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.     

Characterization and mapping of non-S gametophytic self-compatibility in sweet cherry (Prunus avium L.)

Self-incompatibility in Prunus (Rosaceae) species, such as sweet cherry, is controlled by a multiallelic locus (S), in which two tightly linked genes, S-RNase and SFB (S haplotype-specific F-box), determine the specificity of the pollen and the style. Fertilization in these species occurs only if the S-specificities expressed in the pollen and the pistils are different. However, modifier genes have been proposed to be necessary for a full manifestation of the self-incompatibility response. 'Cristobalina' is a spontaneous self-compatible sweet cherry cultivar that originated in Eastern Spain. Previous studies with this genotype suggested that pollen modifier gene(s), not linked to the S-locus, may be the cause of self-incompatibility breakdown. In this work, an F(1) population from 'Cristobalina' that segregates for this trait was used to identify molecular markers linked to self-compatibility by bulked segregant analysis. One simple sequence repeat (SSR) locus (EMPaS02) was found to be linked to self-compatibility in this population at 3.2?cM. Two additional populations derived from 'Cristobalina' were used to confirm the linkage of this marker to self-compatibility. Since EMPaS02 has been mapped to the sweet cherry linkage group 3, other markers located on the same linkage group were analysed in these populations to confirm the location of the self-compatibility locus.     

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.     

The effect of temperature on stigmatic receptivity in sweet cherry (Prunus avium L.)

Plant reproduction is highly vulnerable to environmental conditions such as temperature and, consequently, planet warming may have significant consequences on the reproductive phase with serious implication in agricultural crops. Although pollen tube growth is clearly affected by temperature, little information is available on its effect on the female side and on flower receptivity. In this work, the effect of temperature has been evaluated on stigmatic receptivity of sweet cherry in vivo, in the laboratory, and in planta, in the field. Results herein show that temperature has a clear effect on the duration of stigmatic receptivity. Thus, whereas high temperature reduced stigmatic receptivity, low temperature enlarged it. The stigma lost the capacity to offer support first for pollen penetration, second for pollen germination and, finally, for pollen adhesion. The effect of temperature was more pronounced on pollen germination and penetration than on pollen adhesion. High temperature reduced the germination capacity of the pollen as early as the first day after anthesis, a time when no apparent signs of stigma degeneration are apparent. This clear effect of temperature on stigmatic receptivity and pollen performance may have clear implication in crop performance and in establishing screening criteria of best‐adapted genotypes.     

Comparative analysis of the within-population genetic structure in wild cherry (Prunus avium L.) at the self-incompatibility locus and nuclear microsatellites

Gametophytic self-incompatibility (SI) systems in plants exhibit high polymorphism at the SI controlling S-locus because individuals with rare alleles have a higher probability to successfully pollinate other plants than individuals with more frequent alleles. This process, referred to as frequency-dependent selection, is expected to shape number, frequency distribution, and spatial distribution of self-incompatibility alleles in natural populations. We investigated the genetic diversity and the spatial genetic structure within a Prunus avium population at two contrasting gene loci: nuclear microsatellites and the S-locus. The S-locus revealed a higher diversity (15 alleles) than the eight microsatellites (4-12 alleles). Although the frequency distribution of S-alleles differed significantly from the expected equal distribution, the S-locus showed a higher evenness than the microsatellites (Shannon's evenness index for the S-locus: E = 0.91; for the microsatellites: E = 0.48-0.83). Also, highly significant deviations from neutrality were found for the S-locus whereas only minor deviations were found for two of eight microsatellites. A comparison of the frequency distribution of S-alleles in three age-cohorts revealed no significant differences, suggesting that different levels of selection acting on the S-locus or on S-linked sites might also affect the distribution and dynamics of S-alleles. Autocorrelation analysis revealed a weak but significant spatial genetic structure for the multilocus average of the microsatellites and for the S-locus, but could not ascertain differences in the extent of spatial genetic structure between these locus types. An indirect estimate of gene dispersal, which was obtained to explain this spatial genetic pattern, indicated high levels of gene dispersal within our population (sigma(g) = 106 m). This high gene dispersal, which may be partly due to the self-incompatibility system itself, aids the effective gene flow of the microsatellites, thereby decreasing the contrast between the neutral microsatellites and the S-locus.     

Pollen performance as affected by the pistilar genotype in sweet cherry (Prunus avium L.)

Summary Differences in pollen performance in higher plants can result in significant selective advantages for some particular genotypes leading to both gametophytic and sexual selection. However, the possibility of selection among male gametophytes has been questioned since natural selection could lead to the fixation of alleles for the best competing male genotypes. These two apparently conflicting hypotheses could be reconciled if pollen performance, rather than operating in absolute terms, could be modulated by the pistilar genotype. Thus, pollen performance in vivo and in vitro has been compared in four sweet cherry (Primus avium L.) cultivars. Differences among the cultivars studied have been recorded in the speed and final pollen germination percentages both in vivo and in vitro. The results obtained show that the female genotype also modulates the final result of pollen performance. These two factors are not merely additive but, on the contrary, the interaction between them affects pollen behavior in vivo. This fact has clear implications for gametophytic and sexual selection since the best male-female combinations can be favored and this could explain the variability observed for pollen performance in nature.     

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.     

Somatic embryogenesis in wild cherry (Prunus avium)

Indirect somatic embryogenesis was obtained inPrunus avium L. from either somatic or zygotic embryos. An embryogenic line was established by reinduction of embryogenic calluses from somatic embryos. The line was maintained for more than 3 years through 6 generations of embryogenic cultures. In the last 2 generations, more than 50% of the explants were embryogenic. Embryos at different stages of development were produced. Among cotyledonary-stage embryos, 50% had two cotyledons and a distinct hypocotyl, 43% had one or more than 2 cotyledons and 7% had fused cotyledons. Most of the embryos were translucent and conversion into plantlets was very rare. Secondary embryos could be observed to occur with low frequency from cultured somatic embryos and from embryos emerging from calluses. Indirect somatic embryogenesis was also induced from immature zygotic embryos. From one donor tree, 51% of the explants were embryogenic when cultured on a medium containing 0.9 μM kinetin, 0.9 μM BA and 0.5 μM NAA. This revised version was published online in June 2006 with corrections to the Cover Date.     

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).     

Investigation of tissue structure and xylem anatomy of eight rootstocks of sweet cherry (Prunus avium L.)

Reduction of crop yield from high water stress is a serious agricultural problem worldwide, which is expected to intensify under future global change scenarios. Efforts are underway worldwide to improve crops resistance to drought. One possible way is the careful choice of rootstock-scion combination. It is well known that water stress symptoms of grafted cherry trees to a large extent depend on the rootstock variety. Scions grafted on dwarf rootstocks show more serious stress symptoms, so it is reasonable to hypothesize that these symptoms and the tissue structure of the rootstock stem are connected. This paper examines this connection for 1-year-old seedlings of eight cherry rootstocks with different degree of vigor. Tissue structure was observed via scanning electron microscope images. Investigations were focused on number, size, and distribution of vessels, because they are principally responsible for water transport. Stem porosity (i.e., the total vessel area relative to the stem area, in cross-sectional view) of the dwarfing rootstocks was found to be significantly smaller than that of the others, supporting the notion that stem anatomical characteristics might contribute to water stress sensitivity.     

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.     

In vitro plant regeneration from leaves and internode sections of sweet cherry cultivars (Prunus avium L.)

Regeneration of adventitious shoots from leaves and, for the first time, from internode sections were compared and optimized for five economically important sweet cherry cultivars, i.e. Schneiders, Sweetheart, Starking Hardy Giant, Kordia and Regina (Prunus avium L.). The influence of basal media, carbon source, combination and dosage of phytohormones, ethylene inhibitor such as silver thiosulfate and a 16 h:8 h light:dark photoperiod versus complete darkness were evaluated. Both, DKW/WPM (1:1) and Quoirin/Lepoivre (QL) basal media stimulated organogenesis more than QL/WPM (1:1), Chee and Pool (CP), Murashige Skoog (MS), Driver and Kuniyuki (DKW) or woody plant (WPM) media did. An induction phase in darkness resulted in lower or zero regeneration rates. The best regeneration efficiencies were generally obtained with thidiazuron in combination with indole-3-butyric-acid. The addition of silver thiosulfate resulted in a similar or reduced regeneration efficiency. Significant genotypic variability in adventitious bud formation was evident for both explant sources, leaf and internode section. Adventitious shoots were obtained from 11% of leaf explants and 50% of internode sections indicating that shoot regeneration from internodes was significantly more efficient than from leaves.     

Characterization of novel microsatellites and development of multiplex PCR for large‐scale population studies in wild cherry,Prunus avium

Primers were developed for 14 microsatellite or simple sequence repeat (SSR) loci identified from a Prunus avium‘Charger’ genomic DNA library. In a survey of 16 wild cherry accessions 10 of the loci revealed polymorphisms of between two and six alleles. The remaining loci were found to be monomorphic. Seven polymorphic loci identified in this study and four polymorphic loci previously reported in sweet cherry were mapped and found to be unlinked. Two multiplex polymerase chain reactions (PCR) were optimized to enable the characterization of all 11 unlinked, polymorphic SSR loci.     

Promotion by phloroglucinol of adventitious root formation in micropropagated shoots of adult wild cherry (Prunus avium L.)

Micropropagated shoots of wild cherry (Prunus avium L.) produced roots in auxin-free medium. Phloroglucinol (PG) increased the proportion of shoots that rooted, while phloretic acid reduced this response in medium with or without PG, and cancelled the promotive effect of PG. Concentration of PG also significantly affected rooting in media with and without auxin. The proportion of shoots rooting in media containing auxin, or auxin plus PG, increased with the number of successive subculture, but the proportion that rooted with PG alone was unaffected by the number of subcultures. Before the shoots had become responsive to auxin, 1 mM PG was more effective than auxin in inducing root formation.     

Spatial genetic structure in wild cherry (Prunus avium L.): I. variation among natural populations of different density

Conservation of forest genetic resources requires intensive knowledge of the spatial arrangement of genetic diversity. In this study, we used four natural Prunus avium stands in Germany with contrasting for densities to understand patterns of spatial genetic structure. To this end, we genotyped adults and saplings at eight microsatellite markers, 54 AFLP loci and at the gametophytic incompatibility locus. We estimated levels of clonal propagation, spatial genetic structure and gene dispersal. High mortality occurred among young clonal individuals, as depicted by the lower clonal diversity in saplings. Contrasting levels of spatial genetic structure were observed among markers, ontogenic stages and populations. AFLP were more efficient for detecting spatial autocorrelation but did not allow us to differentiate low and high density populations, while high density populations showed substantially stronger spatial genetic structure at microsatellite loci. Furthermore, kinship decreased with tree age only in low density stands. We discuss the present results in terms of population history, pollen and seed dispersal and population density. Although conspecific density seems to be an interesting indicator of genetic diversity for conservation programmes, we still need to disentangle the relative influence of clonal propagation and density on the strength of spatial genetic structure. Simulation studies are needed to further address this question.