Plant regeneration from root callus protoplasts of sour cherry (Prunus cerasus L.)

Summary Callus protoplasts of sour cherry clone CAB4D entered sustained division in Murashige and Skoog's (1962) medium with 1-naphthaleneacetic acid, 6-beneylaminopurine and zeatin. Further to callusing, organogenesis was induced from the protoplastderived callus, in a basal regeneration medium with these same growth regulators at 0.01 mg/l, 2,0 mg/l and 0.05 mg/l, respectively. The regeneration pathway, from such callus, could be altered by adding different organic compounds to this medium. Casein hydrolysate, added alone, promoted rhizogenesis, with shoot buds regenerated from the protoplast-derived roots, while in a basal regeneration medium with casein hydrolysate and a group B vitamin mixture direct caulogenesis occurred.Abbreviations BAP 6-benzylaminopurine - BR basal regeneration medium - CEH casein enzymatic hydrolysate - FPE final plating efficiency - fwt fresh weight - IPE initial plating efficiency - MES 2-N-morpholinoethane sulfonic acid - MPE intermediate plating efficiency - MS Murashige and Skoog (1962) - NAA 1-naphthaleneacetic acid     

Genetic and molecular characterization of three novel S-haplotypes in sour cherry (Prunus cerasus L.)

Tetraploid sour cherry (Prunus cerasus L.) exhibits gametophytic self-incompatibility (GSI) whereby the specificity of self-pollen rejection is controlled by alleles of the stylar and pollen specificity genes, S-RNase and SFB (S haplotype-specific F-box protein gene), respectively. As sour cherry selections can be either self-compatible (SC) or self-incompatible (SI), polyploidy per se does not result in SC. Instead the genotype-dependent loss of SI in sour cherry is due to the accumulation of non-functional S-haplotypes. The presence of two or more non-functional S-haplotypes within sour cherry 2x pollen renders that pollen SC. Two new S-haplotypes from sour cherry, S(33) and S(34), that are presumed to be contributed by the P. fruticosa species parent, the complete S-RNase and SFB sequences of a third S-haplotype, S(35), plus the presence of two previously identified sweet cherry S-haplotypes, S(14) and S(16) are described here. Genetic segregation data demonstrated that the S(16)-, S(33)-, S(34)-, and S(35)-haplotypes present in sour cherry are fully functional. This result is consistent with our previous finding that 'hetero-allelic' pollen is incompatible in sour cherry. Phylogenetic analyses of the SFB and S-RNase sequences from available Prunus species reveal that the relationships among S-haplotypes show no correspondence to known organismal relationships at any taxonomic level within Prunus, indicating that polymorphisms at the S-locus have been maintained throughout the evolution of the genus. Furthermore, the phylogenetic relationships among SFB sequences are generally incongruent with those among S-RNase sequences for the same S-haplotypes. Hypotheses compatible with these results are discussed.     

An alternative approach to plant regeneration from protoplasts of sour cherry (Prunus cerasus L.)

Mesophyll protoplasts, of sour cherry (Prunus cerasus), clones CAB 4D, CAB 5H and CAB 11E, gave differential cultural responses. Protoplasts in media based on Murashige and Skoog (MS) salts, supplemented with 9% (w/v) mannitol plus growth regulators underwent cell wall regeneration, cell colony and callus formation. Zeatin (Z) was needed in order to induce cell division for all three sour cherry clones. The protoplast-derived calli, of clones CAB 4D and CAB 5H, underwent rhizogenesis as an intermediate step towards shoot bud differentiation. Clone CAB 5H also gave direct shoot bud regeneration from the protoplast callus.     

Identification of incompatibility alleles in the tetraploid species sour cherry

The incompatibility genetics of sour cherry (Prunus cerasus), an allotetraploid species thought to be derived from sweet cherry (diploid) and ground cherry (tetraploid), were investigated by test crossing and by analysis of stylar ribonucleases which are known to be the products of incompatibility alleles in sweet cherry. Stylar extracts of 36 accessions of sour cherry were separated electrophoretically and stained for ribonuclease activity. The zymograms of most accessions showed three bands, some two or four. Of the ten bands seen, six co-migrated with bands that in sweet cherry are attributed to the incompatibility alleles S ₁ , S ₃ , S ₄ , S _6, S ₉ and S ₁₃ . aanski Rubin, Erdi Botermo B, Koro and Ujfehertoi Furto, which showed bands apparently corresponding to S ₁ and S ₄ , were test pollinated with the sweet cherry Merton Late (S ₁ S ₄ ). Monitoring pollen tube growth, and, in one case, fruit set, showed that these crosses were incompatible and that the four sour cherries indeed have the alleles S ₁ and S ₄ . Likewise, test pollination of Marasca Piemonte, Marasca Savena and Morello, Dutch with Noble (S ₆ S ₁₃ ) showed that these three sour cherries have the alleles S ₆ and S ₁₃ . S ₁₃ was very frequent in sour cherry cultivars, but is rare in sweet cherry cultivars, whereas with S ₃ the situation is reversed. It was suggested that the other four bands are derived from ground cherry and one of these, provisionally attributed to S _B , occurred frequently in a small set of ground cherry accessions surveyed. Analysing some progenies from sour by sweet crosses by S allele-specific PCR and monitoring the success of some sweet by sour crosses were informative. They indicated mostly disomic inheritance, with sweet cherry S alleles belonging to one locus and, presumably, the ground cherry alleles to the other, and helped clarify the genomic arrangement of the alleles and the interactions in heteroallelic pollen.Communicated by H.F. Linskens     

Inheritance and interactions of incompatibility alleles in the tetraploid sour cherry

Three progenies of sour cherry (Prunus cerasus) were analysed to correlate self-(in)compatibility status with S-RNase phenotype in this allotetraploid hybrid of sweet and ground cherry. Self-(in)compatibility was assessed in the field and by monitoring pollen tube growth after selfing. The S-RNase phenotypes were determined by isoelectric focusing of stylar proteins and staining for RNase activity and, for the parents, confirmed by PCR. Seedling phenotypes were generally consistent with disomic segregation of S-RNase alleles. The genetic arrangements of the parents were deduced to be ‘Köröser’ (self-incompatible) S ₁ S ₄ .S _B S _D , ‘Schattenmorelle’ (self-compatible) S ₆ S ₁₃ .S _B S _B , and clone 43.87 (self-compatible) S ₄ S ₁₃ .S _B S _B , where “.” separates the two homoeologous genomes. The presence of S ₄ and S ₆ alleles at the same locus led to self-incompatibility, whereas S ₁₃ and S _B at homoeologous loci led to self-compatibility. The failure of certain heteroallelic genotypes in the three crosses or in the self-incompatible seedlings indicates that S ₄ and S ₆ are dominant to S _B . However, the success of S ₁₃ S _B pollen on styles expressing corresponding S-RNases indicates competitive interaction or lack of pollen-S components. In general, the universal compatibility of S ₁₃ S _B pollen may explain the frequent occurrence of S ₁₃ and S _B together in sour cherry cultivars. Alleles S _B and S _D , that are presumed to derive from ground cherry, and S ₁₃ , presumably from sweet cherry, were sequenced. Our findings contribute to an understanding of inheritance of self-(in)compatibility, facilitate screening of progenies for self-compatibility and provide a basis for studying molecular interactions in heteroallelic pollen.     

Cardioprotective mechanisms of Prunus cerasus (sour cherry) seed extract against ischemia-reperfusion-induced damage in isolated rat hearts

The effects of kernel extract obtained from sour cherry (Prunus cerasus) seed on the postischemic cardiac recovery were studied in isolated working rat hearts. Rats were treated with various daily doses of the extract for 14 days, and hearts were then isolated and subjected to 30 min of global ischemia followed by 120 min of reperfusion. The incidence of ventricular fibrillation (VF) and tachycardia (VT) fell from their control values of 92% and 100% to 50% (not significant) and 58% (not significant), 17% (P<0.05), and 25% (P<0.05) with the doses of 10 mg/kg and 30 mg/kg of the extract, respectively. Lower concentrations of the extract (1 and 5 mg/kg) failed to significantly reduce the incidence of VF and VT during reperfusion. Sour cherry seed kernel extract (10 and 30 mg/kg) significantly improved the postischemic recovery of cardiac function (coronary flow, aortic flow, and left ventricular developed pressure) during reperfusion. We have also demonstrated that the extract-induced protection in cardiac function significantly reflected in a reduction of infarct size. Immunohistochemistry indicates that a reduction in caspase-3 activity and apoptotic cells by the extract, beside other potential action mechanisms of proanthocyanidin, trans-resveratrol, and flavonoid components of the extract, could be responsible for the cardioprotection in ischemic-reperfused myocardium.     

Evaluation of the glycoside hydrolase activity of a Brettanomyces strain on glycosides from sour cherry (Prunus cerasus L.) used in the production of special fruit beers

The glycoside hydrolase activity of Saccharomyces cerevisiae and Brettanomyces custersii was examined on sour cherry (Prunus cerasus L.) glycosides with bound volatile compounds. Refermentations by the beta-glucosidase-negative S. cerevisiae strains LD25 and LD40 of sour cherry juice-supplemented beer demonstrated only a moderate increase of volatiles. In contrast, the beta-glucosidase-positive B. custersii strain LD72 showed a more pronounced activity towards glycosides with aliphatic alcohols, aromatic compounds and terpenoid alcohols. Important contributors to sour cherry aroma such as benzaldehyde, linalool and eugenol were released during refermentation as shown by analytical tools. A gradually increasing release was observed during refermentations by B. custersii when whole sour cherries, sour cherry pulp or juice were supplemented in the beer. Refermentations with whole sour cherries and with sour cherry stones demonstrated an increased formation of benzyl compounds. Thus, amygdalin was partially hydrolysed, and a large part of the benzaldehyde formed was mainly reduced to benzyl alcohol and some further esterified to benzyl acetate. These findings demonstrate the importance and interesting role of certain Brettanomyces species in the production of fruit lambic beers such as 'Kriek'.     

Genetic relationships between diploid and allotetraploid cherry species (Prunus avium, Prunus x gondouinii and Prunus cerasus)

Prunus avium L. (diploid, AA, 2n=2x=16), Prunus cerasus L. (allotetraploid, AAFF, 2n=4x=32) species, and their hybrid Prunus x gondouinii Rehd., constitute the most widely cultivated cherry tree species. P. cerasus is supposed to be an hybrid species produced by the union of unreduced P. avium gametes and normal P. fruticosa gametes. A continuum of morphological traits between these three species makes their assignation difficult. The aim of this paper is to study the genetic relationships between tetraploid and diploid cherry species. In all, 114 genotypes belonging to these species were analyzed using 75 AFLP markers. The coordinates of these genotypes on the first axis of a correspondence analysis allowed us to clearly distinguish each species, to identify misclassifications and to assign unknown genotypes to one species. We showed that there are specific alleles in P. cerasus, which are not present in the A genome of P. avium and which probably come from the F genome of P. cerasus. The frequencies of each marker in the A and the F genomes were estimated in order to identify A and F specific markers. We discuss the utility of these specific markers for finding the origin of the A and F genomes in the allopolyploid species.     

Phosphoenolpyruvate carboxykinase in cherry (Prunus avium L.) fruit during development

In this study the abundance and location of phosphoenolpyruvate carboxykinase (PEPCK) was determined in the flesh and skin of the sweet cherry (Prunus avium L.) cultivar Durone Nero II during development. PEPCK was not present in young fruit but appeared in both tissues as the fruit increased in size. In these there was no net dissimilation of malic acid, which accounts for the bulk of their organic acid contents when PEPCK was present. To assist in understanding the function of PEPCK, the abundance of a number of other enzymes was determined. These enzymes were aspartate aminotransferase (AspAT), glutamine synthetase (GS), phosphoenolpyruvate carboxylase (PEPC), pyruvate, orthophosphate dikinase (PPDK), and ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco). A potential role for PEPCK in the regulation of pH and the utilization of malate in gluconeogenesis in the flesh and skin of cherries is presented.     

Characterisation of novel S-alleles from cherry (Prunus avium L.)

In plant populations exhibiting gametophytic self-incompatibility, individuals harbouring rare S alleles are likely to have a reproductive advantage over individuals having more common alleles. Consequently, determination of the self-incompatibility haplotype of individuals is essential for genetic studies and the development of informed management strategies. This study characterises six new S alleles identified in wild cherry (Prunus avium L.). Investigations to determine the S genotype of individuals in recently planted woodland through length polymorphisms of introns associated with the stylar S-RNase gene and the pollen SFB gene revealed six S intron profiles which did not correspond to those of known S alleles. These are now attributed to S ₂₇ to S ₃₂ . Consensus primers, annealing in the S-RNase sequence coding for the signal peptide and C5 regions, were used to isolate the S-RNase alleles associated with the novel S intron profiles. The proteins corresponding to the new alleles were separated by isoelectric focusing from stylar extracts and their pI values determined. Similarities between the deduced amino acid sequence for the new alleles isolated and other cherry S-RNase sequences available on the databases ranged from 40% to 86%. Amplification products for SFB introns ranged from 172 to 208bp. New sequence regions exposed to positive selection were identified and the significance of the PS3 region reinforced. A phylogenetic relationship between P. avium S-RNases for S ₁₀ and S ₁₃ and between corresponding SFB alleles may indicate co-evolution of allele specificities of these two genes. The nucleotide sequences reported in this paper have been submitted to the EMBL/GenBank database under the following accession numbers: S ₂₇ (DQ266439), S ₂₈ (DQ266440), S ₂₉ (DQ266441), S ₃₀ (DQ266442), S ₃₁ (DQ266443), S ₃₂ (DQ266444).     

Metabolic profiling of ethephon-treated sweet cherry (Prunus avium L.)

Increasing costs and decreasing labor availability for sweet cherry harvest in Washington State, USA, has reinvigorated commercial and research interest of mechanized harvest. Ethephon (2-chloroethyl phosphonic acid) can be used to improve fruit abscission for mechanical harvest. Our previous work shows that 3.5 l ha⁻¹ ethephon enhances red color and reduces firmness of the cultivar ‘Bing’. In the current study, we used metabolic profiling of cultivars ‘Bing’, Chelan’, and ‘Skeena’ fruit meso and exocarp tissue to better understand underlying quality-related metabolism associated with ethephon application. Trees were treated using air-blast sprayer 13–14 days prior to harvest and fruit samples were harvested every 7–10 days starting at least 17 days prior to commercial harvest. Nearly 200 identified and partially characterized metabolites from mesocarp and exocarp tissue were characterized and evaluated. Principal component analysis models revealed changes in the metabolome associated with both natural ripening and ethephon-induced changes, including associations to key color, acid, and sugar components, such as cyanidin 3-glucoside, malic acid and sugar metabolism.     

Inheritance and linkage of isozymes in sweet cherry (Prunus avium L.)

Eight polymorphic isozyme loci, 6PGD, G6PD, MDH, PGM, SKDH, FDP, GOT and IDH, in sweet cherry where found to be in one linkage group, with a ninth isozyme locus, GPI, being in another linkage group on a different chromosome. Isozymes were also linked to the incompatibility S locus and this explained the disturbed segregation ratios observed in the first generation from controlled hybridisations between different sweet cherry cultivars. Analysis revealed close linkage between the isozyme and S loci. The results supported a pre-existing theory that the S gene in cherry consists of three linked segments each coding for a different function. Progeny derived from selfing of Stella, the self-fertile cherry cultivar, also showed disturbed segregation ratios and an absence of homozygotes for the isozyme loci assayed. This demonstrated that codominant inheritance of the S alleles had not been effected by the self-fertile mutation.     

Distribution and fine-scale spatial-genetic structure in British wild cherry (Prunus avium L.)

Insights into the within-population spatial-genetic structure (SGS) of forest tree species, where little is known regarding seed and pollen dispersal patterns, enhance understanding of their ecology and provide information of value in conservation and breeding. This study utilised 13 polymorphic simple sequence repeat loci to investigate the impact of asexual recruitment, management regime and tree size on the development of SGS in wild cherry (Prunus avium L). Only 246 genotypes were identified in the 551 trees sampled, reflecting significant levels of clonal reproduction in both managed and unmanaged populations. Naturally regenerated wild cherry was spatially aggregated under both management regimes. However, in the managed population, sexually derived trees accounted for a greater proportion of the smaller size classes, whereas vegetatively produced trees dominated the smaller size classes in the unmanaged population. High overall SGS values (Sp 0.030-Sp 0.045) were observed when considering only sexually derived genets and kinship coefficients were significant up to the 120 m distance class for both populations. The inclusion of clonal ramets in the analysis significantly increased the overall SGS (Sp 0.089-Sp 0.119) as well as kinship coefficients in the 40-80 m distance classes, illustrating the dramatic impact of vegetative propagation on SGS in this species. Increased spatial aggregation and regeneration appeared to be concomitant with increased SGS in the 40 m distance class in the unmanaged population. Neighbourhood size estimates were relatively small for both populations and kinship coefficients were found to decline with distance under both management regimes, suggesting that common mechanisms may restrict gene dispersal in wild cherry.     

Molecular characterisation of sweet cherry (Prunus avium L.) genotypes using peach [Prunus persica (L.) Batsch] SSR sequences

A total of 76 sweet cherry genotypes were screened with 34 microsatellite primer pairs previously developed in peach. Amplification of SSR loci was obtained for 24 of the microsatellite primer pairs, and 14 of them produced polymorphic amplification patterns. On the basis of polymorphism and quality of amplification, a set of nine primer pairs and the resulting 27 informative alleles were used to identify 72 genotype profiles. Of these, 68 correspond to unique cultivar genotypes, and the remaining four correspond to three cultivars that could not be differentiated from the two original genotypes of which they are mutants, and two very closely related cultivars. The mean number of alleles per locus was 3.7 while the mean heterozygosity over the nine polymorphic loci averaged 0.49. The results demonstrate the usefulness of cross-species transferability of microsatellite sequences allowing the discrimination of different genotypes of a fruit tree species with sequences developed in other species of the same genus. UPGMA cluster analysis of the similarity data divided the ancient genotypes studied into two fairly well-defined groups that reflect their geographic origin, one with genotypes originating in southern Europe and the other with the genotypes from northern Europe and North America.     

The draft chromosome-level genome assembly of tetraploid ground cherry (Prunus fruticosa Pall.) from long reads

Cherries are stone fruits and belong to the economically important plant family of Rosaceae with worldwide cultivation of different species. The ground cherry, Prunus fruticosa Pall., is an ancestor of cultivated sour cherry, an important tetraploid cherry species. Here, we present a long read chromosome-level draft genome assembly and related plastid sequences using the Oxford Nanopore Technology PromethION platform and R10.3 pore type. We generated a final consensus genome sequence of 366 Mb comprising eight chromosomes. The N50 scaffold was ~44 Mb with the longest chromosome being 66.5 Mb. The chloroplast and mitochondrial genomes were 158,217 bp and 383,281 bp long, which is in accordance with previously published plastid sequences. This is the first report of the genome of ground cherry (P. fruticosa) sequenced by long read technology only. The datasets obtained from this study provide a foundation for future breeding, molecular and evolutionary analysis in Prunus studies.     

Characterization of the S-RNase promoters from sweet cherry (Prunus avium L.)

Genomic DNA fragments containing the S(3)-, S(4)-, and S(6)-RNase genes were isolated from the sweet cherry (Prunus avium L.) and sequenced. Comparison of the 5'-flanking sequences of these three S-RNases indicated that a highly conserved region (designated CR) existed just upstream from the putative TATA boxes. We postulate that CR contains cis-regulatory element(s) involved in pistil expression. To examine the activity of the isolated S-RNase promoters of sweet cherry in the pistil, we transiently introduced approximately 650-bp fragments of the S(4)- and S(6)-RNase promoters fused to beta-glucuronidase (GUS) gene into the pistil of the petunia using a particle bombardment technique. Histochemical analysis showed that the 5'-flanking region of each S-RNase was active in the pistil. This suggests that cis-regulatory element(s) for pistil-specific expression may exist(s) within the 650-bp region upstream from the TATA box in the sweet cherry S-RNase promoter.