

S-RNase is the female determinant of gametophytic self-incompatibility in apple and is usually considered to be the reason for rejection of pollen. In this study, we investigated the role of microtubules (MTs) in internalization of S-RNases by pollen tubes cultured in vitro. The results showed that S-RNase was imported into the pollen tube where it inhibits pollen tube growth, and that S-RNase is co-localized with the Golgi vesicle during the internalization process. Moreover, MT depolymerization is observed following accumulation of S-RNases in the pollen cytosol. On the other hand, S-RNase was prevented from entering the pollen tube when the pollen was treated with the actin filament (AF) inhibitor latrunculin A (LatA), the MT inhibitor oryzalin, or the MT stabilizer taxol at subtoxic concentrations. These hindered the construction of the MT, with pollen tubes capable of growth under these conditions. Pollen tubes showed improved growth in self-pollinated styles that were pre-treated with taxol. This suggests that cytoskeleton antagonists can prevent S-RNase-mediated inhibition of pollen tubes in vivo by blocking S-RNase internalization. These results suggest that an intact and dynamic cytoskeleton is required for the in vitro internalization of S-RNase, as shown by the effects of various cytoskeleton inhibitors. S-RNase internalization takes place via a membrane/cytoskeleton-based Golgi vesicle system, which can also affect self-incompatibility in apple. [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]

Highlights: [•] We identified the S-genotype of 13 North Western European pear cultivars. [•] All described cultivars differed by their S-genotypes. [•] Several identified S-alleles were rare alleles. [Copyright &y& Elsevier]

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

Highlights: [•] S6ins-RNase was cloned from the self-compatible loquat cultivars. [•] Four S6-SFBs were cloned from homozygote S6 loquat seedlings. [•] A 24-nucleotide insertion was identified within S6ins-RNase open reading frame. [•] No mutation was observed in the S6-SFBs. [•] The mutation in S6ins-RNase was found to be responsible for SC in these cultivars. [ABSTRACT FROM AUTHOR]

The stylar determinant of gametophytic self-incompatibility (GSI) in Solanaceae, Rosaceae, and Plantaginaceae is an S-RNase encoded by a multiallelic S-locus. The primary structure of S-RNases shows five conserved (C) and two hypervariable (HV) regions, the latter forming a domain implicated in S-haplotype-specific recognition of the pollen determinant to SI. All S-RNases are glycosylated at a conserved site in the C2 region, although previous studies have shown that N-linked glycans at this position are not required for S-haplotype-specific recognition and pollen rejection. Here the incompatibility phenotype of three constructs derived from an originally monoglycosylated S11-RNase of Solanum chacoense, that were designed to explore the role of the HV domain in determining pollen recognition and the role of the N-linked glycan in the C2 region, is reported. In one series of experiments, a second glycosylation site was introduced in the HVa region to test for inhibition of pollen-specific recognition. This modification does not impede pollen rejection, although analysis shows incomplete glycosylation at the new site in the HVa region. A second construct, designed to permit complete glycosylation at the HVa site by suppression of the conserved site in the C2 region, did increase the degree of site occupancy, but, again, glycosylation was incomplete. Plants expressing this construct rejected S11 pollen and, surprisingly, also rejected S13 pollen, thus displaying an unusual dual specificity phenotype. This construct differs from the first by the absence of the conserved C2 glycosylation site, and thus the dual specificity is observed only in the absence of the C2 glycan. A third construct, completely lacking glycosylation sites, conferred an ability to reject only S11 pollen, disproving the hypothesis that lack of a conserved glycan would confer a universal pollen rejection phenotype to the plant. [ABSTRACT FROM AUTHOR]

As with other self-incompatible Prunus species, cultivars of Japanese apricot ( Prunus mume Sieb. et Zucc.) display the S-RNase-based gametophytic self-incompatibility system. In this study, S-genotypes of ten Japanese apricot cultivars native to China were subjected to polyacrylamide gel electrophoresis (PAGE) analysis using an efficient Prunus S-RNase primer pair, Pru-C2 and PCE-R. In addition, three new S-RNase genes ( S, S and S -RNase) and six new SFB genes ( PmSFB14, PmSFB18, PmSFB22, PmSFB24, PmSFB31 and PmSFB34) were identified and their sequences were characterized and deposited in the GenBank database. [ABSTRACT FROM AUTHOR]

To avoid endogamy many flowering plants evolved a mechanism to recognize and reject their self-pollen, which is known as self-incompatibility (SI) system. In many cases the SI is genetically controlled by a single and highly polymorphic locus known as the S-locus. This DNA region carries two tightly linked transcriptional units. One of them encodes the female determinant (pistil expressed) and the second one the male determinant (pollen expressed). In this review, we discuss the most relevant advances in the SI mechanism in S-RNase based systems, which are present in Solanaceae, Rosaceae and Plantaginaceae. In these families, the female determinant encodes a ribonuclease named S-RNase and the male determinant encodes a protein called SLF/SFB (5-locus F-Box). We finally describe the role of the role of modifier genes, not coded by S-locus, in the pollen rejection response and propose a model including the main events so far described. [ABSTRACT FROM AUTHOR]

Key message: We report a rapid and reliable method for S genotyping of Rosaceae fruit trees, which would to be useful for successful planting of cross-compatible cultivars in orchards. Abstract: Japanese plum ( Prunus salicina) and sweet cherry ( Prunus avium), belonging to the family Rosaceae, possess gametophytic self-incompatibility controlled by a single polymorphic locus containing at least two linked genes, S- RNase and SFB ( S-haplotype-specific F-box gene). For successful planting of cross-compatible cultivars of Rosaceae fruit trees in commercial orchards, it is necessary to obtain information on S genotypes of cultivars. Recently, a method of dot-blot analysis utilizing allele-specific oligonucleotides having sequences of SFB-HVa region has been developed for identification of S haplotypes in Japanese plum and sweet cherry. However, dot-blot hybridization requires considerable time and skill for analysis even of a small number of plant samples. Thus, a quick and efficient method for S genotyping was developed in this study. In this method, instead of a nylon membrane used for dot-blot hybridization, streptavidin-coated magnetic beads are used to immobilize PCR products, which are hybridized with allele-specific oligonucleotide probes. Our improved method allowed us to identify 10 S haplotypes ( S- a, S- b, S- c, S- d, S- e, S- f, S- h, S- k, S- 7 and S- 10) of 13 Japanese plum cultivars and 10 S haplotypes ( S- 1, S- 2, S- 3, S- 4, S- 4′, S- 5, S- 6, S- 7, S- 9 and S- 16) of 13 sweet cherry cultivars utilizing SFB or S- RNase gene polymorphism. This method would be suitable for identification of S genotypes of a small number of plant samples. [ABSTRACT FROM AUTHOR]

Many species in the families of Rosaceae, Solanaceae, and Scrophulariaceae exhibit gametophytic self-incompatibility, a phenomenon controlled by two polymorphic genes at the S-locus, style- S ( S-RNase) and pollen- S ( SFB). Sequences of both genes show high levels of diversity, characteristic of genes involved in recognition of self-incompatibility systems in plants. In this study, S -RNase and SFB alleles were cloned from Prunus armeniaca cv. Chuanzhihong (Chinese apricot). Sequence comparisons of deduced amino acid sequences revealed that the P. armeniaca S-haplotype has different SFB alleles, but shares a single S-RNase allele with P. armeniaca S-haplotype. Moreover, P. armeniaca S -RNase haplotype has a single and three different alleles with S -RNase of P. tenella (dwarf almond) and S -RNase of P. mira (smooth pit peach), respectively. The functionalities of SFB and SFB have been evaluated by pollen tube growth and controlled field tests of P. tenella and P. mira. Genetic analysis of the two intercrosses showed that progenies segregated 1:1 into two S-genotype classes, which is consistent with the expected ratio for semi-compatibility. These findings imply that the allelic function of the S-haplotype is identical to that of the S-haplotype in a self-incompatibility reaction. Thus, these two Prunus S-haplotypes are in fact two neutral variants of the same S-haplotype. The evolution of the S-allele is also discussed in terms of both functions and differences between S- and S-haplotypes in Prunus. [ABSTRACT FROM AUTHOR]

S-RNase-based self-incompatibility (SI) is an intraspecific reproductive barrier to prevent self-fertilization found in many species of the Solanaceae, Plantaginaceae and Rosaceae. In this system, S- RNase and SLF/SFB ( S- locus F- box) genes have been shown to control the pistil and pollen SI specificity, respectively. Recent studies have shown that the SLF functions as a substrate receptor of a SCF (Skp1/Cullin1/F-box)-type E3 ubiquitin ligase complex to target S-RNases in Solanaceae and Plantaginaceae, but its role in Rosaceae remains largely undefined. Here we report the identification of two pollen-specific SLF-interacting Skp1-like (SSK) proteins, PbSSK1 and PbSSK2, in Pyrus bretschneideri from the tribe Pyreae of Rosaceae. Both yeast two-hybrid and pull-down assays demonstrated that they could connect PbSLFs to PbCUL1 to form a putative canonical SCF (SSK/CUL1/SLF) complex in Pyrus. Furthermore, pull-down assays showed that the SSK proteins could bind SLF and CUL1 in a cross-species manner between Pyrus and Petunia. Additionally, phylogenetic analysis revealed that the SSK-like proteins from Solanaceae, Plantaginaceae and Rosaceae form a monoclade group, hinting their shared evolutionary origin. Taken together, with the recent identification of a canonical SCF complex in Prunus of the tribe Amygdaleae of Rosaceae, our results show that a conserved canonical SCF complex is present in Solanaceae, Plantaginaceae and Rosaceae, implying that S-RNase-based self-incompatibility shares a similar molecular and biochemical mechanism. [ABSTRACT FROM AUTHOR]

Self-incompatibility is an important genetic mechanism that prevents inbreeding and promotes genetic polymorphism and heterosis in flowering plants. Many fruit species in the Rosaceae, including apple, pear, plum, apricot, sweet cherry, Japanese apricot, and almond, exhibit typical gametophytic self-incompatibility (GSI) controlled by an apparently single multi-allelic locus. This locus encodes at least two components from both the pollen and the pistil, and controls recognition of self- and non-self pollen. Recently, the GSI system has been investigated at the molecular and cellular levels in Rosaceae, and findings have provided some important insights as to how these two genes interact within pollen tubes that lead to specific inhibition of germination and/or growth of self-pollen tubes. In this review, molecular features of S-determinants of both pistil and pollen, identification of S-alleles, mechanisms of self-incompatibility break-down, and evolution of S-alleles are presented. Moreover, hypothetical signal transduction models in a self-incompatible system in Rosaceae are proposed based on recent findings that indicate that several signal factors are involved in GSI responses. [ABSTRACT FROM PUBLISHER]

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 ., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2022 Kunming Institute of Botany, Chinese Academy of Sciences. Publishing services by.)

Japanese pear has a genetically controlled self-incompatibility system, but both the pollen-tube growth in a semi in vivo assay and fruit set after self-pollination differ considerably among cultivars. The percentage of styles in which pollen tubes have reached the base ranges from 0 to 36 %, a value determined by culture of styles in vitro, and fruit set ranges from 0.6 to 15.2 %. Based on these data, we have assigned a value for the self-incompatibility weakness to each cultivar. Here, we showed that pollen factors control the degree of self-incompatibility. When the pollen-tube growth of 13 cultivars was compared in a completely compatible 'Hougetsu' ( S S) style, it differed a fair amount among cultivars and showed a significantly positive relation to self-incompatibility weakness ( r = 0.707). The degree of self-incompatibility of pear is, therefore, determined by pollen factor(s) unrelated to the S-locus. Although the fruit set and fruit growth of 'Hougetsu' were not affected by the pollen donor, a positive relationship was also observed between seed number and self-incompatibility weakness ( r = 0.972). However, in a style with no S-RNase production (genotype: S S), the relationship disappeared ( r = 0.341) and pollen-tube growth was promoted by 12-36 % except in one cultivar. These results suggest that S-RNase functions as a cytotoxin on compatible pollen in a cultivar-dependent manner, and that the degree of inhibition is determined by pollen factor(s) unrelated to the S-locus. The pollen factor also functions on S-RNase in incompatible styles, resulting in a different degree of self-incompatibility. [ABSTRACT FROM AUTHOR]

Japanese pear (Pyrus pyrifolia) exhibits gametophytic self-incompatibility (GSI) controlled by a complex and multiallelic S locus. The pistil-part product of the S locus is the polymorphic ribonuclease, S-RNase. Infor-mation on S-genotypes is important for the production and breeding of Japanese pears. Molecular analyses of S-genotypes of Japanese pear have been conducted with the CAPS (cleaved amplified polymorphic se-quence) system; PCR amplification of S-RNase fragments by a common primer pair followed by digestion with restriction enzymes each of which cleaves a specific S haplotype. Here, we show that the separation of S-RNase fragments by Polyacrylamide gel electrophoresis (PAGE) distinguishes four out of nine S haplotypes of Japanese pear without restriction digestion. S3-, S5-, S6- and Ss-RNases were identified as distinct bands by PAGE. S3- and S3-RNases were separated by PAGE despite their identical fragment sizes. Using this sys-tem, three Japanese pear lines with unknown S-genotypes were analyzed. The newly determined S-genotypes of the lines were confirmed by CAPS analysis. [ABSTRACT FROM AUTHOR]

Self-incompatibility, a common attribute of plant development, forms a classical paradigm of balancing selection in natural populations, in particular negative frequency-dependent selection. Under negative frequency-dependent selection population genetics theory predicts that the S-locus, being in command of self-incompatibility, keeps numerous alleles in equal frequencies demonstrating a wide allelic range. Moreover, while natural populations exhibit a higher within population genetic diversity, a reduction of population differentiation and increase of effective migration rate is expected in comparison to neutral loci. Allelic frequencies were investigated in terms of distribution and genetic structure at the gametophytic self-incompatibility locus in five wild cherry ( Prunus avium L.) populations. Comparisons were also made between the differentiation at the S-locus and at the SSR loci. Theoretical expectations under balancing selection were congruent to the results observed. The S-locus showed broad multiplicity (16 S-alleles), high genetic diversity, and allelic isoplethy. Genetic structure at the self-incompatibility locus was almost four times lower than at 11 nSSR loci. Analysis of molecular variance revealed that only 5 % of the total genetic variation concerns differentiation among populations. In conclusion, the wealth of S-allelic diversity found in natural wild cherry populations in Greece is useful not only in advancing basic population genetics research of self-incompatibility systems in wild cherry but also in the development of breeding programs. [ABSTRACT FROM AUTHOR]

The molecular bases of the gametophytic self-incompatibility (GSI) system of species of the subtribe Pyrinae (Rosaceae), such as apple and pear, have been widely studied in the last two decades. The characterization of S-locus genes and of the mechanisms underlying pollen acceptance or rejection have been topics of major interest. Besides the single pistil-side S determinant, the S-RNase, multiple related S-locus F-box genes seem to be involved in the determination of pollen S specificity. Here, we collect and review the state of the art of GSI in the Pyrinae. We emphasize recent genomic data that have contributed to unveiling the S-locus structure of the Pyrinae, and discuss their consistency with the models of self-recognition that have been proposed for Prunus and the Solanaceae. Experimental data suggest that the mechanism controlling pollen–pistil recognition specificity of the Pyrinae might fit well with the collaborative ‘non-self’ recognition system proposed for Petunia (Solanaceae), whereas it presents relevant differences with the mechanism exhibited by the species of the closely related genus Prunus, which uses a single evolutionarily divergent F-box gene as the pollen S determinant. The possible involvement of multiple pollen S genes in the GSI system of Pyrinae, still awaiting experimental confirmation, opens up new perspectives to our understanding of the evolution of S haplotypes, and of the evolution of S-RNase-based GSI within the Rosaceae family. Whereas S-locus genes encode the players determining self-recognition, pollen rejection in the Pyrinae seems to involve a complex cascade of downstream cellular events with significant similarities to programmed cell death. [ABSTRACT FROM AUTHOR]

Abstract: In this study, eight S-RNase alleles were isolated from two Prunus pseudocerasus and two Prunus speciosa accessions by PCR amplification from genomic DNA. Analysis of the amino acid sequences revealed five novel and three published S-alleles. These S-RNases share typical structural features with S-RNases from other Prunus species exhibiting gametophytic self-incompatibility. The deduced amino acid identities ranged from 60.8 to 75.6% among four S-RNase alleles in Prunus speciosa and ranged from 73 to 81.4% among four S-RNase alleles in Prunus pseudocerasus. The size of the first introns ranged from 197 to 341bp, and the size of second introns ranged from 81 to 1182bp. Sequence analysis demonstrated that the deduced amino acid identities, by comparison with other Prunus species, were often higher than those of intraspecific identities. Moreover, exceptionally high identities were found between Pspe-S 1 and Pd-S 28 ; between Pspe-S 31 and Pm-S 6 ; among Pspe-S 51 , Pa-S 29 and Pweb-S 7 ; and among Pps-S 13 , Psim-S 4 and Ps-S b , indicating that the S-RNase alleles evolved before Prunus species divergence. Interestingly, the similarities of the first and second introns were also high between the two S-RNase alleles, which range from 83.63 to 100% among the first introns and from 46.13 to 100% among the second introns. These information could not increase our knowledge on the S-alleles of Prunus species, but is available for molecular breeding of fruit trees, to avoid cross-incompatibility. [Copyright &y& Elsevier]

Abstract: The 5′‐flanking region of the S 12-, S 13-, S 21-RNase with a length of 854bp, 1448bp and 1137bp were successfully isolated by TAIL-PCR from genomic DNA from ‘Jinhua’, ‘Maogong’ (Pyrus pyrifolia) and ‘Yali’ (Pyrus bretschneideri) genomic DNA. Sequence alignment and analysis of S 13-, S 12-, S 21-RNase gene promoter sequences with S 2-, S 3-, S 4-, S 5-RNase 5′-flanking sequences indicated that a homology region of about 240bp exists in the regions just upstream of the putative TATA boxes of the seven Chinese/Japanese pear S-RNase genes. Phylogenetic tree suggests that the homology region between the Chinese/Japanese pear and apple S-RNase gene promoter regions reflects the divergence of S-RNase gene was formed before the differentiation of subfamilies. Full length and a series of 5′-deletion fragments-GUS fusions were constructed and introduced into Arabidopsis thaliana plants. GUS activity were detected in S 12 -pro-(1 to 5)-GUS‐pBll01.2 transgenic pistils and progressively decreased from S 12 -pro-1-GUS-pBI l01.2 to S 12 -pro-5-GUS-pBll01.2. No GUS activity was detected in S 12 -pro-6-GUS-pBll01.2 transgenic pistil and other tissues of non-transformants and all transgenic plants. The result suggested S 12-RNase promoter is pistil specific expression promoter. [Copyright &y& Elsevier]

Background and Aims The integrity of actin filaments (F-actin) is essential for pollen-tube growth. In S-RNase-based self-incompatibility (SI), incompatible pollen tubes are inhibited in the style. Consequently, research efforts have focused on the alterations of pollen F-actin cytoskeleton during the SI response. However, so far, these studies were carried out in in vitro-grown pollen tubes. This study aimed to assess the timing of in vivo changes of pollen F-actin cytoskeleton taking place after compatible and incompatible pollinations in Nicotiana alata. To our knowledge, this is the first report of the in vivo F-actin alterations occurring during pollen rejection in the S-RNase-based SI system. Methods The F-actin cytoskeleton and the vacuolar endomembrane system were fluorescently labelled in compatibly and incompatibly pollinated pistils at different times after pollination. The alterations induced by the SI reaction in pollen tubes were visualized by confocal laser scanning microscopy. Key Results Early after pollination, about 70 % of both compatible and incompatible pollen tubes showed an organized pattern of F-actin cables along the main axis of the cell. While in compatible pollinations this percentage was unchanged until pollen tubes reached the ovary, pollen tubes of incompatible pollinations underwent gradual and progressive F-actin disorganization. Colocalization of the F-actin cytoskeleton and the vacuolar endomembrane system, where S-RNases are compartmentalized, revealed that by day 6 after incompatible pollination, when the pollen-tube growth was already arrested, about 80 % of pollen tubes showed disrupted F-actin but a similar percentage had intact vacuolar compartments. Conclusions The results indicate that during the SI response in Nicotiana, disruption of the F-actin cytoskeleton precedes vacuolar membrane breakdown. Thus, incompatible pollen tubes undergo a sequential disorganization process of major subcellular structures. Results also suggest that the large pool of S-RNases released from vacuoles acts late in pollen rejection, after significant subcellular changes in incompatible pollen tubes. [ABSTRACT FROM AUTHOR]

The gametophytic self-incompatibility system in Rosaceae is controlled by a single highly polymorphic gene complex, termed the S locus, which is comprised of tightly linked stylar-expressed gene, S-RNase, with a pollen-expressed gene. In this study, seven novel S haplotype-specific F-box protein genes, PmSFB to PmSFB in Prunus mume, have been identified and characterised. Similarities amongst the deduced amino acid sequences of these SFBs ranged from 73.2% to 90.9%. Comparisons of two trans-specific pairs of haplotypes, PmS with PspS and PmS/ ParS with PspS, indicated high degrees of polypeptide sequence identity. The deduced amino acid sequences of PmSFB and PspSFB showed similarity scores of 99.1%. In addition, the deduced amino acid sequences of the corresponding S-RNases, PmS-RNase and PspS-RNase exhibited a high similarity score of 99.0%. The similarity scores of the sequences physically positioned between S-RNase and SFB in the PmS haplotype and PspS were 94.6%. The deduced amino acid sequence of PmSFB showed high similarity scores with those of PspSFB and ParSFB at 99.3% and 97.9%, respectively. At the deduced amino acid level, the similarity scores of the PmS-RNase with the PspS-RNase or the ParS-RNase were 99.0% or 99.1%, respectively. Moreover, the similarity score of the sequence separating S-RNase and SFB in PmS was 96.1% compared with that of the PspS haplotype and 99.0% compared with that of the ParS haplotype. Our results suggest that the S haplotypes in P. mume, P. armeniaca and P. spinosa may have a common ancestor. [ABSTRACT FROM AUTHOR]

Abstract: Lopsided apple fruits were found in an orchard consisting of a single commercial cultivar Fuji and pollinizers of crab-apples under conditions of natural pollination. Speculating that it was caused by inadequate partial pollination, we devised an appropriate model. We artificially pollinated one out of five pistils of apple gynoecium, and then investigated the correlations among inadequate partial pollination, seed number, distribution, and fruit shape. It was speculated that a decrease in seed number and an increase in the rate of lopsidedness were caused by inadequate partial pollination. A deviation in seed distribution was also a factor in the occurrence of lopsided fruits. Since many of the main pollinator Osmia cornifrons visited a flower repeatedly at an orchard consisting of a single cultivar and pollinizers, we analyzed the effect of repeated pollination on increasing and improving seed number in fruits and fruit shape, respectively, using artificial pollination of a pollinizer and cultivars with various S-genotypes. Repeated pollination at 24-h intervals did not contribute to fertilization, but those at 4-h intervals contributed significantly as pollen parents of seeds in fruits. The ability of pistil fertilization was maintained until four days after flowering regardless of pollination, but once fertilization occurred, this pistil was not considered to contribute to seed production 24h after initial pollination. Those results suggested that pollinizers in full bloom be shifted more than a day seemed unnecessary at an orchard with single commercial cultivar, and repeated artificial pollination must be done within one day. [Copyright &y& Elsevier]

Apple ( Malus × domestica Borkh.) is a typical Rosaceae species that exhibits gametophytic self-incompatibility (GSI) controlled by polymorphic S-alleles. In this study, the S-alleles of wild Malus species were amplified, sequenced and compared using polymerase chain reaction (PCR) technology. 21 S-alleles were identified in 27 wild Malus species. The results indicated that the overwhelming majority of S-alleles between wild Malus species and cultivars shared identical sequences. Simultaneously, five new S alleles (designated S- S) were identified in wild Malus species. There are the S-RNase in M. angustifolia (Ation) Michaux, S-RNase in M. orientalis Uglitzk. Ex Juz. and M. sylvestris (L.) Mill., S-RNase in M. tschonoskii (Maxim.) C.K. Schneid. and M. sieversii (Ldb.) Roem., S-RNase in M. komarovii (Sarg.) Rehd. and M. kansuensis (Batal.) C. K. Schneid., S-RNase in M. manshurica (Maxim.) V. Komorov wild Malus species. Additionally, an S-RNase was cloned in wild Malus prunifolia var. ringo, which have the same open reading frame as Malus × domestica cv. Fuji, but lacked whole intron. [ABSTRACT FROM AUTHOR]

Pyrus displays gametophytic self-incompatibility controlled by a single highly polymorphic gene complex termed S locus, which comprises a stylar-expressed gene ( S-RNase) tighlty linked with a pollen expressed gene, that determines the specificity of the self-incompatibility locus. Deduced amino acid sequence of 'Meigetsu' S-RNase in Pyrus pyrifolia and 'Kuerlexiangli' S-RNase in P. sinkiangensis showed 100% identity. S-RNase in Malus spectabilis was also found to be similar to S-RNase in P. pyrifolia with 96.9% identity in the deduced amino acid sequence. The intron, which is generally highly polymorphic between alleles, was also remarkably well conserved within these allele pairs. The intron of PpS-RNase showed 95.3 and 91.9% identity with PsS-RNase and MsS-RNase, respectively. Pollen tube growth in styles, pollen tube length in artificial media containing different S-RNases and segregation of S haplotypes in F plants revealed commonality of the recognition specificity between PpS-RNase and PsS-RNase and between PpS-RNase and MsS-RNase. Results suggested that PpS-RNase, PsS-RNase and MsS-RNase have maintained the same recognition specificity after the divergence of the two species and that amino acid substitutions found between PpS-RNase and MsS-RNase do not alter the recognition specificity. [ABSTRACT FROM AUTHOR]

Although RNase-based self-incompatibility (SI) is suspected to operate in a wide group of plant families, it has been characterized as the molecular genetic basis of SI in only three distantly related families, Solanaceae, Plantaginaceae, and Rosaceae, all described over a decade ago. Previous studies found that gametophytic SI, controlled by a multi-allelic S-locus, operates in the coffee family (Rubiaceae). The molecular genetic basis of this mechanism remains unknown, despite the immense importance of coffee as an agricultural commodity. Here, we isolated ten sequences with features of T2-S-type RNases from two Coffea species. While three of the sequences were identified in both species and clearly do not appear to be S-locus products, our data suggest that six sequences may be S-alleles in the self-incompatible C. canephora, and one may be a relict in the self-compatible C. arabica. We demonstrate that these sequences show style-specific expression, display polymorphism in C. canephora, and cluster with S-locus products in a phylogenetic analysis that includes other plant families with RNase-based SI. Although our results are not definitive, in part because the available plant materials were limited and data patterns relatively complex, our results strongly hint that RNase-based SI mechanism operates in the Rubiaceae family. [ABSTRACT FROM AUTHOR]

Apricot (Prunus armeniaca L.) cultivars show a gametophytic self-incompatibility (GSI) system, like other fruit species of Rosaceae family. Thus, it is necessary to determine their S-genotypes in order for stable fruit set in commercial cultivation. S-genotypes of apricots were determined by PCR and test crosses. Three sets of consensus primers designed from Prunus S-RNases were used to amplify fragments containing the first and second S-RNase intron, respectively. Through the results obtained from the 3 PCRs, we could identify SI genotypes of 33apricot cultivars. Several cultivars such as 'Heiwa', 'Yamagata No.3' and 'Shinsuoomi' had the self-compatible (Sc) allele. Self-pollination tests revealed that cultivars with Sc allele were self-compatible. Cross-pollination tests confirmed that there was cross-incompatibility between the cultivars with the same S-genotypes. These results might be very useful for growers for effective pollination and for breeders using these in cross breeding programs. [ABSTRACT FROM AUTHOR]

Loquat ( Eriobotrya japonica Lindl.; Pyrinae, Rosaceae) is an evergreen fruit tree which is acquiring increasing importance as an alternative crop in warm-temperate and subtropical regions. Recent pollination studies suggest that crop intensification has resulted in a reduction of fruit productivity in different locations. Loquat exhibits gametophytic self-incompatibility (GSI) and, consequently, fruit set strongly depends on cross-pollination between intercompatible cultivars. S-genotyping and incompatibility group assignment is therefore essential for adequate loquat orchard design. This study reports the molecular characterization of the S- RNase allele composition of 31 loquat cultivars grown in Spain. PCR with primers developed from loquat S- RNase sequences was used in combination with high-performance capillary polyacrilamide electrophoresis for the fine-evaluation of length polymorphisms at the S- RNase gene. This approach allowed to uncover a new S- RNase allele ( S), whose sequence is reported and to characterize the first six S- RNase-based incompatibility groups (I-VI) in loquat, resulting from the combination of five S-alleles ( S- S). The large number of cultivars that could be assigned to only six groups suggests a narrow genetic base for this crop in Spain and stresses the need for careful determination of the S-genotype of cultivars in an attempt to overcome production problems associated with inadequate pollination. [ABSTRACT FROM AUTHOR]

Background S-RNase-based self-incompatibility (SI) occurs in the Solanaceae, Rosaceae and Plantaginaceae. In all three families, compatibility is controlled by a polymorphic S-locus encoding at least two genes. S-RNases determine the specificity of pollen rejection in the pistil, and S-locus F-box proteins fulfill this function in pollen. S-RNases are thought to function as S-specific cytotoxins as well as recognition proteins. Thus, incompatibility results from the cytotoxic activity of S-RNase, while compatible pollen tubes evade S-RNase cytotoxicity. Scope The S-specificity determinants are known, but many questions remain. In this review, the genetics of SI are introduced and the characteristics of S-RNases and pollen F-box proteins are briefly described. A variety of modifier genes also required for SI are also reviewed. Mutations affecting compatibility in pollen are especially important for defining models of compatibility and incompatibility. In Solanaceae, pollen-side mutations causing breakdown in SI have been attributed to the heteroallelic pollen effect, but a mutation in Solanum chacoense may be an exception. This has been interpreted to mean that pollen incompatibility is the default condition unless the S-locus F-box protein confers resistance to S-RNase. In Prunus, however, S-locus F-box protein gene mutations clearly cause compatibility. Conclusions Two alternative mechanisms have been proposed to explain compatibility and incompatibility: compatibility is explained either as a result of either degradation of non-self S-RNase or by its compartmentalization so that it does not have access to the pollen tube cytoplasm. These models are not necessarily mutually exclusive, but each makes different predictions about whether pollen compatibility or incompatibility is the default. As more factors required for SI are identified and characterized, it will be possible to determine the role each process plays in S-RNase-based SI. [ABSTRACT FROM PUBLISHER]

Background For the Solanaceae-type self-incompatibility, also possessed by Rosaceae and Plantaginaceae, the specificity of self/non-self interactions between pollen and pistil is controlled by two polymorphic genes at the S-locus: the S-locus F-box gene (SLF or SFB) controls pollen specificity and the S-RNase gene controls pistil specificity. Scope This review focuses on the work from the authors' laboratory using Petunia inflata (Solanaceae) as a model. Here, recent results on the identification and functional studies of S-RNase and SLF are summarized and a protein-degradation model is proposed to explain the biochemical mechanism for specific rejection of self-pollen tubes by the pistil. Conclusions The protein-degradation model invokes specific degradation of non-self S-RNases in the pollen tube mediated by an SLF, and can explain compatible versus incompatible pollination and the phenomenon of competitive interaction, where SI breaks down in pollen carrying two different S-alleles. In Solanaceae, Plantaginaceae and subfamily Maloideae of Rosaceae, there also exist multiple S-locus-linked SLF/SFB-like genes that potentially function as the pollen S-gene. To date, only three such genes, all in P. inflata, have been examined, and they do not function as the pollen S-gene in the S-genotype backgrounds tested. Interestingly, subfamily Prunoideae of Rosaceae appears to possess only a single SLF/SFB gene, and competitive interaction, observed in Solanaceae, Plantaginaceae and subfamily Maloideae, has not been observed. Thus, although the cytotoxic function of S-RNase is an integral part of SI in Solanaceae, Plantaginaceae and Rosaceae, the function of SLF/SFB may have diverged. This highlights the complexity of the S-RNase-based SI mechanism. The review concludes by discussing some key experiments that will further advance our understanding of this self/non-self discrimination mechanism. [ABSTRACT FROM PUBLISHER]

The pear cultivar 'Zaoguan' ( S S) is the a self-compatible descendant of 'Yali' ( S S) × 'Qingyun'( S S). Two self-incompatible cultivars 'Xinya' and 'Yaqing', also S-genotyped as S S for the S- RNase gene, were used as controls. Field pollination data revealed that 'Zaoguan' displayed SC, whereas 'Xinya' and 'Yaqing' showed self-incompatibility (SI) upon self-pollination. Reciprocal pollinations between the varieties showed that most of the 'Zaoguan' flowers pollinated with 'Xinya' or 'Yaqing' pollen set fruits but that few of the 'Xinya' or 'Yaqing' flowers set fruit when pollinated with 'Zaoguan' pollen. The pollen performance was monitored with fluorescence microscopy, and we observed that 'Zaoguan' accepted self-pollen as well as 'Xinya' or 'Yaqing' pollen, whereas 'Xinya' or 'Yaqing' rejected self-pollen and 'Zaoguan' pollen. The S- RNase but not the S- RNase could be detected in all selfed progeny of 'Zaoguan'. Comparisons of the 2D-PAGE profiles of the stylar extracts from the three cultivars showed that the S-RNase protein expressed normally, but the S-RNase of 'Zaoguan' was not found. Thus, we concluded that the stylar S products were defective in 'Zaoguan' and that the S-allele functioned normally. The nucleotide sequences of the S- and S- RNase of 'Zaoguan' showed no differences from those of 'Xinya' or 'Yaqing', and they transcribed normally. These results indicate that SC in 'Zaoguan' was due to the loss of the S-RNase caused by unknown post-transcriptional factors. [ABSTRACT FROM AUTHOR]

The recent analysis of the S-locus region of apple and Japanese pear, two species of Pyrinae (Rosaceae), suggested multiple and different F-box genes (called SFBBs) as candidates for the male determinant (pollen S) of RNase-based gametophytic self-incompatibility in these two species. Here, we followed a phylogenetic approach to take advantage of the pattern of molecular evolution of the S-locus of Pyrinae in characterizing SFBB homologs belonging to S-haplotypes of apple and three species of Pyrus (European, Japanese, and Chinese pears). Our results suggested that the S-locus region of Pyrinae contains no less than six SFBB members and that its structure seems to be rather conserved between apple and pear species. In accordance with the prevailing theory on S-haplotype evolution, the pollen S is expected to have coevolved with the S-RNase and to show some common features derived from the long-term evolution under frequency-dependent balancing selection, i.e., high sequence diversity, evidence of positive selection, and shared ancestral polymorphisms. Using this conceptual framework, we present evidence that some SFBB genes may be better candidates for pollen S in Pyrinae than others. Overall, the SFBB genes analyzed exhibited much lower sequence diversity than their associated S-RNases; likewise, they showed little or no evidence of positive selection. However, evidence of coevolution with the S-RNase clearly emerged for two of them. Altogether our results suggested different evolutionary histories for different SFBBs putatively derived from their distinct involvement in self-incompatibility. [ABSTRACT FROM AUTHOR]

Abstract: Previous SDS PAGE gel analysis of the floral nectars from petunia and tobacco plants revealed significant differences in the protein patterns. Petunia floral nectar was shown to contain a number of RNase activities by in gel RNase activity assay. To identify these proteins in more detail, the bands with RNase activity were excised from gel and subjected to trypsin digestion followed by LC–MS/MS analysis. This analysis revealed that S-RNases accumulate in nectar from Petunia hybrida, where they should carry out a biological function different from self-pollen rejection. In addition, other proteins were identified by the LC–MS/MS analysis. These proteins include a peroxidase, an endochitinase, and a putative fructokinase. Each of these proteins contained a secretory signal sequence that marked them as potential nectar proteins. We developed RT-PCR assays for each of these five proteins and demonstrated that each of these proteins was expressed in the petunia floral nectary. A discussion of the role of these proteins in antimicrobial activity in nectar is presented. [Copyright &y& Elsevier]

European pear, as well as its close relatives Japanese pear and apple, exhibits S-RNase-based gametophytic self-incompatibility. The male determinant of this self-incompatibility mechanism is a pollen-expressed protein containing an F-box domain; in the genera Petunia (Solanaceae), Antirrhinum (Plantaginaceae), and Prunus (Rosaceae), a single F-box gene determines the pollen S. In apple and Japanese pear, however, multiple S-locus F-box genes were recently identified as candidates for the pollen S, and they were named S-locus F-Box Brothers. These genes were considered good candidates for the pollen S determinant since they exhibit S-haplotype-specific polymorphisms, pollen-specific expression, and linkage to the S-RNase. In the present study, S-locus F-Box Brothers homologs have been cloned from two of the most agronomically important European pear varieties, 'Abbé Fétel' (S/S) and 'Max Red Bartlett' (S/S), and they have been mapped on a genetic linkage map developed on their progeny. Our results suggest that the number of F-box genes linked to the S-locus of the European pear is higher than expected according with previous reports for apple and Japanese pear, since up to five genes were found to be linked to a single S-haplotype. Moreover, two of these genes exhibited an incomplete linkage to the S-RNase, allowing the identification of low-frequency recombinant haplotypes, generated by a crossing-over event between the two genes. These F-box genes are most likely placed in close proximity of the S-locus but do not belong to it, and they can thus be excluded from being responsible for the determination of pollen S function. [ABSTRACT FROM AUTHOR]

Most fruit trees in the Rosaceae exhibit self-incompatibility, which is controlled by the pistil S gene, encoding a ribonuclease (S-RNase), and the pollen S gene at the S-locus. The pollen S in Prunus is an F-box protein gene (SLF/SFB) located near the S-RNase, but it has not been identified in Pyrus and Malus. In the Japanese pear, various F-box protein genes (PpSFBB-α–γ) linked to the S-RNase are proposed as the pollen S candidate. Two bacterial artificial chromosome (BAC) contigs around the S-RNase genes of Japanese pear were constructed, and 649 kb around S4-RNase and 378 kb around S2-RNase were sequenced. Six and 10 pollen-specific F-box protein genes (designated as PpSFBB4-u1–u4, 4-d1–d2 and PpSFBB2-u1–u5, 2-d1–d5, respectively) were found, but PpSFBB4-α–γ and PpSFBB2-γ were absent. The PpSFBB4 genes showed 66.2–93.1% amino acid identity with the PpSFBB2 genes, which indicated clustering of related polymorphic F-box protein genes between haplotypes near the S-RNase of the Japanese pear. Phylogenetic analysis classified 36 F-box protein genes of Pyrus and Malus into two major groups (I and II), and also generated gene pairs of PpSFBB genes and PpSFBB/Malus F-box protein genes. Group I consisted of gene pairs with 76.3–94.9% identity, while group II consisted of gene pairs with higher identities (>92%) than group I. This grouping suggests that less polymorphic PpSFBB genes in group II are non-S pollen genes and that the pollen S candidates are included in the group I PpSFBB genes. [ABSTRACT FROM PUBLISHER]

The plant genus Lycium (Solanaceae) originated in the Americas and includes approximately 85 species that are distributed worldwide. The vast majority of Old World species occur in southern Africa and eastern Asia. In this study, we examine biogeographic relationships among Old World species using a phylogenetic approach coupled with molecular evolutionary analyses of the S-RNase self-incompatibility gene. The phylogeny inferred from nuclear granule-bound starch synthase I (GBSSI), nuclear conserved ortholog set II (COSII) marker C2_At1g24360, and plastid spacer data (trnH-pbsA, trnDGUC–trnTGGU, rpl32–trnLUAG, and ndhF–rpl32) includes a clade of eastern Asian Lycium nested within the African species, suggesting initial dispersal from the Americas to Africa, with subsequent dispersal to eastern Asia. Molecular dating estimates suggest that these dispersal events occurred relatively recently, with dispersal from the Americas to Africa approximately 3.64 Ma (95% highest posterior density [HPD]: 1.58–6.27), followed by subsequent dispersal to eastern Asia approximately 1.21 Ma (95% HPD: 0.32–2.42). In accordance, the S-RNase genealogy shows that S-RNases isolated from Old World species are restricted to four lineages, a subset of the 14 lineages including S-RNases isolated from New World Lycium species, supporting a bottleneck of S-RNase alleles concomitant with a single dispersal event from the Americas to the Old World. Furthermore, the S-RNase genealogy is also consistent with dispersal of Lycium from Africa to Asia, as eastern Asian alleles are restricted to a subset of the lineages that also include African alleles. Such a multilocus approach, including complementary data from GBSSI, COSII, plastid spacer regions, and S-RNase, is powerful for understanding dispersal histories of closely related species. [ABSTRACT FROM PUBLISHER]

The cherry ( Prunus avium), a self-incompatible diploid species, and the sour cherry ( Prunus cerasus), a self-incompatible or self-compatible allotetraploid species derived from P. avium and Prunus fruticosa, share several S-RNase alleles, including S. An inactive form, S °, is found in some sour cherries. Two (AT) microsatellites are associated with allele S -RNase, one in the first intron and one in the second. Their length polymorphisms were studied in 14 sweet and 17 wild cherries (both P. avium) and in 42 sour cherries. Fluorescent primers amplifying each microsatellite were designed and amplification products sized on an automated sequencer. Variants ranged from 247 to 273 bp for the first intron microsatellite and from 308 to 322 bp for the second. There were 34 combinations and, surprisingly, the lengths of the two microsatellites were correlated. Generally, the sweet, wild and sour cherries had different combinations, and the four examples of S °-RNase were associated with three different combinations. Certain sequences associated with the microsatellites match footprints of transposons. The distribution of combinations indicated little overlap between the three populations analysed and provided useful insights into relationships of some of the accessions allowing some parentages to be checked. In the diploid sweet and wild cherries, S variants presumably resulted from slippage during replication, but in the tetraploid sour cherries, which can have more than one copy of S or S °, intra-allelic crossing over may have generated new variants. The possible involvement of transposable elements in the origin of these microsatellites is considered. [ABSTRACT FROM AUTHOR]