Development of a molecular marker for self‐compatible S4′ haplotype in sweet cherry (Prunus avium L.) using high‐resolution melting

Sweet cherry (Prunus avium L.) has stylar gametophytic self‐incompatibility, which is controlled by the multi‐allelic S‐locus and encompasses the highly polymorphic genes for the S‐ribonuclease (S‐RNase) and S‐haplotype‐specific F‐box (SFB), which are female and male determinants, respectively. The self‐compatible mutant SFB4′ corresponds to an allele variant of SFB4 and presents a frameshift mutation. Even though male‐determinant molecular markers can discriminate between SFB4 and SFB4′ alleles, the methods required are laborious, time‐consuming and expensive, and not suitable for massive analysis and integration into breeding programmes. Our aim was to develop molecular markers for the evaluation of self‐compatibility alleles in sweet cherry, that could be used as a high‐throughput screening strategy to identify SFB4 and SFB4′ alleles, based on a marker for male determinacy. Our results were consistent using primers flanking the mutation responsible for the SFB4′ allele. We designed a specific molecular marker and confirmed it in sweet cherry commercial varieties. This new molecular marker is feasible for self‐compatibility alleles in the male determinant in sweet cherry‐assisted breeding programs.     

Improved discrimination of self-incompatibility S-RNase alleles in cherry and high throughput genotyping by automated sizing of first intron polymerase chain reaction products

A novel polymerase chain reaction (PCR) approach to determine and confirm the self‐incompatibility (S) genotype of cherries is reported. The method involves PCR amplification with a new pair of consensus primers that immediately flank the first intron of cherry S‐RNases, one of which is fluorescently labelled. Fluorescent amplification products range from 234 to c. 460 bp and can be sized accurately on an automated sequencer. Thirteen S alleles reported in sweet cherry can be distinguished, except for S₂ and S₇, which have an amplification product of exactly the same size. S₁₃, which is also amplified, gives a microsatellite‐like trace which shows minor intra‐allelic length variation. This method gives fast and accurate results and should be especially useful for medium/high‐throughput genotyping of wild and cultivated cherries.     

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.     

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.     

Determination of self-incompatible genotypes in sweet cherry (Prunus avium L.) accessions and cultivars of the German Fruit Gene Bank and from private collections

Sweet cherries are self-incompatible because of a gametophytic self-incompatibility system. S alleles in the style and pollen determine the crossing relationships. Knowledge of the S allele constitution of cultivars is very important for cherry growers and breeders, and recently, molecular methods have been developed to distinguish the S alleles in sweet cherries. The S allele genotypes of 149 sweet cherry cultivars and clones, including 126 not previously genotyped, were determined by using PCR analysis. Thirteen different S alleles in 40 combinations were distinguished and nine new incompatibility groups were documented. Two new S alleles were identified in five local sweet cherry processing cultivars from southwestern Germany using the second intron primers. The sequence of these alleles was determined and compared to all known sequences available in the NCBI database. The sequences obtained showed high similarities to the alleles S ₁₉ and S ₂₂, previously described only in wild cherries, Prunus avium L.     

Determination of incompatibility genotypes in almond using first and second intron consensus primers: detection of new S alleles and correction of reported S genotypes

The work aimed to develop a reliable and convenient PCR approach for determining incompatibility S genotypes in almond. Initially, genomic DNAs of 24 accessions of known S genotype were amplified with novel consensus primers flanking the first and second introns of the S‐RNase gene. The PCR products separated on agarose showed length polymorphisms and correlated well with the reference alleles S1‐S₂3 and Sf. In addition, to improve discrimination between alleles of similar sizes, the same sets of primers but fluorescently labelled were used, and the products sized on an automated sequencer. These fluorescent primers were particularly informative in the case of the first intron, variation in the length of which has not been used previously for S genotyping in almond. Some reference alleles showed the same patterns with first and second intron primers, and others showed a microsatellite‐like trace. Subsequently, the S genotypes of 26 cultivars not genotyped previously and of four of uncertain genotype were determined. An allele described in Australian work as putative S₁₀ was shown to be a ‘new’ allele and ascribed to S₂₄ and evidence of five more ‘new’S alleles was found, for which the labels S₂₅‐S₂₉ are proposed. This PCR approach should be useful for genotyping in other 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.     

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.     

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.     

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.     

Primers amplifying a range of Prunus S-alleles

Although various consensus polymerase chain reaction (PCR) primers have been reported for identifying Prunus S‐alleles, they have been developed from and optimized on a limited set of alleles, which may limit their applicability to a broader allele range. To develop a primer set for use across the genus, degenerate consensus primers were designed from conserved regions of 27 S‐RNase sequences available from five Prunus species. The primers were tested in 15 previously genotyped cultivars of cherry, almond and apricot, representing alleles S₁ to S₆ in each crop and also S_c in apricot. Comparisons were made with previously published primers tested in the same 15 cultivars under reported reaction conditions. The new primers generated an amplification product for each of the 19 S‐alleles whereas those previously available amplified no more than 14. The primers will be useful for genotyping and genetic studies in cultivars and wild populations.     

Genotyping apricot cultivars for self-(in)compatibility by means of RNases associated with S alleles

In previous work the existence of proteins with RNase activity associated with S alleles in apricot was demonstrated. These proteins were inherited as described previously for the inheritance of self‐compatibility in this species. In this study, new cultivars have been genotyped for self‐compatibility using this method and it has been demonstrated that in all self‐compatible cultivars examined, the self‐compatibility allele is the same and is associated with an RNase with high activity. Homozygous self‐compatible individuals have been detected among established cultivars as well as among seedlings following breeding activity. This germplasm is of great value within the breeding programme because only self‐compatible seedlings will be produced. The number of S alleles in apricot appears to be low and only eight different alleles have been found in the large number of different cultivars screened. Furthermore, there are alleles present in the Spanish population that are also found in the genetic pool of North American cultivars. The screening of a progeny from the cross between the American cultivar ‘Goldrich’ and the Spanish cultivar ‘Pepito’ demonstrated the existence of the common allele S₂ (detected previously by examining RNases), which was confirmed by the segregation of self‐compatibility in the progeny.     

Identification of cherry incompatibility alleles by microarray

We have developed a microarray for identification of sweet cherry incompatibility alleles. Using intron sequence information of the S-RNase gene, we have created a microarray chip that allows the specific recognition of the incompatibility alleles present in a cultivar. Most of the probes designed showed high specificity towards their alleles. In the original set of probes, cross-hybridization was observed between a few alleles with high sequence similarity. As our identification system is based on the combined hybridization information from both introns, we were able to identify false positive and unspecific probes which could be eliminated from our microarray. The optimized microarray was tested on cultivars with known alleles. The chip correctly identified all alleles tested. Furthermore, it was also possible to identify alleles in other cultivars where, so far, only one allele has been determined and also to determine in sour cherry the alleles originating from the sweet cherry parent. Our results demonstrate the great promise of microarray technology for this novel application.     

Analysis of S‐allele genetic diversity in Sicilian almond germplasm comparing different molecular methods

Italian almond germplasm is characterized by a wide diversity in several growing areas among which Sicily is one of the most important. Analysis with consensus and specific primers and DNA sequencing was performed to investigate S‐RNase genetic diversity and to elucidate the homology rate within a genetic pool of 27 Italian accessions. Interestingly, some of the self‐compatible cultivars did not show the presence of S_f allele. Amplicons from consensus and allele‐specific PCR primers revealed a high level of variability. Sequencing of all the S‐RNase amplicons derived from consensus primers allowed the identification of two new S‐RNase alleles (S₅₁ and S₅₂). Surprisingly, despite the AA replacement mutation, S₅₁ did not exhibit any change of its S‐RNase function. Additionally, several mutations, with no effect on amino acid composition, were detected in the intron and/or in the ORF of four known alleles (S_g, S₁₀, S₃₁ and S₃₅). Genetic variation, regarding point mutations and only detected by sequencing, was revealed among 11 of 27 tested cultivars. The new sources of variability might have an interest for product traceability.     

Development of self-incompatible Brassica napus: (III) B. napus genotype effects on S-allele expression

Use of self‐incompatibility (SI) as a pollination control method for Brassica napus hybrid production requires the development of a sufficient number of S‐alleles that are expressed consistently in a range of B. napus lines. Self‐incompatibility (SI) alleles have been transferred from Brassica oleracea and Brassica rapa into B. napus var. oleifera. An understanding of expression of these alleles in B. napus is essential for their commercial use. Four SI B. napus doubled haploids containing the B. oleracea S‐alleles S2, S5, S13 and S24 were crossed to three B. napus cultivars to measure the B. napus genetic background effect on S‐allele expression. A line x tester analysis indicated that the largest source of variation in the expression rate of SI was the S‐allele itself. The B. napus genotypes tested contained modifier gene(s), some that enhanced SI expression and others that inhibited SI expression. The B. napus Canadian cultivar ‘Westar’ generally had a negative effect on SI expression while the European cultivar ‘Topas’ had a positive effect on the B. oleracea S‐allele expression. The B. oleracea S‐allele S24 was very similar in expression to the B. rapa allele W1. The application of these results for the use of B. oleracea S‐alleles for hybrid production in B. napus is discussed.     

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.     

SFB‐based S‐haplotyping of apricot (Prunus armeniaca) with DHPLC

Most cultivars that belong to the Rosaceae are self‐incompatible and depend on cross‐pollination. The pollen donor and pollen recipient have to flower synchronously and must be genetically compatible. Genetic compatibility is governed by the S‐locus, which holds the S‐RNase and S‐haplotype‐specific F‐Box (SFB) genes. Thus, the S‐genotype of cultivars is an important feature and is characterized molecularly by the S‐RNase and SFB alleles which are distinctive for each S‐haplotype. Here, we report the usage of DNA chromatography (denaturing high‐performance liquid chromatography – DHPLC) for identifying the S‐genotypes of European apricots on the basis of their SFB alleles. DHPLC is amenable to high‐throughput automation, and therefore is valuable for breeding and for high‐quality plant typing in the nursery.     

Development of a molecular CAPS marker for the self-incompatibility locus in Brassica napus and identification of different S alleles

Using primers annealing to S locus sequences the cleaved amplified polymorphic sequences (CAPS) method was applied to develop a marker and to characterize different alleles at the self‐incompatibility locus in Brassica napus. A segregating F₂ population from a cross of a self‐incompatible (SI) and a self‐compatible parent, as well as seven SI lines representing four different S alleles were used. Several primers specific to the S locus in B. oleracea and B. campestris, chosen from the literature, allow polymerase chain reaction (PCR) amplification of genomic DNA. However, only one primer pair amplified a single specific and reproducible PCR fragment of the expected length in B. napus. Digestion with restriction endonucleases revealed polymorphisms for two CAPS markers absolutely linked to the S locus. Using the codominant marker efMboI it was possible to discriminate all three F₂ genotypes. With this marker and an additional marker using another primer pair it was possible to distinguish between three of the four different S alleles and five of the seven SI lines, respectively.     

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.     

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.