Analysis of the S-locus structure in Prunus armeniaca L. Identification of S-haplotype specific S-RNase and F-box genes

The gametophytic self-incompatibility (GSI) system in Rosaceae has been proposed to be controlled by two genes located in the S-locusan S-RNase and a recently described pollen expressed S-haplotype specific F-box gene (SFB). However, in apricot (Prunus armeniaca L.) these genes had not been identified yet. We have sequenced 21kb in total of the S-locus region in 3 different apricot S-haplotypes. These fragments contain genes homologous to the S-RNase and F-box genes found in other Prunusspecies, preserving their basic gene structure features and defined amino acid domains. The physical distance between the F-boxand the S-RNase genes was determined exactly in the S ₂-haplotype (2.9kb) and inferred approximately in the S ₁-haplotype (< 49kb) confirming that these genes are linked. Sequence analysis of the 5 flanking regions indicates the presence of a conserved region upstream of the putative TATA box in the S-RNase gene. The three identified S-RNase alleles (S ₁, S ₂ and S ₄) had a high allelic sequence diversity (75.3 amino acid identity), and the apricot F-box allelic variants (SFB₁, SFB₂ and SFB₄) were also highly haplotype-specific (79.4 amino acid identity). Organ specific-expression was also studied, revealing that S ₁- and S ₂-RNases are expressed in style tissues, but not in pollen or leaves. In contrast, SFB ₁ and SFB ₂ are only expressed in pollen, but not in styles or leaves. Taken together, these results support these genes as candidates for the pistil and pollen S-determinants of GSI in apricot.     

Identification of <Emphasis Type="Italic">S</Emphasis>-haplotype-specific F-box gene in Japanese plum (<Emphasis Type="Italic">Prunus salicina</Emphasis> Lindl.)

Self-incompatibility has been studied extensively at the molecular level in Solanaceae, Rosaceae and Scrophulariaceae, all of which exhibit gametophytic self-incompatibility controlled by a single polymorphic locus containing at least two linked genes, i.e., the S-RNase gene and the pollen-expressed SFB/SLF (S-haplotype-specific F-box/S-locus F-box) gene. However, the SFB gene in Japanese plum (Prunus salicina Lindl.) has not yet been identified. We determined eight novel sequences homologous to the SFB genes of other Prunus species and named these sequences PsSFB. The gene structure of the SFB genes and the characteristic domains in deduced amino acid sequences were conserved. Three sequences from 410 to 2,800 bp of the intergenic region between the PsSFB sequences and the S-RNase alleles were obtained. The eight identified PsSFB sequences showed S-haplotype-specific polymorphism, with 74–83% amino acid identity. These alleles were exclusively expressed in the pollen. These results suggest that the PsSFB alleles are the pollen S-determinants of GSI in Japanese plum. Nucleotide sequence data reported are available in the NCBI database under the accession numbers DQ849084–DQ849090 and DQ849118.     

<Emphasis Type="Italic">S</Emphasis> genotyping and <Emphasis Type="Italic">S</Emphasis> screening utilizing <Emphasis Type="Italic">SFB</Emphasis> gene polymorphism in Japanese plum and sweet cherry by dot-blot analysis

Most Rosaceae fruit trees such as Japanese plum and sweet cherry have a gametophytic self-incompatibility (GSI) system controlled by a single S locus containing at least two linked genes with multiple alleles, i.e., S-RNase as a pistil determinant and SFB (S-haplotype-specific F-box gene) as a candidate for the pollen S determinant. For identification of S genotypes, many methods based on polymerase chain reaction (PCR) utilizing polymorphism in length of the S-RNase and SFB gene have been developed. In this study, we developed two dot-blot analysis methods for S-haplotype identification utilizing allele-specific oligonucleotides based on the SFB-HVa region, which has high sequence polymorphism. Dot-blotting of allele-specific oligonucleotides hybridized with digoxigenin-labeled PCR products allowed S genotyping of plants with nine S haplotypes (S-a, S-b, S-c, S-e, S-f, S-h, S-k, S-7 and S-10) in Japanese plum and ten S haplotypes (S-1, S-2, S-3, S-4, S-4′, S-5, S-6, S-7, S-9 and S-16) in sweet cherry (dot-blot-S-genotyping). In addition, dot-blotting of PCR products of SFB probed with the allele-specific oligonucleotides, occasionally utilizing competitive hybridization, was successful in screening for a desirable S haplotype in sweet cherry (dot-blot-S-screening).     

Inheritance of Hetero-Diploid Pollen S-Haplotype in Self-Compatible Tetraploid Chinese Cherry (Prunus pseudocerasus Lindl)

The breakdown of self-incompatibility, which could result from the accumulation of non-functional S-haplotypes or competitive interaction between two different functional S-haplotypes, has been studied extensively at the molecular level in tetraploid Rosaceae species. In this study, two tetraploid Chinese cherry (Prunus pseudocerasus) cultivars and one diploid sweet cherry (Prunus avium) cultivar were used to investigate the ploidy of pollen grains and inheritance of pollen-S alleles. Genetic analysis of the S-genotypes of two intercross-pollinated progenies showed that the pollen grains derived from Chinese cherry cultivars were hetero-diploid, and that the two S-haplotypes were made up of every combination of two of the four possible S-haplotypes. Moreover, the distributions of single S-haplotypes expressed in self- and intercross-pollinated progenies were in disequilibrium. The number of individuals of the two different S-haplotypes was unequal in two self-pollinated and two intercross-pollinated progenies. Notably, the number of individuals containing two different S-haplotypes (S₁- and S₅-, S₅- and S₈-, S₁- and S₄-haplotype) was larger than that of other individuals in the two self-pollinated progenies, indicating that some of these hetero-diploid pollen grains may have the capability to inactivate stylar S-RNase inside the pollen tube and grow better into the ovaries.     

Genetic and molecular characterization of three novel S-haplotypes in sour cherry (Prunus cerasus L.)

Tetraploid sour cherry (Prunus cerasus L.) exhibits gametophytic self-incompatibility (GSI) whereby the specificity of self-pollen rejection is controlled by alleles of the stylar and pollen specificity genes, S-RNase and SFB (S haplotype-specific F-box protein gene), respectively. As sour cherry selections can be either self-compatible (SC) or self-incompatible (SI), polyploidy per se does not result in SC. Instead the genotype-dependent loss of SI in sour cherry is due to the accumulation of non-functional S-haplotypes. The presence of two or more non-functional S-haplotypes within sour cherry 2x pollen renders that pollen SC. Two new S-haplotypes from sour cherry, S(33) and S(34), that are presumed to be contributed by the P. fruticosa species parent, the complete S-RNase and SFB sequences of a third S-haplotype, S(35), plus the presence of two previously identified sweet cherry S-haplotypes, S(14) and S(16) are described here. Genetic segregation data demonstrated that the S(16)-, S(33)-, S(34)-, and S(35)-haplotypes present in sour cherry are fully functional. This result is consistent with our previous finding that 'hetero-allelic' pollen is incompatible in sour cherry. Phylogenetic analyses of the SFB and S-RNase sequences from available Prunus species reveal that the relationships among S-haplotypes show no correspondence to known organismal relationships at any taxonomic level within Prunus, indicating that polymorphisms at the S-locus have been maintained throughout the evolution of the genus. Furthermore, the phylogenetic relationships among SFB sequences are generally incongruent with those among S-RNase sequences for the same S-haplotypes. Hypotheses compatible with these results are discussed.     

Deletion of a 236 kb region around S 4-RNase in a stylar-part mutant S 4 sm -haplotype of Japanese pear

Japanese pear (Pyrus pyrifolia Nakai) has a gametophytic self-incompatibility (GSI) mechanism controlled by a single S-locus with multiple S-haplotypes, each of which contains separate genes that determine the allelic identity of pistil and pollen. The pistil S gene is the S-ribonuclease (S-RNase) gene, whereas good candidates for the pollen S gene are the F-box protein genes. A self-compatible (SC) cultivar, ‘Osa-Nijisseiki’, which is a bud mutant of ‘Nijisseiki’ (S ₂ S ₄), has a stylar-part mutant -haplotype, which lacks the S ₄-RNase gene but retains the pollen S gene. To delineate the deletion breakpoint in the -haplotype, we constructed a bacterial artificial chromosome (BAC) library from an S ₄-homozygote, and assembled a BAC contig of 570 kb around the S ₄-RNase. Genomic PCR of DNA from S ₄- and -homozygotes and the DNA sequence of the BAC contig allowed the identification of a deletion of 236 kb spanning from 48 kb upstream to 188 kb downstream of S ₄-RNase. The -haplotype lacks 34 predicted open reading frames (ORFs) including the S ₄-RNase and a pollen-specific F-box protein gene (termed as S ₄ F-box0). Genomic PCR with a primer pair designed from the deletion junctions yielded a product specific for the -haplotype. The product could be useful as a maker for early selection of SC cultivars harboring the -haplotype. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterisation of novel S-alleles from cherry (Prunus avium L.)

In plant populations exhibiting gametophytic self-incompatibility, individuals harbouring rare S alleles are likely to have a reproductive advantage over individuals having more common alleles. Consequently, determination of the self-incompatibility haplotype of individuals is essential for genetic studies and the development of informed management strategies. This study characterises six new S alleles identified in wild cherry (Prunus avium L.). Investigations to determine the S genotype of individuals in recently planted woodland through length polymorphisms of introns associated with the stylar S-RNase gene and the pollen SFB gene revealed six S intron profiles which did not correspond to those of known S alleles. These are now attributed to S ₂₇ to S ₃₂ . Consensus primers, annealing in the S-RNase sequence coding for the signal peptide and C5 regions, were used to isolate the S-RNase alleles associated with the novel S intron profiles. The proteins corresponding to the new alleles were separated by isoelectric focusing from stylar extracts and their pI values determined. Similarities between the deduced amino acid sequence for the new alleles isolated and other cherry S-RNase sequences available on the databases ranged from 40% to 86%. Amplification products for SFB introns ranged from 172 to 208bp. New sequence regions exposed to positive selection were identified and the significance of the PS3 region reinforced. A phylogenetic relationship between P. avium S-RNases for S ₁₀ and S ₁₃ and between corresponding SFB alleles may indicate co-evolution of allele specificities of these two genes. The nucleotide sequences reported in this paper have been submitted to the EMBL/GenBank database under the following accession numbers: S ₂₇ (DQ266439), S ₂₈ (DQ266440), S ₂₉ (DQ266441), S ₃₀ (DQ266442), S ₃₁ (DQ266443), S ₃₂ (DQ266444).     

Genetic features of a pollen-part mutation suggest an inhibitory role for the <Emphasis Type="Italic">Antirrhinum</Emphasis> pollen self-incompatibility determinant

Self-incompatibility (SI), an important barrier to inbreeding in flowering plants, is controlled in many species by a single polymorphic S-locus. In the Solanaceae, two tightly linked S-locus genes, S-RNase and SLF (S-locus F-box)/SFB (S-haplotype-specific F-box), control SI expression in pistil and pollen, respectively. The pollen S-determinant appears to function to inhibit all but self S-RNase in the Solanaceae, but its genetic function in the closely-related Plantaginaceae remains equivocal. We have employed transposon mutagenesis in a member of the Plantaginaceae (Antirrhinum) to generate a pollen-part SI-breakdown mutant Pma1 (Pollen-part mutation in Antirrhinum1). Molecular genetic analyses showed that an extra telocentric chromosome containing AhSLF-S ₁ is present in its self-compatible but not in its SI progeny. Furthermore, analysis of the effects of selection revealed positive selection acting on both SLFs and SFBs, but with a stronger purifying selection on SLFs. Taken together, our results suggest an inhibitor role of the pollen S in the Plantaginaceae (as represented by Antirrhinum), similar to that found in the Solanaceae. The implication of these findings is discussed in the context of S-locus evolution in flowering plants. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Yongbiao Xue, Yijing Zhang, and Qiuying Yang contributed equally to this work.     

Molecular and Genetic Analyses of Four Nonfunctional S Haplotype Variants Derived from a Common Ancestral S Haplotype Identified in Sour Cherry (Prunus cerasus L.)

Tetraploid sour cherry (Prunus cerasus) has an S-RNase-based gametophytic self-incompatibility (GSI) system; however, individuals can be either self-incompatible (SI) or self-compatible (SC). Unlike the situation in the Solanaceae, where self-compatibility accompanying polyploidization is often due to the compatibility of heteroallelic pollen, the genotype-dependent loss of SI in sour cherry is due to the compatibility of pollen containing two nonfunctional S haplotypes. Sour cherry individuals with the S₄S₆S_36aS_36b genotype are predicted to be SC, as only pollen containing both nonfunctional S_36a and S_36b haplotypes would be SC. However, we previously found that individuals of this genotype were SI. Here we describe four nonfunctional S₃₆ variants. Our molecular analyses identified a mutation that would confer loss of stylar S function for one of the variants, and two alterations that might cause loss of pollen S function for all four variants. Genetic crosses showed that individuals possessing two nonfunctional S₃₆ haplotypes and two functional S haplotypes have reduced self-fertilization due to a very low frequency of transmission of the one pollen type that would be SC. Our finding that the underlying mechanism limiting successful transmission of genetically compatible gametes does not involve GSI is consistent with our previous genetic model for Prunus in which heteroallelic pollen is incompatible. This provides a unique case in which breakdown of SI does not occur despite the potential to generate SC pollen genotypes.GAMETOPHYTIC self-incompatibility (GSI) is a widespread mechanism in flowering plants that prevents self-fertilization and promotes out-crossing (De Nettancourt 2001). In GSI plants, pollen tube growth is arrested if there is a match between the genes at the S-locus that control pollen and stylar specificity. The gene controlling stylar specificity in the Solanaceae, Rosaceae, and Plantaginaceae is known to encode a ribonuclease (S-RNase) (for a review see McClure 2009), while the gene controlling pollen specificity encodes an F-box protein [S haplotype-specific F-box protein (SFB) or S-locus F-box protein (SLF)] (Lai et al. 2002; Entani et al. 2003; Ushijima et al. 2003; Sijacic et al. 2004). As these two specificity genes are tightly linked and recombination between these two genes has never been observed (Ikeda et al. 2005), these two S-locus specificity genes are collectively termed the S haplotype.Characterization of the S haplotype is most advanced in Prunus (Rosaceae) due to the small physical size of the S haplotype region and the close proximity of the stylar S (S-RNase) and pollen S (SFB) genes (Entani et al. 2003; Ushijima et al. 2003; Yamane et al. 2003b; Ikeda et al. 2005). Within Prunus, sweet cherry (Prunus avium) and sour cherry (P. cerasus) represent a model diploid–tetraploid series that has been used to investigate the effects of polyploidy on GSI. Tetraploid sour cherry is considered to have arisen through hybridization between sweet cherry and tetraploid ground cherry (P. fruticosa) (Olden and Nybom 1968). Like sweet cherry, sour cherry exhibits an S-RNase-based GSI system (Yamane et al. 2001; Hauck et al. 2002; Tobutt et al. 2004) and interspecific crossing studies have demonstrated that sour cherry shares eight sweet cherry S haplotypes: S₁, S₄, S₆, S₉, S₁₂, S₁₃, S₁₄, and S₁₆ (Bošković et al. 2006; Hauck et al. 2006a,b; Tsukamoto et al. 2006, 2008). However, in contrast to sweet cherry, natural sour cherry selections include both self-incompatible (SI) and self-compatible (SC) types. A genetic model demonstrating that the genotype-dependent loss of SI in sour cherry is due to the accumulation of a minimum of two nonfunctional S haploytpes within a single individual was developed and validated (Hauck et al. 2006b). These nonfunctional S haplotypes were characterized as either pollen-part mutants or stylar-part mutants, depending on whether the pollen S or stylar S specificity was disrupted. In Prunus, pollen-part and stylar-part mutants are denoted by a prime symbol “′” or a subscribed “m,” respectively, following the S haplotype number (Tsukamoto et al. 2006). Molecular characterizations of five of the nonfunctional S haplotypes from sour cherry characterized to date support the genetic results because mutations were identified that affected the S-RNase and/or SFB. These changes in coding or regulatory regions included mutations within the S-RNase and/or SFB causing premature stop codons, transposable element insertions within SFB and upstream of the S-RNase, and a 23-bp deletion in a conserved region of the S-RNase (Yamane et al. 2003a; Hauck et al. 2006a; Tsukamoto et al. 2006).According to the genetic model, termed the “one-allele-match model,” sour cherry pollen is rejected if one or both of the functional S haplotypes in the 2x pollen grain match an S haplotype in the style (Hauck et al. 2006b). Therefore, only pollen containing two nonfunctional S haplotypes would be SC; thus, a sour cherry genotype is SC if it has a minimum of two nonfunctional S haplotypes. We previously tested the one-allele-match model using 92 sour cherry selections from four progeny populations (Hauck et al. 2006b). For all the progeny except three, their S genotype correctly predicted whether they were SI or SC. The three progeny individuals that were the exception all had the same genotype: S₄S₆S_aS_d. These individuals were predicted to be SC as the S_a and S_d haplotypes were shown to be nonfunctional in genetic studies and therefore S_aS_d pollen should be SC. However, these progeny were classified as SI on the basis of observations of self-pollen tube growth in the styles. The S_a and S_d haplotypes were originally distinguished on the basis of different RFLP fragment sizes using an S-RNase probe; the HindIII fragment sizes for S_a and S_d differed by ∼200 bp, 6.4-kb and 6.2-kb, respectively (Yamane et al. 2001; Hauck et al. 2002). However, partial S-RNase and SFB sequences from the S_a and S_d haplotypes were identical (N. R. Hauck and A. F. Iezzoni, unpublished results), suggesting that S_a and S_d represented different mutations of the same S haplotype. Therefore, we hypothesized that the SI phenotype of the S₄S₆S_aS_d individuals resulted from complementary pistil S and pollen S mutations in the nonfunctional S_a and S_d haplotypes, thus behaving genetically as one functional S haplotype.We previously reported that heteroallelic sour cherry pollen containing two different functional pollen S haplotypes is incompatible (Hauck et al. 2006b). This finding is counter to the well-documented phenomenon in the Solanaceae where SC accompanying polyploidization is frequently due to the SC of heteroallelic pollen (Lewis 1943; Golz et al. 1999, 2001; Tsukamoto et al. 2005; Xue et al. 2009). Therefore, models explaining the molecular basis of self-recognition in Prunus and the Solanaceae must be consistent with these differing genetic expectations. Recently, Huang et al. (2008) reported competitive interaction in a SC selection of tetraploid P. pseudocerasus, raising the possibility that the SC mechanism between these two tetraploid Prunus species could be different. However, although the data in Huang et al. (2008) are consistent with heteroallelic pollen being SC, homoallelic pollen (e.g., S₁S₁, S₅S₅, or S₇S₇) was not shown to be successful in compatible crosses and unsuccessful in incompatible ones. Therefore, it is possible that the SC in P. pseudocerasus could be caused by mutations in other genes critical for the SI reaction. Because of the importance of these differing genetic expectations for understanding S-RNase-based GSI, we sought to investigate our previously identified exceptions to the one-allele-match model. Specifically, our objective was to test our prior hypothesis that the nonfunctional S_a and S_d haplotypes interact in a complementary manner and therefore behave together genetically as a single functional S haplotype. In this work, the S_a and S_d haplotypes were renamed S_36a and S_36b, respectively, following the order of previously published S haplotypes (Tsukamoto et al. 2008; Vaughan et al. 2008) for reasons explained in the results.     

Trans-specific <Emphasis Type="Italic">S</Emphasis>-RNase and SFB alleles in <Emphasis Type="Italic">Prunus</Emphasis> self-incompatibility haplotypes

Self-incompatibility in the genus Prunus is controlled by two genes at the S-locus, S-RNase and SFB. Both genes exhibit the high polymorphism and high sequence diversity characteristic of plant self-incompatibility systems. Deduced polypeptide sequences of three myrobalan and three domestic plum S-RNases showed over 97% identity with S-RNases from other Prunus species, including almond, sweet cherry, Japanese apricot and Japanese plum. The second intron, which is generally highly polymorphic between alleles was also remarkably well conserved within these S-allele pairs. Degenerate consensus primers were developed and used to amplify and sequence the co-adapted polymorphic SFB alleles. Sequence comparisons also indicated high degrees of polypeptide sequence identity between three myrobalan and the three domestic plum SFB alleles and the corresponding Prunus SFB alleles. We discuss these trans-specific allele identities in terms of S-allele function, evolution of new allele specificities and Prunus taxonomy and speciation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Two Different Prunus SFB Alleles Have the Same Function in the Self-incompatibility Reaction

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 ₂₄ -RNase and SFB ₂₄ alleles were cloned from Prunus armeniaca cv. Chuanzhihong (Chinese apricot). Sequence comparisons of deduced amino acid sequences revealed that the P. armeniaca S ₂₄ -haplotype has different SFB alleles, but shares a single S-RNase allele with P. armeniaca S ₄ -haplotype. Moreover, P. armeniaca S ₂₄ -RNase haplotype has a single and three different alleles with S ₁ -RNase of P. tenella (dwarf almond) and S ₁ -RNase of P. mira (smooth pit peach), respectively. The functionalities of SFB ₂₄ and SFB ₄ 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 ₂₄ -haplotype is identical to that of the S ₄ -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 ₂₄ - and S ₄ -haplotypes in Prunus.     

The use of the S haplotype-specific F-box protein gene,SFB, as a molecular marker for S-haplotypes and self-compatibility in Japanese apricot (Prunus mume)

Japanese apricot (Prunus mume) exhibits the S-RNase-based gametophytic self-incompatibility system as do other self-incompatible Prunus species. This report identifies the S haplotype-specific F-box protein gene (SFB), a candidate gene for pollen-S, of Japanese apricot, which leads to the development of a molecular typing system for S-haplotype in this fruit species. Both 5- and 3-RACE (rapid amplification of cDNA ends) were performed with SFB gene-specific oligonucleotide primers to clone Pm-SFB¹ and Pm-SFB⁷ of 'Nanko (S¹S⁷)'. As in the case of SFB of other Prunus species, Pm-SFB¹ and Pm-SFB⁷ showed a high level of S-haplotype-specific sequence polymorphism and their expression was specific to pollen. Genomic DNA-blot analyses of 11 Japanese apricot cultivars with the Pm-SFB probes under low stringency conditions yielded RFLP bands specific to the S¹- to S⁸-haplotypes as well as a self-compatible S^f-haplotype. A practical usage of SFB as a molecular marker for S-haplotypes and self-compatibility in Japanese apricot is discussed.Communicated by H.F. LinskensThe nucleotide sequences reported in this paper have been submitted to the EMBL/GenBank/DDBJ database under accession numbers, AB101440 and AB101441, for SFB¹ and SFB⁷, respectively     

A segmental duplication encompassing S-haplotype triggers pollen-part self-compatibility in Japanese pear (Pyrus pyrifolia)

Self-compatible mutants of self-incompatible crops have been extensively studied for research and agricultural purposes. Until now, the only known pollen-part self-compatible mutants in Rosaceae subtribe Pyrinae, which contains many important fruit trees, were polyploid. This study revealed that the pollen-part self-compatibility of breeding selection 415-1, a recently discovered mutant of Japanese pear (Pyrus pyrifolia) derived from γ-irradiated pollen, is caused by a duplication of an S-haplotype. In the progeny of 415-1, some plants had three S-haplotypes, two of which were from the pollen parent. Thus, 415-1 was able to produce pollen with two S-haplotypes, even though it was found to be diploid: the relative nuclear DNA content measured by flow cytometry showed no significant difference from that of a diploid cultivar. Inheritance patterns of simple sequence repeat (SSR) alleles in the same linkage group as the S-locus (LG 17) showed that some SSRs closely linked to S-haplotypes were duplicated in progeny containing the duplicated S-haplotype. These results indicate that the pollen-part self-compatibility of 415-1 is not caused by a mutation of pollen S factors in either one of the S-haplotypes, but by a segmental duplication encompassing the S-haplotype. Consequently, 415-1 can produce S-heteroallelic pollen grains that are capable of breaking down self-incompatibility (SI) by competitive interaction between the two different S factors in the pollen grain. 415-1 is the first diploid pollen-part self-compatible mutant with a duplicated S-haplotype to be discovered in the Pyrinae. The fact that 415-1 is not polyploid makes it particularly valuable for further studies of SI mechanisms.     

Self-compatibility and incompatibility in tetraploid sour cherry (Prunus cerasus L.)

. Gametophytic self-incompatibility (GSI) typically "breaks down" due to polyploidy in many Solanaceous species, resulting in self-compatible (SC) tetraploid individuals. However, sour cherry (Prunus cerasus L.), a tetraploid species resulting from hybridization of the diploid sweet cherry (P. avium L.) and the tetraploid ground cherry (P. fruticosa Pall.), is an exception, consisting of both self-incompatible (SI) and SC individuals. Since sweet cherry exhibits GSI with 13 S-ribonucleases (S-RNases) identified as the stylar S-locus product, the objectives were to compare sweet and sour cherry S-allele function, S-RNase sequences and linkage map location as initial steps towards understanding the genetic basis of SI and SC in sour cherry. S-RNases from two sour cherry cultivars that were the parents of a linkage mapping population were cloned and sequenced. The sequences of two S-RNases were identical to those of sweet cherry S-RNases, whereas three other S-RNases had unique sequences. One of the S-RNases mapped to the Prunus linkage group 6, similar to its location in sweet cherry and almond, whereas two other S-RNases were linked to each other but were unlinked to any other markers. Interspecific crosses between sweet and sour cherry demonstrated that GSI exists in sour cherry and that the recognition of common S-alleles has been maintained in spite of polyploidization. It is hypothesized that self-compatibility in sour cherry is caused by the existence of non-functional S-RNases and pollen S-genes that may have arisen from natural mutations.     

An S-Locus Independent Pollen Factor Confers Self-Compatibility in ‘Katy’ Apricot

Loss of pollen-S function in Prunus self-compatible cultivars has been mostly associated with deletions or insertions in the S-haplotype-specific F-box (SFB) genes. However, self-compatible pollen-part mutants defective for non-S-locus factors have also been found, for instance, in the apricot (Prunus armeniaca) cv. ‘Canino’. In the present study, we report the genetic and molecular analysis of another self-compatible apricot cv. termed ‘Katy’. S-genotype of ‘Katy’ was determined as S ₁ S ₂ and S-RNase PCR-typing of selfing and outcrossing populations from ‘Katy’ showed that pollen gametes bearing either the S ₁- or the S ₂-haplotype were able to overcome self-incompatibility (SI) barriers. Sequence analyses showed no SNP or indel affecting the SFB ₁ and SFB ₂ alleles from ‘Katy’ and, moreover, no evidence of pollen-S duplication was found. As a whole, the obtained results are compatible with the hypothesis that the loss-of-function of a S-locus unlinked factor gametophytically expressed in pollen (M’-locus) leads to SI breakdown in ‘Katy’. A mapping strategy based on segregation distortion loci mapped the M’-locus within an interval of 9.4 cM at the distal end of chr.3 corresponding to ∼1.29 Mb in the peach (Prunus persica) genome. Interestingly, pollen-part mutations (PPMs) causing self-compatibility (SC) in the apricot cvs. ‘Canino’ and ‘Katy’ are located within an overlapping region of ∼273 Kb in chr.3. No evidence is yet available to discern if they affect the same gene or not, but molecular markers seem to indicate that both cultivars are genetically unrelated suggesting that every PPM may have arisen independently. Further research will be necessary to reveal the precise nature of ‘Katy’ PPM, but fine-mapping already enables SC marker-assisted selection and paves the way for future positional cloning of the underlying gene.     

Self-compatible peach (<Emphasis Type="Italic">Prunus persica</Emphasis>) has mutant versions of the <Emphasis Type="Italic">S</Emphasis> haplotypes found in self-incompatible <Emphasis Type="Italic">Prunus</Emphasis> species

This study demonstrates that self-compatible (SC) peach has mutant versions of S haplotypes that are present in self-incompatible (SI) Prunus species. All three peach S haplotypes, S ¹ , S ² , and S ^2m , found in this study encode mutated pollen determinants, SFB, while only S ^2m has a mutation that affects the function of the pistil determinant S-RNase. A cysteine residue in the C5 domain of the S ^2m -RNase is substituted by a tyrosine residue, thereby reducing RNase stability. The peach SFB mutations are similar to the SFB mutations found in SC haplotypes of sweet cherry (P. avium) and Japanese apricot (P. mume). SFB ¹ of the S ¹ haplotype, a mutant version of almond (P. dulcis) S ^k haplotype, encodes truncated SFB due to a 155 bp insertion. SFB ² of the S ² and S ^2m haplotypes, both of which are mutant versions of the S ^a haplotype in Japanese plum (P. salicina), encodes a truncated SFB due to a 5 bp insertion. Thus, regardless of the functionality of the pistil determinant, all three peach S haplotypes are SC haplotypes. Our finding that peach has mutant versions of S haplotypes that function in almond and Japanese plum, which are phylogenetically close and remote species, respectively, to peach in the subfamily Prunoideae of the Roasaceae, provides insight into the SC/SI evolution in Prunus. We discuss the significance of SC pollen part mutation in peach with special reference to possible differences in the SI mechanisms between Prunus and Solanaceae.     

Convergent Evolution at the Gametophytic Self-Incompatibility System in Malus and Prunus

S-RNase-based gametophytic self-incompatibility (GSI) has evolved once before the split of the Asteridae and Rosidae. This conclusion is based on the phylogenetic history of the S-RNase that determines pistil specificity. In Rosaceae, molecular characterizations of Prunus species, and species from the tribe Pyreae (i.e., Malus, Pyrus, Sorbus) revealed different numbers of genes determining S-pollen specificity. In Prunus only one pistil and pollen gene determine GSI, while in Pyreae there is one pistil but multiple pollen genes, implying different specificity recognition mechanisms. It is thus conceivable that within Rosaceae the genes involved in GSI in the two lineages are not orthologous but possibly paralogous. To address this hypothesis we characterised the S-RNase lineage and S-pollen lineage genes present in the genomes of five Rosaceae species from three genera: M. × domestica (apple, self-incompatible (SI); tribe Pyreae), P. persica (peach, self-compatible (SC); Amygdaleae), P. mume (mei, SI; Amygdaleae), Fragaria vesca (strawberry, SC; Potentilleae), and F. nipponica (mori-ichigo, SI; Potentilleae). Phylogenetic analyses revealed that the Malus and Prunus S-RNase and S-pollen genes belong to distinct gene lineages, and that only Prunus S-RNase and SFB-lineage genes are present in Fragaria. Thus, S-RNase based GSI system of Malus evolved independently from the ancestral system of Rosaceae. Using expression patterns based on RNA-seq data, the ancestral S-RNase lineage gene is inferred to be expressed in pistils only, while the ancestral S-pollen lineage gene is inferred to be expressed in tissues other than pollen.     

Primary structural features of the<Emphasis Type="Italic"> S</Emphasis> haplotype-specific F-box protein,SFB, in<Emphasis Type="Italic"> Prunus</Emphasis>

The gene SFB encodes an F-box protein that has appropriate S-haplotype-specific variation to be the pollen determinant in the S-RNase-based gametophytic self-incompatibility (GSI) reaction in Prunus (Rosaceae). To further characterize Prunus SFB, we cloned and sequenced four additional alleles from sweet cherry (P. avium), SFB ¹ , SFB ² , SFB ⁴ , and SFB ⁵ . These four alleles showed haplotype-specific sequence diversity similar to the other nine SFB alleles that have been cloned. In an amino acid alignment of Prunus SFBs, including the four newly cloned alleles, 121 out of the 384 sites were conserved and an additional 65 sites had only conservative replacements. Amino acid identity among the SFBs ranged from 66.0% to 82.5%. Based on normed variability indices (NVI), 34 of the non-conserved sites were considered to be highly variable. Most of the variable sites were located at the C-terminal region. A window-averaged plot of NVI indicated that there were two variable and two hypervariable regions. These variable and hypervariable regions appeared to be hydrophilic or at least not strongly hydrophobic, which suggests that these regions may be exposed on the surface and function in the allele specificity of the GSI reaction. Evidence of positive selection was detected using maximum likelihood methods with sites under positive selection concentrated in the variable and hypervariable regions.K. Ikeda and B. Igic contributed equally to this paperNucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ nucleotide sequence databases under the accession numbers AB111518, AB111519, AB111520, and AB111521, for SFB ¹, SFB ², SFB ⁵, and SFB ⁴, respectively     

Genetic and molecular analysis in Cristobalina sweet cherry,a spontaneous self-compatible mutant

Self-compatibility in a naturally self-incompatible species like sweet cherry is a highly interesting trait for breeding purposes and a powerful tool with which to investigate the basis of the self-incompatible reaction in gametophytic systems. However, natural self-compatibility in sweet cherry is a very rare phenomenon. Cristobalina is a local Spanish sweet cherry cultivar that has proven to be spontaneously self-compatible. In this work, the nature of the self-compatibility in Cristobalina has been studied using genetic and molecular approaches. Pollination studies and microscopic observations of pollen tube growth were carried out to confirm the self-compatible character and the results obtained indicate that self-compatibility is caused by a failure of the pollen and not the style factor. Polymerase chain reaction (PCR) analysis of progenies derived from Cristobalina revealed that self-compatibility in this genotype is not related uniquely to one of the two pollen S alleles, but that pollen grains carrying either of the two haplotypes can overcome the incompatibility barrier. Moreover, PCR analysis and microscopic observation of pollen tube growth in progeny derived from Cristobalina also confirmed that the self-compatible descendants can carry either of the two S haplotypes of their progenitor. Isolation and sequencing of the style S-RNases and pollen SFBs revealed that the DNA sequences of these factors are the same as those described in other self-incompatible sweet cherry cultivars with the same S alleles. Possible mechanisms to explain self-compatibility in Cristobalina are discussed.