Characterization of self-compatibility in sweet cherry varieties by crossing experiments and molecular genetic analysis

Self-compatibility is a major breeding objective in sweet cherry. The identification and characterization of new sources of self-compatibility will be useful for breeding and research purposes. In this work, self-compatibility of four local Spanish sweet cherry varieties was investigated by crossing experiments and molecular genetic analysis of two self-incompatibility loci. Crossing experiments included self- and cross-pollinations in the laboratory followed by microscopic observation of pollen tube growth and fruit set assay in the field. After crossing experiments, two accessions, ‘Son Miró’ and ‘Talegal Ahín’, were self-compatible while the other two were self-incompatible. Inheritance of S-locus and microsatellite EMPaS02 (linked to self-compatibility, Sc) were investigated in self-pollination progeny of both self-compatible genotypes. Results indicate that self-compatibility in ‘Talegal Ahín’ is similar to self-compatibility described in sweet cherry ‘Cristobalina’ and may be caused by the same mutation. That is a pollen part mutation not linked to the S-locus but linked to microsatellite EMPaS02 in cherry LG3. In ‘Son Miró’ self-compatibility seems more complex, affecting pollen and style function, and probably involving more than one mutation not described previously in sweet cherry. Together with ‘Cristobalina’, the newly described self-compatible varieties ‘Son Miró’ and ‘Talegal Ahín’ confirm the existence of unique self-compatible plant material in local germplasm from Spain that should be conserved and characterized for its use in breeding and research.     

核果类果树自交不亲和性研究进展

综述了核果类果树甜樱桃(Prunus avium L.)、杏(P. armeniaca L.)、扁桃(P. dulcis (Mill.) D. A.Webb)和梅(P. mume Sieb)等自交不亲和性的研究进展。着重讨论了S-RNase基因(S基因)和SLF基因(S-locus　F-box基因,或称SFB基因),S基因在杂交后代群体中的遗传规律,利用S基因的遗传特性选育自交亲和品种和确定S基因型的主要方法及其特点以及自交亲和机制的几种可能的类型。     

A new self-compatibility haplotype in the sweet cherry 'Kronio', S5', attributable to a pollen-part mutation in the SFB gene

'Kronio' is a Sicilian cultivar of sweet cherry (Prunus avium), nominally with the incompatibility genotype S(5)S(6), that is reported to be naturally self-compatible. In this work the cause of its self-compatibility was investigated. Test selfing confirmed self-compatibility and provided embryos for analysis; PCR with consensus primers designed to amplify S-RNase and SFB alleles showed that the embryos were of two types, S(5)S(5) and S(5)S(6), indicating that S(6) pollen failed, but S(5) succeeded, perhaps because of a mutation in the pollen or stylar component. Stylar RNase analysis indicated active S-RNases for both S(5) and S(6). The S-RNase alleles were cloned and sequenced; and sequences encode functional proteins. Cloning and sequencing of SFB alleles showed that S(6) was normal but S(5) had a premature stop codon upstream of the variable region HVa resulting in a truncated protein. Therefore, the self-compatibility can be attributed to a pollen-part mutation of S(5), designated S(5)', the first reported case of breakdown of self-incompatibility in diploid sweet cherry caused by a natural mutation at the S-locus. The second intron of the S-RNase associated with S(5)' contained a microsatellite smaller than that associated with S(5); primers designed to amplify across this microsatellite effectively distinguished S(5) from S(5)'. Analysis of some other Sicilian cherries with these primers indicated that S(5)' is also present in the Sicilian cultivar 'Maiolina a Rappu', and this proved to be self-compatible.     

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.     

Loss of gametophytic self-incompatibility with evolution of inbreeding depression

Gametophytic self-incompatibility (SI) in plants is a widespread mechanism preventing self-fertilization and the ensuing inbreeding depression, but it often evolves to self-compatibility. We analyze genetic mechanisms for the breakdown of gametophytic SI, incorporating a dynamic model for the evolution of inbreeding depression allowing for partial purging of nearly recessive lethal mutations by selfing, and accounting for pollen limitation and sheltered load linked to the S-locus. We consider two mechanisms for the breakdown of gametophytic SI: a nonfunctional S-allele and an unlinked modifier locus that inactivates the S-locus. We show that, under a wide range of conditions, self-compatible alleles can invade a self-incompatible population. Conditions for invasion are always less stringent for a nonfunctional S-allele than for a modifier locus. The spread of self-compatible genotypes is favored by extremely high or low selfing rates, a small number of S-alleles, and pollen limitation. Observed parameter values suggest that the maintenance of gametophytic SI is caused by a combination of high inbreeding depression in self-incompatible populations coupled with intermediate selfing rates of the self-compatible genotypes and sheltered load linked to the S-locus.     

Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea.

In cruciferous plants, self-pollination is prevented by the action of genes situated at the self-incompatibility locus or S-locus. The self-incompatibility reaction is associated with expression of stigma glycoproteins encoded by the S-locus glycoprotein (SLG) gene. Only a few cases of self-compatible plants derived from self-incompatible lines in the crucifer Brassica have been reported. In these cases, self-compatibility was generally ascribed to the action of single genes unlinked to the S-locus. In contrast, we report here a line of Brassica oleracea var acephala with a self-compatible phenotype linked to the S-locus. By means of both biochemical and immunochemical analyses, we showed that this self-compatible (Sc) line nonetheless possesses stigmatic SLGs (SLG-Sc) that are expressed with a similar spatial and temporal pattern to that described for the SLGs of self-incompatible Brassica plants. Moreover, the SLG-Sc products segregate with the self-compatibility phenotype in F2 progeny, suggesting that changes at the S-locus may be responsible for the occurrence of the self-compatibility character. A cDNA clone encoding the SLG-Sc product was isolated, and the deduced amino acid sequence showed this glycoprotein to be highly homologous to the pollen recessive S2 allele glycoprotein. Hence, self-compatibility in this Brassica Sc line correlates with the expression of a pollen recessive-like S allele in the stigma.     

Identification of quantitative trait loci associated with self-compatibility in a Prunus species

Self-compatibility in Rosaceous fruit species is based on a single-locus qualitative trait. However, the evidence observed in different species has indicated the presence of modifier genes outside the S locus affecting the expression of self-compatibility/self-incompatibility. The study of a progeny obtained from the cross of the almond genotypes ‘Vivot’× ‘Blanquerna’ has allowed the construction of a genetic map based on microsatellite markers and the identification for the first time in the Rosaceae family of two additional loci located outside the S locus and affecting the expression of self-compatibility/self-incompatibility. A quantitative trait locus (QTL) was located relatively close to the S locus, on linkage group 6 (G6), whereas the second one was located on G8. These QTLs appear to be involved in conferring self-compatibility to genotypes not possessing the S _f allele. These results are consistent with almond being a self-incompatible species with a genetic background of pseudo-self-compatibility controlled by modifier genes. The effect of the S _f allele and the two QTLs may contribute to explain the wide range of fruit sets observed when self-pollinating different almond genotypes.     

核果类果树自交不亲和性研究进展

综述了核果类果树甜樱桃(PFunus avium L.)、杏(P.armeniaca L.)、扁桃(P.dulcis(Mill.)D.A.Webb)和梅(P.mume Sieb)等自交不亲和性的研究进展.着重讨论了S-RNase基因(s基因)和SLF基因(S-locus F-box基因,或称SFB基因),S基因在杂交后代群体中的遗传规律,利用S基因的遗传特性选育自交亲和品种和确定S基因型的主要方法及其特点以及自交亲和机制的几种可能的类型.     

Linkage disequilibrium in French wild cherry germplasm and worldwide sweet cherry germplasm

A basic knowledge on linkage disequilibrium (LD) is necessary in order to determine resolution of association studies. We investigated the extent and patterns of LD in a self-incompatible species (Prunus avium L.), in 3 groups (wild cherry, sweet cherry landraces and sweet cherry modern varieties), using a set of 35 microsatellite markers and the gametophytic self-incompatibility locus. Since population structure might create spurious LD, we thus used the information provided by a structure analysis published in a previous study to perform the LD analysis. In the current study, we detected a greater LD extent in sweet cherry than in wild cherry, which is plausibly due to the bottleneck associated with domestication and breeding. Higher LD values in sweet cherry sub-groups may be explained by smaller sample sizes. We also showed that the remaining structure in the groups of sweet cherry, in particular landraces, is responsible for a part of the LD extent. Intra-group relatedness may also account for extensive LD in two sub-groups. These results demonstrate, if ever necessary, the importance of controlling the genetic structure and relatedness when estimating LD. Moreover, LD decays very rapidly with genetic linkage distance in both wild and sweet cherries, which seems promising for future association studies.     

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.     

Characterization of microsatellites in wild and sweet cherry (Prunus avium L.)--markers for individual identification and reproductive processes.

Nuclear microsatellites were characterized in Prunus avium and validated as markers for individual and cultivar identification, as well as for studies of pollen- and seed-mediated gene flow. We used 20 primer pairs from a simple sequence repeat (SSR) library of Prunus persica and identified 7 loci harboring polymorphic microsatellite sequences in P. avium. In a natural population of 75 wild cherry trees, the number of alleles per locus ranged from 4 to 9 and expected heterozygosity from 0.39 to 0.77. The variability of the SSR markers allowed an unambiguous identification of individual trees and potential root suckers. Additionally, we analyzed 13 sweet cherry cultivars and differentiated 12 of them. An exclusion probability of 0.984 was calculated, which indicates that the seven loci are suitable markers for paternity analysis. The woody endocarp was successfully used for resolution of all microsatellite loci and exhibited the same multilocus genotype as the mother tree, as shown in a single seed progeny. Hence, SSR fingerprinting of the purely maternal endocarp was also successful in this Prunus species, allowing the identification of the mother tree of the dispersed seeds. The linkage of microsatellite loci with PCR-amplified alleles of the self-incompatibility locus was tested in two full-sib families of sweet cherry cultivars. From low recombination frequencies, we inferred that two loci are linked with the S locus. The present study provides markers that will significantly facilitate studies of spatial genetic variation and gene flow in wild cherry, as well as breeding programs in sweet cherry.     

Interhaplotypic heterogeneity and heterochromatic features may contribute to recombination suppression at the S locus in apple (Malusxdomestica)

Gametophytic self-incompatibility (GSI) is controlled by a complex S locus containing the pistil determinant S-RNase and pollen determinant SFB/SLF. Tight linkage of the pistil and pollen determinants is necessary to guarantee the self-incompatibility (SI) function. However, multiple probable pollen determinants of apple and Japanese pear, SFBBs (S locus F-box brothers), exist in each S haplotype, and how these multiple genes maintain the SI function remains unclear. It is shown here by high-resolution fluorescence in situ hybridization (FISH) that SFBB genes of the apple S ( 9 ) haplotype are physically linked to the S ( 9 ) -RNase gene, and the S locus is located in the subtelomeric region. FISH analyses also determined the relative order of SFBB genes and S-RNase in the S ( 9 ) haplotype, and showed that gene order differs between the S ( 9 ) and S ( 3 ) haplotypes. Furthermore, it is shown that the apple S locus is located in a knob-like large heterochromatin block where DNA is highly methylated. It is proposed that interhaplotypic heterogeneity and the heterochromatic nature of the S locus help to suppress recombination at the S locus in apple.     

Segregation distortion of T-DNA markers linked to the self-incompatibility (S) locus in Petunia hybrida

In plants with a gametophytic self-incompatibility system the specificity of the pollen is determined by the haploid genotype at the self-incompatibility (S) locus. In certain crosses this can lead to the exclusion of half the gametes from the male parent carrying a particular S-allele. This leads to pronounced segregation distortion for any genetic markers that are linked to the S-locus. We have used this approach to identify T-DNA insertions carrying a maize transposable element that are linked to the S-locus of Petunia hybrida. A total of 83 T-DNA insertions were tested for segregation distortion of the selectable marker used during transformation with Agrobacterium. Segregation distortion was observed for 12 T-DNA insertions and at least 8 of these were shown to be in the same linkage group by intercrossing. This indicates that differential transmission of a single locus (S) is probably responsible for all of these examples of T-DNA segregation distortion. The identification of selectable markers in coupling with a functional S-allele will allow the preselection of recombination events around the S-locus in petunia. Our approach provides a general method for identifying transgenes that are linked to gametophytic self-incompatibility loci and provides an opportunity for transposon tagging of the petunia S-locus.     

Molecular mechanisms of self-incompatibility in Brassica

In Brassica species, self-incompatibility has been mapped genetically to a single chromosomal location. In this region several closely linked genes have been identified. One of them, S-locus receptor kinase (SRK), determines S haplotype specificity of the stigma and it's the key protein for SI reaction. The role of the S locus glycoprotein (SLG) gene remains unclear. In the last decade approximately 15 additional genes linked to S-locus have been found. Recently, a gene has been identified (SCR) that encodes a small cysteine-rich protein which is a candidate for the pollen ligand. In addition to S locus linked genes there are unlinked SLRgenes (S-locus related genes). In this review, we discuss the role of these genes and the current view on the self-incompatibility mechanism in Brassica.     

Molecular characterization of the S locus in two self-incompatible Brassica napus lines.

In Brassica species, self-incompatibility has been mapped genetically to a single chromosomal location. In this region, there are two closely linked genes coding for the S locus glycoprotein (SLG) and S locus receptor kinase (SRK). They appear to comprise the pistil component of the self-incompatibility reaction. SLG and SRK are thought to recognize an unknown pollen component on the incompatible pollen, and the gene encoding this pollen component must also be linked to the SLG and SRK genes. To further our understanding of self-incompatibility, the chromosomal region carrying the SLG and SRK genes has been studied. The physical region between the SLG-910 and the SRK-910 genes in the Brassica napus W1 line was cloned, and a search for genes expressed in the anther revealed two additional S locus genes located downstream of the SLG-910 gene. Because these two genes are novel and are conserved at other S alleles, we designated them as SLL1 and SLL2 (for S locus-linked genes 1 and 2, respectively). The SLL1 gene is S locus specific, whereas the SLL2 gene is not only present at the S locus but is also present in other parts of the genomes in both self-incompatible and self-compatible Brassica ssp lines. Expression of the SLL1 gene is only detectable in anthers of self-incompatible plants and is developmentally regulated during anther development, whereas the SLL2 gene is expressed in anthers and stigmas in both self-incompatible and self-compatible plants, with the highest levels of expression occurring in the stigmas. Although SLL1 and SLL2 are linked to the S locus region, it is not clear whether these genes function in self-incompatibility or serve some other cellular roles in pollen-pistil functions.     

THE EVOLUTION OF DOMINANCE IN SPOROPHYTIC SELF-INCOMPATIBILITY SYSTEMS. II. MATE AVAILABILITY AND RECOMBINATION

Sporophytic self-incompatibility (SSI) is a self-pollen recognition system that enforces outcrossing in plants. Recognition in SSI systems is typically controlled by a complex locus ( S -locus) with separate genes that determine pollen and stigma specificity. Experimental studies show that S -alleles can be dominant, recessive, or codominant, and that the dominance level of a given S -allele can depend upon whether pollen or stigma specificity is examined. Here and in the companion paper by Llaurens and colleagues, the evolution of dominance in single-locus SSI is explored using numerical models and simulation. Particular attention is directed at factors that can cause S -allele dominance to differ in pollen versus stigma. The effect of recombination between the S -locus and modifier locus is also examined. The models predict that limitation in the number of compatible mates is required for the evolution of S -allele dominance in the stigma but not in the pollen. Tight linkage between the S -locus and modifier promotes the evolution of S -allele dominance hierarchies. Model results are interpreted with respect to published information on the molecular basis of dominance in SSI systems, and reported S -allele dominance relationships in a variety of species. These studies show that dominant S -alleles are more common in the pollen than in the stigma, a pattern that when interpreted in light of model predictions, suggests that mate limitation may be relatively infrequent in natural populations with SSI.     

Inheritance and dominance of self-incompatibility alleles in polyploid Arabidopsis lyrata

Natural populations of diploid Arabidopsis lyrata exhibit the sporophytic type of self-incompatibility system characteristic of Brassicaceae, in which complicated dominance interactions among alleles in the diploid parent determine self-recognition phenotypes of both pollen and stigma. The purpose of this study was to investigate how polyploidy affects this already complex system. One tetraploid population (Arabidopsis lyrata ssp kawasakiana from Japan) showed complete self-compatibility and produced viable selfed progeny for at least three generations subsequent to field collection. In contrast, individuals from a second tetraploid population (A. lyrata ssp petraea from Austria) were strongly self-incompatible (SI). Segregation of SI genotypes in this population followed Mendelian patterns based on a tetrasomic model of inheritance, with two to four alleles per individual, independent segregation of alleles, and little evidence of dosage effects of alleles found in multiple copies. Similar to results from diploids, anomalous compatibility patterns involving particular combinations of individuals occurred at a low frequency in the tetraploids, suggesting altered dominance in certain genetic backgrounds that could be due to the influence of a modifier locus. Overall, dominance relationships among S-alleles in self-incompatible tetraploid families were remarkably similar to those in related diploids, suggesting that this very important and complicated locus has not undergone extensive modification subsequent to polyploidization.