Correlation of ribonuclease zymograms and incompatibility genotypes in almond

Proteins were extracted from styles of 29 self-incompatible cultivars of almond and separated using non-equilibrium pH gradient electro-focusing, and the gels were stained for ribonuclease activity. Mutually incompatible cultivars had similar banding patterns and, for the 24 cultivars already genotyped in France or California, the bands correlated well with the reported alleles. The band corresponding to S₁ of the French labelling system was indistinguishable from that corresponding to S_b of the Californian labelling system, and a controlled cross confirmed that these alleles are identical. The band corresponding to the Californian S_a was distinct from the bands corresponding to French alleles and, to harmonise the allele labels, it was redesignated S₅. The genotypes of five uncharacterised self-incompatible cultivars were inferred from zymograms as follows: ‘Desmayo Largueta’ and ‘Glorieta’, S₁S₅, ‘Masbovera’, S₁S₉, ‘Tarragones’, S₂S₉, and ‘Tokyo’, S₆S₇. The alleles designated S₆ and S₉ have not previously been reported. Nine self-compatible cultivars or selections were analysed, and each showed a band corresponding to an incompatibility allele as well as a common band; however, the correspondence of this common band to S_f, the allele for self-compatibility, is unproven. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of S-alleles in almond using multiplex PCR

The S-genotypes of eight almond (Prunus dulcis Miller (D.A. Webb)) cultivars from different geographical origins and of nine new selections from the CEBAS-CSIC (Murcia, Spain) breeding program were determined using single and multiplex PCR with different sets of specific oligonucleotide primers. The results of PCR using the AS1II- and AmyC5R-specific primers showed amplification in a single reaction of 10 different self-incompatibility alleles and of the self-compatibility allele S _f. However, the amplified fragments of the S _f allele were of similar sizes to those amplified from the S ₃ self-incompatibility allele. For this reason, a specific PCR primer CEBASf was designed from the intron sequence of S _f. A multiplex-PCR reaction using the AS1II, CEBASf and AmyC5R primers permitted unequivocal identification of the 10 self-incompatibility alleles and of the self-compatibility allele. Multiplex PCR opens the possibility to identify new S-alleles using different sets of primers. The applications of these PCR markers in the almond-breeding programs are discussed.     

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.     

Molecular and phenotypic characterization of the <Emphasis Type="Italic">S</Emphasis>-locus and determination of flowering time in new ‘Marcona’ and ‘Desmayo Largueta’-type almond (<Emphasis Type="Italic">Prunus dulcis</Emphasis>) selections

‘Marcona’ and ‘Desmayo Largueta’ are the most widely cultivated almonds in Spain, representing around 27% of the total production and 34% of the entire almond growing surface. The excellent quality of their kernels makes them highly appreciated and demanded worldwide. However, due to their self-incompatibility, they should be grown along with cross-compatible cultivars, whose lower kernel quality often reduces the economic benefits of the plantation. In addition, although they are cross-compatible, are not good candidates to share the orchard since ‘Desmayo Largueta’ usually flowers earlier than ‘Marcona’. Therefore, to optimize orchard yield, genotypes with overlapping flowering times, cross-compatible with these cultivars and of similar fruit and ripening characteristics are desirable. In order to find suitable pollinators of these cultivars, five ‘Marcona’ and four ‘Desmayo Largueta’-type selections from “Instituto Técnico Agronómico Provincial (ITAP) de Albacete” (Spain) were characterized for flowering time and for self and cross-incompatibility. The results obtained showed that the nine ITAP selections were self-incompatible, and that three and one were promising candidates as pollinators of ‘Marcona’ and ‘Desmayo Largueta’, respectively. The S-haplotypes of two ITAP selections were characterized by cloning and sequencing their pistil S (S-RNase) and pollen S (SFB) genes. The results also showed that S _f RNase does not have a mutated histidine in C2 region, and detected differences with other previously published sequences for S ₂₃ RNase and SFB ₂₃ allele. Moreover, the sequence for almond SFB ₂₇ allele is reported here for the first time.     

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 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.     

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.     

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.     

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.     

Genetics of self-compatibility in dihaploids of Solanum tuberosum L. 2. Detection and identification of all possible incompatibility and compatibility genotypes in six F1'S from interdihaploid crosses

Summary Two self-compatible (sc) dihaploids, G254 and B16, and one self-incompatible (si) dihaploid, G609, from Solanum tuberosum L. were intercrossed reciprocally. Segregation ratios sc : si : pc (pseudo-compatible) were determined in all 6 F₁'s in three successive years and critically tested and discussed. Genotypes at the S-locus could be assigned to the dihaploid parents and the S-allele on the translocation in sc G254 identified as S ₁. Using these genotypes all sc and si genotypes were derived which could be expected in the F₁'s.Incompatibility groups were detected in each F₁ from the results of complete diallels involving si plants. The genotype of each group was identified by test crosses. Compatibility groups could be both detected and identified by crossing in each F₁ the sc plants as females with the already identified si sibs. In this way a complete series of 6 si testers and corresponding sc genotypes was obtained involving four alleles at the S-locus and S ₁ and S _x on the translocation.Certative disadvantage of pollen carrying the translocation could be ruled out as a possible cause of unexpected ratios. The hypothesis of an S-bearing translocation as the cause of self-compatibility could account for all results on the assumption that translocation homozygotes are lethal and the S-allele on the translocation is active in the pollen only.The following bachelor students have contributed to the experimental data used in this article: Janny Olsder, J. Marelis, H. v.d. Brink, J. Sonneveld, D. Vreugdenhil, Digna van Ballegooijen and Els Staas-Ebregt.     

Genetic analysis of four self-incompatible lines in Brassica napus

Reciprocal hybridization between four self-incompatible lines of Brassica napus: 271, 181, 184 and ‘White Flower’, revealed incompatibility. The reciprocal F₁s obtained by bud pollination showed self-incompatible reactions, and no segregation for self-incompatibility was observed in all the reciprocal F₂ populations, indicating that lines 271, 181, 184 and ‘White Flower’ were genetically identical with regard to self-incompatibility. Observations of self-incompatibility in 17 hybrids from crosses between line 271 and 17 varieties of B. napus showed 10 of the F₁ hybrids to be self-compatible, while four were partially self-compatible and three were self-incompatible. Genetic analysis based on F₂ and BC₁ populations from five self-compatible F₁ hybrids and two self-incompatible F₁ hybrids suggested the existence of at least two loci controlling the self-incompatibility of line 271: one is the S locus, with dominant and recessive relationships between the S alleles, and the other is the suppressor (sp) of the S locus. The sp locus is genetically different from the S locus, and also shows dominant and recessive relationships between the sp alleles.     

Improved <Emphasis Type="Italic">S</Emphasis>-genotyping and new incompatibility groups in Japanese plum

The selection of cross-compatible cultivars is essential to ensure fruit set in self-incompatible species like Japanese plum and thus the S-genotype must be determined in order to establish incompatibility groups. In this study an improved Japanese plum S-genotyping method, based in polymerase chain reaction and capillary electrophoresis detection of intron polymorphisms of S-locus genes, S-RNase and SFB, has been assayed and validated in a wide sample of cultivars. This method allows a more precise determination of amplified fragment sizes and therefore a better differentiation of self-incompatibility alleles. The assayed methodology was proven effective in the detection of 13 different S-alleles of S-RNases and SFBs and was used to S-genotype 105 Japanese plum cultivars, 32 of which are described by first time in this work. Analysed cultivars were assigned into 11 incompatibility groups and two new incompatibility groups (XX and XXI) were identified, increasing to 21 the number of incompatibility groups described in this crop.     

Independence of self-compatibility and potentiality for self-pollination in peach x almond hybrids

Summary The almond of commerce (Prunus dulcis (Mill.) D.A. Webb) is self-incompatible (SI) and requires honey-bees to effect the transfer of pollen among cultivars that flower simultaneously. Four year old trees from the F₂ generation of several peach x almond hybrids were studied to determine whether self-compatibility (SC) and the potentiality for natural, i.e., abiotic, self pollination (NSP) are genetically related or are inherited independently. Both SC and the high potentiality for NSP are characteristic of peach (Prunus persica (L.) Batsch) but not almond. Forty percent of SC genotypes exhibited adequate NSP (SC, +NSP) for good fertility i.e., without insect-mediated pollination. The remaining 60% of SC genotypes (SC,-NSP) exhibited an average 61% reduction in fruit set on limbs bagged to exclude honeybees during anthesis relative to fruit set on open pollinated limbs. Our data are consistent with the concept that fertility is dependent upon the load of compatible pollen deposited on the stigma. Fruit set reduction on bagged limbs, compared with bagged and self-pollinated limbs, was presumably due to a) lack of/insufficient pollination for fertilization and/or b) post-zygotic abortion of genetically inferior recombinants. Selection following manual self-pollination may result in SC genotypes with or without the capacity for NSP. In contrast, significant fruit set on limbs enclosed during pistil receptivity necessitates that the genotype selected express both SC and the potentiality for NSP.     

'Francolí', a late flowering almond cultivar re-classified as self-compatible

To verify the compatibility behaviour of the almond cultivar ‘Francolí’ and to clarify its S genotype a combination of pollination tests, stylar ribonuclease and allele specific PCR analysis was used. ‘Francolí’ was released from IRTA's breeding programme in 1994, having been putatively raised from the cross ‘Cristomorto’ (S₁S₂) × ‘Gabaix’ (S₁₀S₂₅). This cultivar was also reported to be self‐incompatible but revealing only one S band in the zymograms after S‐RNases analysis. ‘Francolí’ sets nuts after test crossing with two S₁S₂₅ cultivars, having a different genotype from that earlier reported. ‘Francolí’ was also observed to be self‐compatible after selfing flowers in the field and in the laboratory. ‘Francolí’ was re‐assigned the S₁S_f genotype after test crossing, stylar ribonuclease and PCR data analysis. After microsatellite analysis, the self‐compatible ‘Tuono’ (S₁S_f) cultivar is suggested as the male parent of ‘Francolí’ instead of the earlier reported ‘Gabaix’.     

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 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.     

Rapid detection of S-glycoproteins of self-incompatible crucifers using Con-A reaction

Summary As S-specific glycoproteins react with concanavalin A (ConA), they could be stained sensitively with ConA-FITC after cellulose acetate isoelectric focusing of stigma proteins in self-incompatible crucifers. By this method the S genotype of an individual plant could be inferred within 4 h using 4 flowers. This simple and rapid method may be applicable for describing S genotypes in practical F₁ breeding.     

Expression of S-locus inhibitor gene (Sli) in various diploid potatoes

Summary S-locus inhibitor gene (Sli), which can inhibit gametophytic self-incompatibility in diploid potatoes and alter self-incompatible to self-compatible plants, was introduced by crossing into 32 diploid genotypes as females and its expression in the F₁ and S₁ progenies was investigated. We found that the expression of self-compatibility in the F₁ hybrid progeny depended largely upon the female genotypes and partly upon the male genotypes (=Sli gene donor clones). Successful females produced hybrid plants, in which 67.1% of self-pollinated plants set S₁ seeds. By second selfing upon the S₁ plants, an average of 44.2% of self-pollinated plants were self-compatible. Unsuccessful females produced hybrids, most of which were self-incompatible or male-sterile. Restriction fragment patterns of chloroplast DNA (ctDNA) were able to distinguish successful females (S- or A-type ctDNA) from unsuccessful females (W- or T-type ctDNA). A ctDNA high-resolution marker analysis using seven microsatellites and H3 marker supported a higher degree of differentiation between the two groups of ctDNA types and implied a possible interaction between the cytoplasm and Sli gene function. However, it has been known that the cytoplasm having T-type ctDNA and that derived from Solanum demissum (haplotype 26 of W-type ctDNA) cause male sterility, and the present case with unsuccessful females were likely caused by male sterility rather than the failure of Sli gene function.     

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.     

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.