Loss of pollen-S function in two self-compatible selections of Prunus avium is associated with deletion/mutation of an S haplotype-specific F-box gene

Recently, an S haplotype-specific F-box (SFB) gene has been proposed as a candidate for the pollen-S specificity gene of RNase-mediated gametophytic self-incompatibility in Prunus (Rosaceae). We have examined two pollen-part mutant haplotypes of sweet cherry (Prunus avium). Both were found to retain the S-RNase, which determines stylar specificity, but one (S3' in JI 2434) has a deletion including the haplotype-specific SFB gene, and the other (S4' in JI 2420) has a frame-shift mutation of the haplotype-specific SFB gene, causing amino acid substitutions and premature termination of the protein. The loss or significant alteration of this highly polymorphic gene and the concomitant loss of pollen self-incompatibility function provides compelling evidence that the SFB gene encodes the pollen specificity component of self-incompatibility in Prunus. These loss-of-function mutations are inconsistent with SFB being the inactivator of non-self S-RNases and indicate the presence of a general inactivation mechanism, with SFB conferring specificity by protecting self S-RNases from inactivation.     

The mutated S1-haplotype in sour cherry has an altered S-haplotype-specific F-box protein gene

Gametophytic self-incompatibility (GSI) is an outcrossing mechanism in flowering plants that is genetically controlled by 2 separate genes located at the highly polymorphic S-locus, termed S-haplotype. This study characterizes a pollen part mutant of the S(1)-haplotype present in sour cherry (Rosaceae, Prunus cerasus L.) that contributes to the loss of GSI. Inheritance of S-haplotypes from reciprocal interspecific crosses between the self-compatible sour cherry cultivar Ujfehértói Fürt?s carrying the mutated S(1)-haplotype (S(1)'S(4)S(d)S(null)) and the self-incompatible sweet cherry (Prunus avium L.) cultivars carrying the wild-type S(1)-haplotype revealed that the mutated S(1)-haplotype confers unilateral incompatibility with a functional pistil component and a nonfunctional pollen component. The altered sour cherry S(1)-haplotype pollen part mutant, termed S(1)', contains a 615-bp Ds-like element within the S(1)-haplotype-specific F-box protein gene (SFB(1)'). This insertion generates a premature in-frame stop codon that would result in a putative truncated SFB(1) containing only 75 of the 375 amino acids present in the wild-type SFB(1). S(1)' along with 2 other previously characterized Prunus S-haplotype mutants, S(f) and S(6m), illustrate that mobile element insertion is an evolutionary force contributing to the breakdown of GSI.     

Characterization and mapping of non-S gametophytic self-compatibility in sweet cherry (Prunus avium L.)

Self-incompatibility in Prunus (Rosaceae) species, such as sweet cherry, is controlled by a multiallelic locus (S), in which two tightly linked genes, S-RNase and SFB (S haplotype-specific F-box), determine the specificity of the pollen and the style. Fertilization in these species occurs only if the S-specificities expressed in the pollen and the pistils are different. However, modifier genes have been proposed to be necessary for a full manifestation of the self-incompatibility response. 'Cristobalina' is a spontaneous self-compatible sweet cherry cultivar that originated in Eastern Spain. Previous studies with this genotype suggested that pollen modifier gene(s), not linked to the S-locus, may be the cause of self-incompatibility breakdown. In this work, an F(1) population from 'Cristobalina' that segregates for this trait was used to identify molecular markers linked to self-compatibility by bulked segregant analysis. One simple sequence repeat (SSR) locus (EMPaS02) was found to be linked to self-compatibility in this population at 3.2?cM. Two additional populations derived from 'Cristobalina' were used to confirm the linkage of this marker to self-compatibility. Since EMPaS02 has been mapped to the sweet cherry linkage group 3, other markers located on the same linkage group were analysed in these populations to confirm the location of the self-compatibility locus.     

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.     

Development and Evaluation of a Genome-Wide 6K SNP Array for Diploid Sweet Cherry and Tetraploid Sour Cherry

High-throughput genome scans are important tools for genetic studies and breeding applications. Here, a 6K SNP array for use with the Illumina Infinium® system was developed for diploid sweet cherry (Prunus avium) and allotetraploid sour cherry (P. cerasus). This effort was led by RosBREED, a community initiative to enable marker-assisted breeding for rosaceous crops. Next-generation sequencing in diverse breeding germplasm provided 25 billion basepairs (Gb) of cherry DNA sequence from which were identified genome-wide SNPs for sweet cherry and for the two sour cherry subgenomes derived from sweet cherry (avium subgenome) and P. fruticosa (fruticosa subgenome). Anchoring to the peach genome sequence, recently released by the International Peach Genome Initiative, predicted relative physical locations of the 1.9 million putative SNPs detected, preliminarily filtered to 368,943 SNPs. Further filtering was guided by results of a 144-SNP subset examined with the Illumina GoldenGate® assay on 160 accessions. A 6K Infinium® II array was designed with SNPs evenly spaced genetically across the sweet and sour cherry genomes. SNPs were developed for each sour cherry subgenome by using minor allele frequency in the sour cherry detection panel to enrich for subgenome-specific SNPs followed by targeting to either subgenome according to alleles observed in sweet cherry. The array was evaluated using panels of sweet (n = 269) and sour (n = 330) cherry breeding germplasm. Approximately one third of array SNPs were informative for each crop. A total of 1825 polymorphic SNPs were verified in sweet cherry, 13% of these originally developed for sour cherry. Allele dosage was resolved for 2058 polymorphic SNPs in sour cherry, one third of these being originally developed for sweet cherry. This publicly available genomics resource represents a significant advance in cherry genome-scanning capability that will accelerate marker-locus-trait association discovery, genome structure investigation, and genetic diversity assessment in this diploid-tetraploid crop group.     

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。     

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.     

Recognition of S-RNases by an S locus F-box like protein and an S haplotype-specific F-box like protein in the Prunus-specific self-incompatibility system

Key message

S-RNase was demonstrated to be predominantly recognized by an S locus F-box-like protein and an S haplotype-specific F-box-like protein in compatible pollen tubes of sweet cherry.

Abstract

Self-incompatibility (SI) is a reproductive barrier that rejects self-pollen and inhibits self-fertilization to promote outcrossing. In Solanaceae and Rosaceae, S-RNase-based gametophytic SI (GSI) comprises S-RNase and F-box protein(s) as the pistil and pollen S determinants, respectively. Compatible pollen tubes are assumed to detoxify the internalized cytotoxic S-RNases to maintain growth. S-RNase detoxification is conducted by the Skp1-cullin1-F-box protein complex (SCF) formed by pollen S determinants, S locus F-box proteins (SLFs), in Solanaceae. In Prunus, the general inhibitor (GI), but not pollen S determinant S haplotype-specific F-box protein (SFB), is hypothesized to detoxify S-RNases. Recently, SLF-like proteins 1–3 (SLFL1–3) were suggested as GI candidates, although it is still possible that other proteins function predominantly in GI. To identify the other GI candidates, we isolated four other pollen-expressed SLFL and SFB-like (SFBL) proteins PavSLFL6, PavSLFL7A, PavSFBL1, and PavSFBL2 in sweet cherry. Binding assays with four PavS-RNases indicated that PavSFBL2 bound to PavS^{1, 6}-RNase while the others bound to nothing. PavSFBL2 was confirmed to form an SCF complex in vitro. A co-immunoprecipitation assay using the recombinant PavS⁶-RNase as bait against pollen extracts and a mass spectrometry analysis identified the SCF complex components of PavSLFLs and PavSFBL2, M-locus-encoded glutathione S-transferase (MGST), DnaJ-like protein, and other minor proteins. These results suggest that SLFLs and SFBLs could act as predominant GIs in Prunus-specific S-RNase-based GSI.

     

Characterization of SLFL1, a pollen-expressed F-box gene located in the Prunus S locus

The S locus and its flanking regions in the genus Prunus (Rosaceae) contain four pollen-expressed F-box genes. These genes contain the S locus F-box genes with low allelic sequence polymorphism genes 1, 2, and 3 (SLFL1, SLFL2, and SLFL3) as well as the putative pollen S gene, named the S haplotype-specific F-box protein gene (SFB). As much less information is available on the function of SLFLs than that of SFB, we analyzed the SLFLs of six S haplotypes of sweet cherry (Prunus avium) in this study. Genomic DNA blot analysis and the isolation of SLFL1 showed that the SLFL1 gene in a functional self-incompatible S ³ haplotype is deleted and only a partial sequence resembling SLFL1 is left in the S ³ locus region, suggesting that SLFL1 by itself is not directly involved in either the GSI reaction or pollen-tube growth. Genomic DNA blot analysis showed that there was no substantial modification or mutation in SLFL2 and SLFL3. A phylogenic analysis of F-box genes in the rosaceous S locus and its border regions showed that Prunus SLFLs were more closely related to maloid S locus F-box brothers than to Prunus SFBs. The functions of SLFLs and the evolution of self-incompatibility in Prunus are discussed based on these results. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequence data reported appear in the DDBJ, EMBL, and GenBank Nucleotide Sequence Databases under the accession numbers, AB360339, AB360340, AB360341, and AB360342, for SLFL1-S ¹ , SLFL1-S ² , SLFL1-S ⁵ , and SLFL1-S ⁶ , respectively.     

Identification of a Skp1-like protein interacting with SFB, the pollen S determinant of the gametophytic self-incompatibility in Prunus

Many species in Rosaceae, Solanaceae, and Plantaginaceae exhibit S-RNase-based self-incompatibility (SI). In this system, the pistil and pollen specificities are determined by S-RNase and the S locus F-box protein, respectively. The pollen S determinant F-box protein in Prunus (Rosaceae) is referred to by two different terms, SFB (for S-haplotype-specific F-box protein) and SLF (for S locus F box), whereas it is called SLF in Solanaceae and Plantaginaceae. Prunus SFB is thought to be a molecule indispensable for its cognate S-RNase to exert cytotoxicity and to arrest pollen tube growth in incompatible reactions. Although recent studies have demonstrated the molecular function of SCF(SLF) in the SI reaction of Solanaceae and Plantaginaceae, how SFB participates in the Prunus SI mechanism remains to be elucidated. Here we report the identification of sweet cherry (Prunus avium) SFB (PavSFB)-interacting Skp1-like1 (PavSSK1) using a yeast (Saccharomyces cerevisiae) two-hybrid screening against the pollen cDNA library. Phylogenetic analysis showed that PavSSK1 belongs to the same clade as Antirrhinum hispanicum SLF-interacting Skp1-like1 and Petunia hybrida SLF-interacting Skp1-like1 (PhSSK1). In yeast, PavSSK1 interacted not only with PavSFBs from different S haplotypes and Cullin1-likes (PavCul1s), but also with S-locus F-box-likes. A pull-down assay confirmed the interactions between PavSSK1 and PavSFB and between PavSSK1 and PavCul1s. These results collectively indicate that PavSSK1 could be a functional component of the SCF complex and that PavSFB may function as a component of the SCF complex. We discuss the molecular function of PavSFB in self-/nonself-recognition in the gametophytic SI of Prunus.     

QTL analysis and candidate gene mapping for skin and flesh color in sweet cherry fruit (<Emphasis Type="Italic">Prunus avium</Emphasis> L.)

Sweet cherry (Prunus avium L.) skin and fruit colors vary widely due to differences in red and yellow pigment profiles. The two major market classes of sweet cherry represent the two color extremes, i.e., yellow skin with red blush and yellow flesh and dark mahogany skin with mahogany flesh. Yet, within these extremes, there is a continuum of skin and flesh color types. The genetic control of skin and flesh color in sweet cherry was investigated using a quantitative trait locus (QTL) approach with progeny derived from a cross between cherry parents representing the two color extremes. Skin and flesh colors were measured using a qualitative color-card rating over three consecutive years and also evaluated quantitatively for darkness/lightness (L*), red/green (a*), and yellow/blue (b*). Segregations for the color measurements (card, L*, a*, and b*) did not fit normal distributions; instead, the distributions were skewed towards the color of the dark-fruited parent. A major QTL for skin and flesh color was identified on linkage group (LG) 3. Two QTLs for skin and flesh color were also identified on LG 6 and LG 8, respectively, indicating segregation for minor genes. The significance and magnitude of the QTL identified on LG 3 suggests the presence of a major regulatory gene within this QTL interval. A candidate gene PavMYB10, homologous to apple MdMYB10 and Arabidopsis AtPAP1, is within the interval of the major QTL on LG 3, suggesting that PavMYB10 could be the major determinant of fruit skin and flesh coloration in sweet cherry.     

A cherry protein and its gene, abundantly expressed in ripening fruit, have been identified as thaumatin-like.

A 29-kD polypeptide is the most abundant soluble protein in ripe cherry fruit (Prunus avium L); accumulation begins at the onset of ripening as the fruit turns from yellow to red. This protein was extracted from ripe cherries and purified by size-exclusion and ion-exchange chromatography. Antibodies to the purified protein were used to screen a cDNA library from ripe cherries. Numerous recombinant plaques reacted positively with the antibodies; the DNA sequence of representative clones encoded a polypeptide of 245 amino acid residues. A signal peptide was indicated, and the predicted mature protein corresponded to the purified protein in size (23.3 kD, by mass spectrometry) and isoelectric point (4.2). A search of known protein sequences revealed a strong similarity between this polypeptide and the thaumatin family of pathogenesis-related proteins. The cherry thaumatin-like protein does not have a sweet taste, and no antifungal activity was seen in preliminary assays. Expression of the protein appears to be regulated at the gene level, with mRNA levels at their highest in the ripe fruit.     

Expression of a sweet cherry DREB1/CBF ortholog in Arabidopsis confers salt and freezing tolerance

Dehydration responsive element binding protein 1 (DREB1)/C-repeat binding factor (CBF) induces the expression of many stress-inducible genes in Arabidopsis. We have previously reported the identification of three DREB1/ICBF homologs from sweet cherry (Prunus avium). To identify the function of these homologs, one of the genes, CIG-B, was transformed into Arabidopsis. In one of the transgenic plant lines, the DREB1/CBF target gene cor15a was induced in the absence of stress treatment. The cor15a-overexpressing transgenic plant exhibited mild growth retardation and had greater salt and freezing tolerance than did the wild-type and the transgenic lines in which cor15a was not induced. These results suggest that this sweet cherry DREB1/CBF homolog has a function similar to that of DREB1/CBF.     

GmCOI1, a soybean F-box protein gene, shows ability to mediate jasmonate-regulated plant defense and fertility in Arabidopsis

The F-box protein gene COI1 from Arabidopsis plays a fundamental role in response to jasmonates, which regulate plant root growth, pollen fertility, wounding and healing, and defense against pathogens and insects. Null mutations in COI1 were previously found to abolish all the jasmonate responses, and the Arabidopsis coil-1 mutant is male sterile and susceptible to pathogen infection. In this study, we isolated an F-box protein gene from soybean, which shares significant homology with the Arabidopsis COI1 and similarly contains an F-box motif and leucine rich repeats (LRR), here designated GmCOI1 (Glycine max L. (Merr.) COI1). To test whether the sequence homology and structural similarity are indicative of functional conservation, we expressed GmCOI1 in the Arabidopsis coil-1 mutant. The transgenic coil-1 plants with expression of the GmCOI1 gene were found to exhibit normal jasmonate responses, including jasmonate-regulated plant defense and fertility. In addition, the chimerical proteins with swapped domain of the F-box motif or LRR between GmCOI1 and COI1 were shown to functionally complement the coil-1 mutation. Furthermore, GmCOI1 was found to assemble into the Skpl-Cullin-F-box (SCF) complexes, similar to the formation of the Arabidopsis SCF(COO1). These data demonstrate the soybean F-box protein gene GmCOI1 is able to mediate jasmonate-regulated plant defense and fertility in Arabidopsis, which implies a generic jasmonate pathway with conserved signal components in different plant species.     

PCR markers for mutated <Emphasis Type="Italic">S</Emphasis>-haplotypes enable discrimination between self-incompatible and self-compatible sour cherry selections

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, the 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. We previously determined that sour cherry has non-functional S-haplotypes for the S ₁ -, S ₆ - and S ₁₃ -haplotypes that are also present in diploid sweet cherry (P. avium L.). The mutations underlying these non-functional S-haplotypes have been determined to be structural alterations of either the S-RNase or SFB. Based on these structural alterations we designed derived cleaved amplified polymorphic sequence (dCAPS) markers and S-haplotype specific primer pairs that took advantage of either the length polymorphisms between S-haplotypes, differential S-haplotype sequences, or differential restriction enzyme cut sites. These primer pairs can discriminate among the mutant and wild-type S-haplotypes thereby enabling the identification of the S-haplotypes present in a sour cherry individual. This information can be used to determine whether the individual is either SC or SI. In a sour cherry breeding program, the ability to discriminate between SI and SC individuals at the seedling stage so that SI individuals can be discarded prior to field planting, dramatically increases the program’s efficiency and cost-effectiveness.     

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.