S-allele identification by PCR analysis in sweet cherry cultivars

Gametophytic self‐incompatibility, governed by the S‐locus, operates in sweet cherry. The knowledge of the S‐genotype of sweet cherry cultivars is therefore essential to establish productive orchards by defining compatible combinations. The isolation of sweet cherry S‐R Nases has allowed the use of different molecular techniques to characterize the S‐genotypes of sweet cherry cultivars. Previously, incompatibility group assignment could only be carried out on mature trees through pollination tests. In this work, PCR analysis with primers designed on the conserved sequences of sweet cherry S‐R Nases has been used to characterize the S‐genotype of 71 sweet cherry cultivars, including 26 cultivars whose S‐allele constitution had not been previously described. This approach has allowed the detection of alleles that had not been amplified by PCR before, to identify six putative new S‐alleles, to define three new self‐incompatibility groups and to compile the standards for a PCR‐based S‐allele typing method in sweet cherry.     

S-RNase genotyping and incompatibility group assignment by PCR and pollination experiments in Japanese plum

Most Japanese plum-type cultivars are self-incompatible and cross pollination is necessary to ensure fruit set. In this study, the S -RNase genotype and the incompatibility group of 68 Japanese plum-type cultivars were determined by PCR amplification of the S-RNase gene. The S -RNase genotype of 50 cultivars is first reported here and five new Japanese plum S -RNase alleles ( So , Sp , Sq , Sr , Ss ) were identified. The results obtained, together with information compiled from previous studies, allowed describing 12 new incompatibility groups (VIII–XIX). The self-incompatibility of several cultivars and the cross-compatibility among different incompatibility groups were verified by self- and cross-pollination experiments followed by observation of pollen tube growth. Five cultivars behaved as self-compatible, but two of them do not have the Se allele, which has been correlated with self-compatibility. Thus, additional sources of self-compatibility different from Se appear to be involved in Japanese plum self-compatibility.     

Correlation of stylar ribonuclease zymograms with incompatibility alleles in sweet cherry

Summary Protein stylar extracts of 16 cultivars of sweet cherry (Prunus avium), from the 10 different incompatibility groups to which incompatibility alleles have been assigned, were separated on acrylamide gels using isoelectric focusing (IEF) and were stained for ribonuclease activity. When two cultivars from the same incompatibility group were analyzed they gave identical zymograms and the cultivars of the 10 different incompatibility groups gave in all eight distinct zymograms. The ribonuclease polymorphism could be correlated with the reported S allele constitutions of the cultivars. Three ribonuclease bands were identified that each consistently corresponded to one of the six known incompatibility alleles (S ₁, S₂ and S ₆), a fourth band apparently corresponded to S ₃ and to the combination of S ₄ and S ₅, and a fifth band to S ₄ and S ₅ in other combinations. Thus, it seems that S alleles of cherry have ribonuclease activity and that IEF is useful for distinguishing S allele constitutions. The ribonuclease pattern of Summit, a cultivar of unknown incompatibility group, indicated its incompatibility genotype to be S ₁S₂, and this was confirmed by controlled pollination. The same band corresponded to S ₄ and S ₄', the mutant allele in self-compatible cultivars. IEF and ribonuclease staining promise to be useful tools for exploring the incompatibility relationships of cherry cultivars and perhaps of other self-incompatible Prunus crops.     

Identification of self-incompatibility alleles and pollen incompatibility groups in sweet cherry by PCR based s-allele typing and controlled pollination

A total of 17 pollen incompatibility groups in sweet cherry (Prunusavium L.) were identified among 46 accessions by PCR based S-alleletyping analysis and by controlled test pollinations. Two putativeS-alleles different from S ₁ to S ₆,S _z and S _y were identified. Five S-genotypes, S ₁ S ₅, S ₁ S ₆,S ₂ S ₆, S ₄ S ₆, andS ₅ S ₆, combinations of S ₁ toS ₆ alleles that had not previously been identified from cultivars in NYSAES, were positively confirmed by PCR based S-genotyping analysis. Also, the S-genotypes of cultivars in some pollen incompatibility groups that had previously been incorrectly reported have been clarified. Several popular cultivars, which were previously used as testers for S-allele typing analysis, were found to have been inaccurately genotyped. In addition, the S-genotypes and self-incompatibility groups of some relatively recentlyintroduced cultivars were identified. The molecular typing system ofS-genotypes based on PCR is a useful and rapid method for identifying newS-alleles and incompatibility groups in sweet cherry.     

S‐locus diversity and cross‐compatibility of wild Prunus avium for timber breeding

Prunus avium is primarily cultivated for its fruit, sweet cherries. However, it is also used to produce high‐quality timber. In a P. avium seed orchard, gametophytic self‐incompatibility is a restriction for free pollen flow and should be considered when establishing basic forest materials. In this study, S‐locus diversity and cross‐incompatibility of wild cherry individuals in clonal banks established for breeding for timber production were investigated. Wild cherry trees (140) with outstanding forest growth habit, collected in northern Spain, grafted and planted in two clonal banks, were genotyped at the S‐locus. The self‐incompatibility S‐locus genes, S‐RNase and SFB, were analysed by PCR. Twenty‐two S‐haplotypes, resulting in 72 different S‐genotypes, were identified. The genotypes were grouped into 33 incompatibility groups and 39 unique genotypes. This initial S‐locus analysis revealed large genetic diversity of wild cherry trees from the Spanish northern deciduous forest, and provides useful information for seed orchard design. Wild P. avium displays significantly more genetic diversity than what is detected in local cultivars, revealing a narrowing of genetic diversity during local domestication.     

S-genotype identification based on allele-specific PCR in Japanese pear

Gametophytic self-incompatibility in Japanese pear (Pyrus pyrifolia Nakai) is controlled by the single, multi-allelic 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 S^k) 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 S^4sm/S^4sm) 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.     

S-genotyping of sweet cherry varieties from Spain and S-locus diversity in Europe

Sweet cherry is a traditional fruit crop in Spain. The introduction of modern cultivars is replacing the local varieties and genetic diversity is lost. To conserve this plant material, local varieties are being collected and characterized. As part of this objective we investigated the S-genotype of 73 local varieties. S-locus analysis was carried out by PCR analysis of S-RNase and SFB genes. PCR was done using conserved primers and fragments detection was carried out by capillary electrophoresis. Fifty-six cultivars were S-genotyped for first time and 17 had been S-RNase typed previously. The S-genotype of the 73 varieties was unambiguously determined. Ten different S-haplotypes were identified: S ₁ , S ₂ , S ₃ , S ₄ , S ₅ , S ₆ , S ₉ , S ₁₃ , S ₁₆ and S ₂₂ . The varieties were assigned to 17 incompatibility groups, to Group ‘0’ of universal donors, and to Group ‘SC’ of self-compatible varieties. The results provide cross-compatibility information for cross design and orchard management. The results also reveal the S-locus diversity of this plant material. S-haplotypes S ₃ , S ₆ and S ₂₂ were the most frequent and S ₁₆ was only found in the Balearic Islands. Comparison of S-haplotype frequencies worldwide and within Europe showed that S ₂₂ is almost exclusive of southern Europe. Other S-haplotypes that are common in northern Europe, like S ₂ , S ₄ , S ₅ , were rare in the southern plant material. This geographic distribution of S-haplotypes across Europe may indicate a common origin or genetic relationship of varieties from close areas. Alternatively, there may be an association of certain S-haplotypes with adaptive traits correlated to climatic conditions in the different areas.     

Incompatibility and resistance to woolly apple aphid in apple

The study investigated the reported linkage of the locus for resistance to woolly apple aphid with the locus for incompatibility. Apple seedlings from the cross ‘Northern Spy’(heterozygous for resistance) בTotem’(susceptible) were scored for resistance, and for incompatibility genotype, by analysis of stylar ribonucleases, and for Got‐1, the isoenzyme marker for incompatibility. Cosegregation analysis provided no evidence that the loci for resistance and incompatibility are linked. Two rootstock cultivars,‘M9’and ‘Merton 789′, which in early work had been reported to give poor set in crosses with ‘Northern Spy’, were found to have the same incompatibility genotype as ‘Northern Spy’, namely S₁S₃.‘M4’and ‘Irish Peach’, two other cultivars that had given poor set when crossed on to ‘Northern Spy’, appeared to be homozygous at the incompatibility locus and to have the genotypes S₃S₃ and S₁S₁, respectively.     

Identification of self-incompatibility alleles in pear cultivars (Pyrus communis L.)

Self and cross-incompatibility determination by means of fruit and seed set experiments or pollen tube growth observations in the style has been frequently reported to be unclear in pear (Pyrus communis L.). Thus,in order to develop a reliable in vivo method to test pollen-pistil incompatibility in pear, pollen tube performance has been studied along the pistil following self and cross-pollinations. Results show that, while pollen tube growth in the style may be an unclear test, ovule observation at the microscope for the presence of pollen tube in the nucellus is a proper method to test incompatibility in this crop. With this analysis we could identify S-alleles of ‘Williams’ (S₁S₂) and ‘Coscia’(S₃S₄), and three of the four possible S-genotypes resulting from the ‘Williams’ × ‘Coscia’ cross, as represented by ‘Butirra Precoz Morettini’ (S₁S₃), ‘Santa Maria Morettini’ (S₂S₃)and ‘Tosca’ (S₁S₄). This result demonstrates that ‘Williams’ and ‘Coscia’ cultivars do not share any allele in common. We also established two new inter-incompatibility groups in pear. Furthermore, the presence of a common allele between ‘Williams’ and ‘Agua de Aranjuez’,and ‘Coscia’ and ‘Agua de Aranjuez’, three apparently unrelated old cultivars, may indicate a narrower genetic base than expected for European pear. This finding together with the fact that 40% of new released cultivars have direct or indirect parental relationship with the cultivars ‘Coscia’ and/or ‘Williams’, anticipates the possibility of new cases of cross-incompatibility for this crop in the future. Both the method described and the determination of the S-genotypes will facilitate the characterisation of self and cross-incompatibility relationships in this species. This revised version was published online in August 2006 with corrections to the Cover Date.     

Stylar ribonucleases in almond: correlation with and prediction of incompatibility genotypes

To clarify incompatibility relationships among almond cultivars, 35 were analysed for stylar ribonucleases, which have previously been shown to correlate with incompatibility S alleles. Stylar proteins were extracted and separated electrophoretically and the zymograms compared with ladders of ribonucleases corresponding to the 12 S alleles previously reported. Sixteen cultivars showed a band corresponding to two of the known ribonucleases, 17 showed one known ribonuclease and one ‘new’ band, and two showed two new bands. Twelve new ribonucleases were detected; 11 were attributed to new S alleles (S₁₃ to S23) and a mutant form of S₇ was attributed to S_7A. Genotypes were proposed for nine cultivars of five incompatibility groups that had not been genotyped previously, VII, X, XI, XII and XIII. Twenty‐four cultivars of unknown incompatibility relationships were provisionally genotyped: six of these could be assigned to existing groups and two new groups were established, XIV and XV, along with group O of cultivars with unique genotypes. Test crosses confirmed that eight pairs of cultivars showing similar zymograms were indeed cross‐incompatible, including the two representatives of each of the two new groups. Virtually all self‐incompatible cultivars of known genotype are listed in a table. The data should be useful for planning cultivar combinations for orchards and for designing crosses for breeding programmes.     

Identification and characterization of new S‐alleles associated with self‐incompatibility in almond

Almond is a highly heterozygous species with a high number of S‐alleles controlling its gametophytic self‐incompatibility system (GSI). In this work, we have analysed 14 Spanish local almond cultivars for S‐RNase allele diversity. Five new S‐RNase alleles were identified by cloning and sequencing, S₃₁ (804 bp) in ‘Pou de Felanitx’ and ‘Totsol’, S₃₂ (855 bp) in ‘Taiatona’, S₃₃ (1165 bp) in ‘Pou d’Establiments’ and ‘Muel’, S₃₄ (1663 bp) in ‘Pané‐Barquets’ and S₃₅ (1658 bp) in ‘Planeta de les Garrigues’. Additionally, seven already known almond alleles could be recognized in the local cultivars studied. The high number of new alleles identified reveals the wide diversity of almond germplasm still existing and requiring characterization, and points to the possibility of new findings by a wider study focusing on other provenances. The almond S‐RNases have been compared to those of other Prunus species, showing a high identity and confirming that the S‐RNase gene in this genus presents a probable common ancestor.     

The myrobalan (<Emphasis Type="Italic">Prunus cerasifera</Emphasis> L.): a useful diploid model for studying the molecular genetics of self-incompatibility in plums

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

Identification and characterization of genomic DNA sequences of the S-ribonuclease gene associated with self-incompatibility alleles S1 to S5 in European pear

The stylar products of the S‐locus for the gametophytic self‐incompatibility (GSI) system in the Rosaceae are ribonucleases (S‐RNases). Recently, sequences for 13 pear S‐RNase alleles have been published and named following a letter–symbol nomenclature (S_a to S_d and S_h to S_p). To establish the correspondence between these sequences and the self‐incompatibility alleles we have described previously (S₁ to S₅), we have amplified genomic DNA with consensus primers from the cultivars, ‘Williams’ (S₁S₂), ‘Coscia’ (S₃S₄), ‘Butirra Precoce Morettini’ (S₁S₃), ‘Santa Maria Morettini’ (S₂S₃) and ‘Doyenne du Comice’ (S₄S₅) and identified PCR products specifically associated with each S allele. Cloning and sequencing of the amplification products has revealed that they correspond to European pear sequences already deposited in the database. This allowed us to link S‐RNase sequences with S allele phenotypes and to determine a correspondence between the symbol–letter nomenclature used to name S‐RNase sequences and the number‐based nomenclature used to name S alleles. Based on this result the prediction of new cross‐incompatibilities among pear cultivars is discussed. Finally, we propose a unified number‐based nomenclature to avoid future confusion denominating S alleles in pear.     

New self-incompatibility alleles in apricot (Prunus armeniaca L.) revealed by stylar ribonuclease assay and S-PCR analysis

Apricot (Prunus armeniaca L.) shows gametophytic self-incompatibility controlled by a single locus with several allelic variants. An allele for self-compatibility (S_C) and seven alleles for self-incompatibility (S₁–S₇) were described previously. Our experiments were carried out to ascertain whether the number of allelic variants of apricot S-locus was indeed so small. Twenty-seven apricot accessions were analysed for stylar ribonucleases by non-equilibrium pH gradient electrofocusing (NEpHGE) to determine their S-genotype. To validate the results of electrofocusing, the applicability of the S-gene-specific consensus PCR primers designed from sweet cherry sequences was tested. NEpHGE revealed 12 bands associated with distinct S-alleles in newly genotyped cultivars. Cherry consensus primers amplified 11 alleles out from 16 ones, which indicated that these primers could also recognize most of the S-RNase sequences in apricot, and provided an efficient tool to confirm or reject NEpHGE results. By combining the protein and DNA-based methods, complete or partial S-genotyping was achieved for 23 apricot accessions and nine putatively new alleles (provisionally labelled S₈–S₁₆) were found. Their identity needs to be confirmed by pollination tests or S-allele sequencing. This study provides evidence that similarly to other Prunus species, the S-locus of apricot is more variable than previously believed.     

self-(in)compatibility almond genotypes: A review

To compile self-(in)compatibility almond genotypes, a review of 133 commercial cultivars of wide geographical origin was made. The information gathered from own and mainly published work will be useful for both grower's cultivar choice when planting and for breeder's cross design when planning. The almond S genotypes compiled were identified using five different methods: biological (pollination tests in the field and in the laboratory) and molecular (RNases, PCR and sequencing). In most cases, genotypes were assigned after combining more than one technique. Cultivars were classified into three categories: self-incompatible (99), self-compatible (16) and doubtful self-incompatible (18). The database is divided in 9 fields (name, origin, parentage, obtention year (crossing, selection or release), S genotype, technique used, reference, consensus genotype, and cross incompatibility group). A study of the 27 S alleles already identified and their geographical distribution within the cultivated almond is also presented. The study was divided into cultivars of known and unknown parentage and the distribution of S alleles frequencies was uneven among the 133 cultivars. S allele frequencies are related to geographical origin. Some alleles (S ₁, S ₅, S ₇ and S ₈) are more frequently observed than the others among cultivars of both known and unknown parentage. In the cultivated almond, the S f allele is only found in the Puglia region, Italy. The S _f frequency is three times higher in cultivars released from breeding programmes than in cultivars selected by growers. From the 351 resulting possible genotypes by combination of the 27 S alleles identified only 20 CIG (0-XIX) have been established, which represents a small fraction of the whole genetic diversity of this polymorphic gene in almond.     

Distribution of S haplotypes in commercial cultivars of Brassica rapa

Self‐incompatibility in Brassicaceae plants is sporophytically controlled by a single multi‐allelic locus (S locus), which contains at least three highly polymorphic genes expressed in the stigma (SLG and SRK) and in the pollen (SCR/SP11). Using polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) analysis with SXG‐specific primer pairs, the S haplotypes of F₁ hybrid and open‐pollinated commercial cultivars of Brassica rapa were identified. The number of S haplotypes detected in the F₁ hybrid cultivars of Chinese cabbage, komatsuna, pak‐choi, turnip, open‐pollinated cultivars of Chinese cabbage and turnip were 9, 9, 4, 11, 13 and 12, respectively. Nine of them had different PCR‐RFLP profiles from those of the S‐tester lines that determined the SLG sequences. Four SLG sequences in the F₁ hybrid cultivars were determined and named S⁵³, S⁵⁴, S⁵⁵ and S⁵⁶, respectively. It is demonstrated that the PCR‐RFLP analysis using specific primer pairs of SLG and SRK is useful for identification of the S haplotypes, in both, S homozygous and S heterozygous plants of B. rapa. The possibility of using this method routinely in breeding programmes, and in the evaluation of F₁ hybrid seed purity, is discussed.     

Development of PCR-based markers for the identification of the <Emphasis Type="Italic">S</Emphasis>-<Emphasis Type="Italic">RNase</Emphasis> alleles in wild potato species

Sexual self-incompatibility in wild diploid potato species is controlled by a single multiallelic S-locus encoding a polymorphic stylar ribonuclease (S-RNase) that is responsible for the female function in pollen–pistil recognition. In this study, an approach using PCR-based markers were originally developed to amplify the S-RNase alleles in Solanum chacoense. Subsequently, to investigate their general applicability in Solanum, this molecular approach was successfully tested on S. spegazzinii and S. kurtzianum. Application of PCR-SSCP approach revealed potentially new S-RNase alleles in the three species, demonstrating for the first time the existence of S-RNase genetic variability within and between populations of wild diploid potato species. Species-specific SSCP markers that may be successfully used in gene flow studies was also detected in this investigation.     

Sequence of the S 9-RNase cDNA and PCR-RFLP system for discriminating S 1- to S 9-allele in Japanese pear

A new S ₉-allele was discovered in 6 Japanese pear cultivars, ‘Shinkou’, ‘Shinsei’, ‘Niitaka’, ‘Amanogawa’, ‘Nangetsu’ and ‘Nansui’. cDNA encoding S ₉-RNase, a stylar product of S ₉-allele, was cloned from pistils of ‘Shinkou’ and ‘Shinsei’ by 3' and 5' RACE. The S ₉-RNase gene had an open reading frame of 684 nucleotides encoding 228 amino acid residues. S ₉-RNase had a hypervariable (HV) region different from S ₁- to S ₈-RNase and shared higher similarity (95.2%) with apple S ₃-RNase than with 8 Japanese pear S-RNases (from 61.0% to 70.7%). Genomic PCR with primers ‘FTQQYQ’ and ‘anti-(I/T) IWPNV’ provided S ₁- to S ₉-amplicon (product), but could not discriminate the S ₂ from the S ₉ of ca. 1.3 kb. The S ₂ and S ₉ were distinguished by digestion with AflII and BstBI, respectively. The digestion with nine S-allele-specific restriction endonucleases, SfcI, AflII, PpuMI, NdeI,AlwNI, HincII, AccII, NruI and BstBI, distinguished S ₁ to S ₉, establishing that this PCR-RFLP system is useful for S-genotype assignments in Japanese pear harboring S ₁- to S ₉-allele. ‘Shinkou’, ‘Shinsei’, ‘Nangetsu’ and ‘Nansui’ assigned as S ₄ S ₉ were determined to be cross incompatible. This revised version was published online in July 2006 with corrections to the Cover Date.     

Resistance of almond cultivars to Plum pox virus (sharka)

Sharka, a disease caused by Plum pox virus (PPV), mainly affects some Prunus species, including apricot, peach and plum, and to a lesser degree, sweet cherry and sour cherry. In almond, different PPV isolates have been transmitted experimentally to the ‘Aï’ cultivar. In this study, the resistance of 10 almond cultivars to a Dideron PPV isolate was evaluated in controlled conditions by grafting the cultivars on to inoculated GF305 peach rootstocks. The results demonstrated a high level of resistance to PPV in all the almond cultivars assayed. They did not show any symptoms and were ELISA and RT‐PCR negative, despite the strong symptoms observed in their GF305 rootstocks. The implications of these results for the dispersion of PPV, and the potential role of almond as a source of resistance to PPV in other Prunus species such as peach, are also discussed.     

Identification of self-incompatibility (S) alleles in Latvian and Swedish sweet cherry genetic resources collections by PCR based typing

The Latvian and the Swedish sweet cherry (Prunus avium L.) genetic resources collections comprise valuable material for breeding. The collections represent local Latvian and Scandinavian genetic resources: semi-wild samples, landraces, and cultivars developed in local breeding programmes, as well as diverse germplasm from the northern temperate zone. The objective of this investigation was to determine which S ₁ –S ₆ alleles are most important in the sweet cherry genetic resources collections and to compare the identified allelic and genotypic frequencies in material of different origin. Accessions in the two collections were screened for the presence of the self-incompatibility (S) S ₁ to S ₆ alleles, using PCR based typing. Significant differences (P < 0.05) between screened collections were found in frequencies of S ₄ and S ₅ alleles. Analysis of allele combinations identified the high occurrence of selections with the S-genotype S ₃ S ₆ in both collections. Compared to the S-allele frequencies published for over 250 sweet cherry cultivars from Western and Southern Europe, the Latvian and Swedish germplasm appeared to have a high frequency of the S ₆ allele in both collections, and a relatively high frequency of the S ₅ allele in Latvian germplasm. This study represents the first comprehensive S-allele screening for the sweet cherry genetic resources collections in Latvia and Sweden. Both sweet cherry collections contain high proportion of accessions adapted to north central European growing conditions, not typical for the majority of the documented sweet cherry genetic resources, which explains differences in certain S-allele occurrence.