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.     

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.     

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.     

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.     

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.     

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.     

A gene for susceptibility to the fungicide azoxystrobin in apple and a tightly linked microsatellite marker

Various apple cultivars, including 'McIntosh' and 'Cox', are reported to be susceptible to the strobilurin fungicide azoxystrobin whereas others, including 'Delicious' and 'Golden Delicious', are resistant. To investigate the genetic control, progenies from various crosses between these four cultivars were raised and the seedlings tested for response by painting three expanded leaves with 200 mg/l of azoxystrobin solution and noting symptoms about a week later. From the segregations, it was concluded that susceptibility to azoxystrobin is due to a dominant gene, Azs , for which 'McIntosh' and 'Cox' are heterozygous. By scoring the segregation in the mapping progeny 'Fiesta' × 'Totem' and comparing it with the segregation of microsatellite [simple sequence repeat (SSR)] markers, Azs was found to co-segregate with SSR-GD127 on linkage group 12 and this tight linkage was confirmed in the progeny from the cross of 'Golden Delicious' × 'Cox' and 'McIntosh'   ×   'Golden Delicious'. The susceptibility of 'McIntosh', attributed to allele Azs-m , was greater than that of 'Cox', Azs-c , and in the 'McIntosh'   ×   'Cox' progeny the GD127 marker showed that Azs-c is dominant to Azs-m .     

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.     

Comparison of isoenzymes in Prunus avium separated by two different electrophoretic techniques

Isoenzyme variation for seven systems revealed by two different electrophoretic procedures was compared in Prunus avium. Fourteen cultivars and 14 wild selections were analysed for acid phosphatase (ACP), isocitrate dehydrogenase (IDH), leucine aminopeptidase (LAP), malate dehydrogenase (MDH), phosphoglucomutase (PGM), shikimate dehydrogenase (SKD) and superoxide dismutase (SOD). Extracts were separated by isoelectric focusing (IEF) and by polyacrylamide gel electrophoresis (PAGE). For the eight loci that had been described previously in these enzyme systems on the basis of IEF analysis, we compared the variation revealed with IEF and PAGE. Similar variation was revealed for Acp‐1 and Pgm‐1, and the alleles revealed by PAGE could be identified directly with those reported for IEF. For Lap‐1, Mdh‐1 and Skd‐1, variation was seen with IEF but not with PAGE. For Mdh‐2, PAGE revealed additional variation not revealed by IEF. For Idh‐1, different patterns of variation were revealed by PAGE and IEF, and both procedures would be needed to genotype cherry accessions. We were unable to detect variation corresponding to that reported previously for Sod‐1 with either technique. The implications of these findings for allele labelling, for studies of genetic diversity and for linkage analysis are discussed.     

Identification of marker alleles linked to fire blight resistance QTLs in apple genotypes

Molecular marker analysis can be an effective tool when searching for new fire blight resistance donors. It can speed up the breeding process as well, even though many of the available markers linked to fire blight resistance QTLs have not yet been tested by screening a large number of cultivars. The aim of this study was to search for alternate sources of the three major QTLs of fire blight resistance; FBF7, FB_MR5 and FB_E, as well as to test the efficiency of some markers linked to minor QTLs. Altogether, nine primer pairs were used on 77 genotypes including new Hungarian cultivars and old apple cultivars from the Carpathian basin. Several marker alleles of FB resistance QTLs have been detected in the screened genotypes, most importantly the alleles coupling with FB_MR5 in the old cultivars ‘Kéresi muskotály’, ‘Szabadkai szercsika’ and ‘Batul’. We propose these cultivars as the first available resistance donors of FB_MR5 instead of the crabapple Malus × robusta 5. The results also bring new information regarding the resistance alleles of new Hungarian cultivars and selections.     

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.     

Combining apetalous parthenocarpy with columnar growth habit in apple

Summary ‘Wijcik’, a sport of ‘McIntosh’ with columnar growth habit, was crossed with ‘Wellington Bloomless’, which has apetalous flowers and bears parthenocarpic seedless fruit if not pollinated. The seedlings segregated for columnar versus normal habit but all had normal flowers and fruit. Four columnar seedlings were crossed with ‘Spencer Seedless’, another apetalous cultivar, and the resulting seedlings segregated not only for plant habit but also for apetalous versus normal flowers, approximately 1 : 1. Thus apetaly is controlled by a recessive gene, for which the symbolape is proposed. Apetalous columnar apples may be useful for planting in very high density orchards, cropping without pollination and thus not dependent on bees, pollinator varieties and warm weather at flowering time; moreover, being seedless they may avoid biennial bearing tendencies that are attributable to developing seeds inhibiting fruit bud formation.     

Screening for scab resistance by RAPD markers in cultivars of apple (Malus spp.)

Genetic markers are a much faster and more practical alternative to classical methods for the identification of genes for scab resistance present in different apple cultivars. In our study, 28 scab-resistant cultivars, four wild sources of the resistance genes and 10 susceptible cultivars were screened for the presence of the RAPD fragments OPM18/900, OPD20/600 and OPA15/900, which are reported to be linked to the Vf gene. All three marker fragments were successfully amplified with different protocols in Vf-resistant cultivars including ‘M. floribunda 821’. No marker fragments were amplified in susceptible cultivars, three out of four Va-resistant cultivars, three out of four Vm-resistant cultivars, two Vr-resistant cultivars, ‘Antonovka PI 172612’ and ‘M. pumila R 12740-7A’. All three markers were found in the cv. ‘Nova Easygro’, reported to possess the Vr gene, and the cv. ‘Reglindis’, reported to be Va-resistant. M. atrosanguinea of unknown origin showed the presence of OPD20/600 and OPA 15/900 marker bands. The cvs. ‘Nova Easygro’, ‘Reglindis’ and M. atrosanguinea are probably carriers of the VF gene.     

Inbred lines with dominant incompatibility alleles for use as parents of F1 hybrid brussels sprouts

Summary To improve the chances of obtaining highly self-incompatible inbred lines for use as parents of F₁ hybrid Brussels sprouts and to extend the range of mutually cross-compatible combinations available, new inbred lines are being produced from plants selected for the presence of dominant S-alleles. The material comprises 42 different inbred families representing 15 cultivars and contains 12 dominant S-alleles of uncommon occurrence in Brussels sprouts. Data on S-allele interactions in the material are presented.Tests showed that whilst many of the parent plants containing dominant S-alleles were highly self-incompatible, a few had only weak self-incompatibility. Although the chances of obtaining strongly self-incompatible inbreds may be increased by using material with dominant S-alleles, it remains necessary to test and select for strong self-incompatibility during the breeding programme.Production of such inbred lines requires the application of two independent selection procedures, one for agronomic type and one for S-allele constitution. Only a small proportion of the plants of a parent cultivar are acceptable on both counts. Thus large populations of the cultivar and large numbers of selections are necessary: this in turn involves much expensive S-allele screening. To alleviate this problem a wide-based panmix containing only dominant S-alleles is being produced. It is hoped that from this it will be possible to extract inbred lines which carry only dominant S-alleles, so avoiding the need to screen each parent plant for its S-allele content.     

The use of a modified bulk segregant analysis to identify a molecular marker linked to a scab resistance gene in apple

Almost two-hundred random sequence decamer-primers were used to screen a pair of bulked samples and the donor parent Malus floribunda clone 821 for markers linked to the V_f gene conferring resistance to apple scab (Venturia inaequalis (Cke.) Wint.). A single primer was identified which generated a PCR fragment, OPK16/1300, from the donor parent M. floribunda clone 821 and the scab-resistant selections/cultivars bulk, but not from the scab-susceptible recurrent parent bulk. Co-segregation analysis using a segregating apple progeny and polymorphism analysis of individual scab-resistant Coop selections/cultivars confirmed that this marker was linked to the scab-resistance gene V_f with a recombination frequency of 4.3%. OPK16/1300 was then cloned and sequenced. Sequence-specific primers of 25 oligonucleotides based on the marker were synthesized, and used in turn to screen M. floribunda clone 821, scab-susceptible apple cultivars, scab-resistant apple cultivars, and scab-resistant Coop selections. A pair of sequence-specific primers of clone OPK16/1300 amplified a distinct single band of the same size as the RAPD clone. Thus, a sequence characterized amplified region (SCAR) marker was developed which can be used to identify polymorphisms of OPK16/1300 based on the presence or absence of a single band. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetics and histology of powdery mildew resistance in apple

Summary Eight scab-resistant cultivars and selections along with eight commercial apple cultivars were evaluated for powdery mildew resistance in greenhouse and nursery tests. Dayton, Liberty, Delicious and Tolman Sweet were rated moderately resistant to infection in both greenhouse and nursery tests. Segregation of seedlings among 14 progenies for mildew reaction indicated that mildew resistance is polygenically controlled in this material with additive gene effects. Recovery of mildew resistant seedlings from crosses involving a scab-resistant parent(s) suggested that this material can be useful in developing scab- and mildew-resistant apple cultivars. Histological investigations were conducted to describe mildew symptoms of infected leaves.     

Inheritance of peroxidase isozymes in mulberry (Morus spp.)

Summary Electrophoretic variants of peroxidase in mulberry (Morus spp.) were demonstrated by thin-layer gel isoelectric focusing. Of these variants, three isozyme band groups were found to be controlled by codominant alleles at a single locus. The gene symbol Px ₁ was given to this locus, with alleles Px ₁ ¹ and Px ₁ ² assigned to the A6-A7-A8 and A7-A8-A9 band groups, respectively. The A6-A7-A8-A9 band group proved to be controlled by the Px ₁ ¹ and Px ₁ ² heterozygote.Additional experiments showed that among the three banding types, there were no statistically significant differences in leaf blade length, leaf blade width, length-width ratio of leaf blade, internode length, phyllotaxis, leaf shape, tree vigor and resistance to powdery mildew, but there were significant differences in leafstalk length.     

Pollination with gamma-irradiated pollen and development of fruits,seeds and parthenogenetic plants in apple

Summary Four apple (Malus X domestica) genotypes, Erovan, Golden Delicious, R1-49 and X6677, were pollinated with a marked pollen irradiated by -rays at doses ranging from 125 to 1000 Gy. Pollination with such irradiated pollen affected fruit set, seed number and seed contents, and induced the formation of parthenocarpic fruits and the development of parthenogenetic embryos. The immature embryos extracted from seeds. 2 and 3 months after pollination, were cultured in vitro and germinated after 2 months of cold treatment (3°C). Haploid plants were obtained in all 4 genotypes, after pollination with pollen irradiated at doses from 200 to 500 Gy. The optimum conditions for induction of apple haploids, by irradiated pollen approach, have been established.