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.     

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.     

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.     

Inheritance of stylar ribonucleases in cherry progenies, and reassignment of incompatibility alleles to two incompatibility groups

Stylar proteins were extracted from parents and seedlings of six progenies of cherry (Prunus avium), separated using isoelectric focusing, and the gels stained for ribonuclease activity. The zymogram of each plant showed two main ribonuclease bands in the region pI 8.3 to 9.6. Progenies from crosses of parents with one band in common segregated into just two classes, whereas progenies from crosses of parents with no common bands segregated into four classes, the two types of segregation corresponding to those expected from semi-compatible and fully-compatible crosses respectively. This behaviour was consistent either with the ribonuclease locus being tightly linked with the self-incompatibility, S, locus, or else with the S locus coding for the ribonuclease variants. Evidence favouring the latter hypothesis is discussed. An apparently anomalous segregation led us to assign to ‘Bradbourne Black’ a genotype different from that previously reported, and analysis of some other cultivars in the same incompatibility group, Group VII, led us to conclude the genotype of this group is S₃S₅, and not S₄S₅ as previously reported. Correspondingly, we suggest the genotype of Group V is S₄S₅, and not S₃S₅. Five new S alleles, S₇, S₈, S₉, S₁₀ and S₁₁ were proposed in parental cultivars and selections that had not previously been assigned a genotype. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

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.     

The self-incompatible RNase S-alleles of Brazilian apple cultivars

Apple plants are self-incompatible because a genetic mechanism allows the female reproductive organ to recognize and reject self-pollen or pollen from genetic related individuals and allows non-self pollen to effect fertilization. Thus, there are implications to both breeding strategies and orchard management for fruit production. The purpose of this study was to identify and to characterize the S-RNase alleles of the gametophytic incompatibility among apple cultivars developed in Brazil, seeking to give support for choosing right combinations of parent in the apple breeding programs. It also sought to identify correct combinations of scion/pollinator cultivars of commercial apple orchards. A total of 16 specific S-RNase alleles primers were tested against DNA extracted from 12 Brazilian cultivars and their parents. The molecular analysis confronted to the reference cultivars, showed that the cultivars Daiane, Imperatriz and Princesa have the same incompatibility S3 and S5 alleles, while ‘Lisgala’ showed the alleles S2 and S5; ‘Suprema’, S1 and S9; ‘Catarina’, S1 and S19; ‘Joaquina’ and ‘Fred Hough’, S5 and S19; ‘Baronesa’, S3 and S9; ‘Duquesa’, S2 and S3. For ‘Primícia’ and ‘Condessa’ it was only possible to identify one of the S-alleles, namely S24 and S2, respectively, with the second allele remaining to be identified. Progeny test indicated the Mendelian inheritance for RNase alleles. Results of this study will be helpful to judiciously choose parents in apple breeding programs to improve compatibility.     

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.     

Location of the self-incompatibility gene on the almond linkage map

A progeny obtained from the almond cross ‘Ferragnès’בTuono’ (Prunus amygdalus Batsch) was used to study the self-incompatibility trait in three different ways: fruit set, pollen tube growth and stylar ribonuclease activity. As expected from the genotypes of the parents, all progeny appeared phenotypically as self-compatible. However, the progeny could be scored for the segregation of stylar ribonuclease isozymes and thus allowed the incompatibility locus to be placed on the almond linkage map.     

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.     

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.     

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.     

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.     

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.     

Leaf proteins as markers useful in the genetic improvement of coconut palms

There is a need to identify genetic markers that can assist coconut (Cocos nucifera L.) breeding programs. With that objective, electrophoretic patterns of leaf peroxidases, endopeptidases and coomassie blue stained proteins were analysed in four cultivars (‘West African Tall’, ‘Rennell Tall’, ‘Malayan Yellow Dwarf’, ‘Cameroon Red Dwarf’), and in the hybrids PB121 (‘Malayan Yellow Dwarf’ × ‘West African Tall’) and PB111 (‘Cameroon Red Dwarf’ × ‘West African Tall’). The polymorphisms detected fit the expression of two alleles of a dimeric peroxidase, two alleles of a monomeric endopeptidase, and a pair of active and null alleles of a coomassie blue stained protein. Four distinctive genotypes were identified. One for each of the tall cultivars, another for both of the dwarf cultivars, and the last for both of the hybrids. Applications of the markers in breeding programs, and in research concerning reproductive biology and phylogeny, are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Identification of incompatibility genotypes in almond (Prunus dulcis Mill.) using specific primers based on the introns of the S-alleles

Identification of the incompatibility genotypes of almond cultivars is important in breeding programmes for designing crosses and for selecting progeny. This paper describes a novel molecular technique for the identification of S‐alleles in almond based on the use of PCR primers designed from the sequences of the introns without the need for restriction enzyme digestion. Nine specific pairs of primers have been designed for the S1, S2, S5, S7, S8, S9, S10 (putative), S23 and Sf alleles, and these confirmed the S‐allele specificities for 22 of the 23 accessions for which published information is available. This technique provides a precise method for identifying S‐alleles from the genomic DNAs of almond cultivars, and will be useful for confirming the segregation of alleles in breeding progeny.     

Breeding behavior for tuber protein in Solanum tuberosum and Tuberosum-phureja hybrids

Summary True protein content among tubers within a plant of Solanum tuberosum cv. Oneida was found to be negatively correlated with tuber size. A similar study of S. phureja genotype 148-17 revealed no correlation. Tuber protein was determined for 250 genotypes in each of four hybrid potato populations in a factorial mating design with four potato cultivars as stylar parents mated to four groups of pollen parents (4x cultivars, 4x high protein selections, 2x S. phureja, 2x high protein S. phureja). The hybrid population derived from the 4x high protein selections was significantly higher in protein content than the other populations. High estimates of general combining ability for tuber protein content were found for both stylar and pollen parents. Poor photoperiodic adaptation to growing conditions in northern latitudes was a possible explanation for the variable protein phenotypes typical of S. phureja and the lack of transmission of the high protein character in phureja to 4x-2x hybrids. Although protein content was negatively correlated with total yield, high protein segregates with good yield potential were identified in all four populations.Scientific Journal Series Article 11, 616 of the Minnesota Agricultural Experiment Station.     

Stability of esterases and total proteins during seed storage of perennial ryegrass (Lolium perenne L.) and their use for cultivar discrimination

Polymorphisms in electrophoretic patterns obtained by isoelectric focusing (IEF) were examined to evaluate their suitability for cultivar identification in perennial ryegrass (Lolium perenne L.). It was possible to discriminate 64 (94%) of 68 cultivars by combining results from esterase and total protein analysis. Discrimination was based on quantitative differences (relative band intensity) rather than on qualitative differences (presence or absence of bands). Esterase patterns from different recent (fresh to seven years old) seed lots of the same perennial ryegrass cultivars were very stable. Occasionally, minor differences in band intensity were observed between recent and old (up to 30 years old) seed lots of a cultivar. Storage of meal samples up to two years at −20 ^°C had no effect on the total protein patterns. No correlation was found between esterase patterns and ploidy level, cultivar type (pasture or turf), heading date or breeding company. Esterase patterns appeared to be unsuitable markers for the selection of reference cultivars for distinctiveness, uniformity, and stability (DUS) testing, because no correlation was found between cultivars on the basis of esterase banding patterns and morphological characteristics. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic diversity,structure and fruit trait associations in Greek sweet cherry cultivars using microsatellite based (SSR/ISSR) and morpho-physiological markers

It is important to couple phenotypic analysis with genetic diversity for germplasm conservation in gene bank collections. The use of molecular markers supports the study of genetic marker-trait associations of biological and agronomic interest on diverse genetic material. In this report, 19 Greek traditional sweet cherry cultivars and two international cultivars, which were used as controls, were grown in Greece and characterized for 17 morpho-physiological traits, 15 simple sequence repeat (SSR) loci and 10 inter simple sequence repeat (ISSR) markers. To our knowledge, this is the first report on molecular genetic diversity studies in sweet cherry in Greece. Principal component analysis (PCA) of nine qualitative and eight quantitative morphological parameters explain over 77.33% of total variability in the first five axes. The SSR markers yielded a combined matching probability ratio (MPR) of 9.569 × e−12. The 15 SSR loci produced a total of 92 alleles. Ten ISSR primers generated 91 bands, with an average of 9.1 bands per primer. Expected heterozygosity (gene diversity) values of 15 SSR loci and 10 ISSR markers averaged at 0.683 and 0.369, respectively. Based on stepwise multiple regression analysis (MRA), SSR alleles were found associated with harvest time and fruit polar diameter. Furthermore, three ISSR markers were correlated with fruit harvest and soluble solids and four ISSR markers were correlated with fruit skin color. Stepwise MRA identified six SSR alleles associated with harvest time with a high correlation (P < 0.001), with linear associations with high F values. Hence, data analyzed by the use of MRA could be useful in marker-assisted breeding programs when no other genetic information is available.