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.     

Dormancy in peach (Prunus persica L.) flower buds

Morphological studies were carried out with peach flower buds collected monthly in 1989 and 1990, from two months before leaf fall (7 March) until two to three weeks before bloom (7/8 August). Chilled (2–4°C for 30 days) and unchilled buds were exposed to 20 to 25°C, 100% RH and continuous light. Gibberellin A₃ (3 ng or 30 ng) was applied to some of the non-chilled cuttings at three days intervals. Then, 12, 19, and 26 days after they were planted, the buds were sampled and processed for histological studies. Cultured flower buds (chilled or unchilled) had accelerated anther and gynoecium morphogenesis after 12 days under controlled conditions, compared to buds processed immediately after collection from the field. Chilling treatment augmented the bud culture effect, while Gibberellin A₃ applications to the excised buds retarded bud morphogenesis to a stage comparable to that of buds collected directly from the field. This, suggests that the comparatively high levels of Gibberellin A_1/3 we previously found in mid winter [15, 18] could be at least one of the factors that controls floral bud dormancy by retarding anther and gynoecium development.     

Dormancy in peach (Prunus persica L.) flower buds

Summary Peach buds (floral and vegetative) were periodically collected from midsummer until the spring flowering and sprouted under continuous light, 100% relative humidity and 20–25°C. Treatments with 200 ppm gibberellin A₃ (GA₃) or chilling (2–4°C for 30 days before planting) were applied. Vegetative buds showed well-defined phenological stages: pre-dormancy, true dormancy, and end of dormancy. Both GA₃ and chilling treatments shortened the sprouting times of vegetative dormant buds close to those in predormancy. Isolated floral buds were irresponsive under all conditions and did not sprout even with the GA₃ or chilling treatments. In a comparative study with buds immediately after collection anatomical analysis demonstrated that vegetative buds were almost completely developed by midsummer/early automn and remained in a resting state until the end of winter. Floral buds developed continuously over the same period. Both types of verticils began to differentiate in midsummer. Sepals and petals developed mainly in late summer, androecious floral parts developed throughout the resting period, while gynoecious floral parts showed differentiation in late winter. The flower was completely formed a few days prior to blossoming. Thus, in isolated peach buds fertile verticils are not sufficiently developed during the resting time to allow sprouting.     

桃品种'八月香'花粉的超低温保存

桃品种‘八月香’的花粉经4℃硅胶干燥8h或(20±1)℃下玻璃化液PVS4(35%甘油 20%乙二醇 0.6mol·L-1蔗糖)处理60min后直接投入液氮保存,解冻后的萌发率都可以达到70%以上,萌发率与保存时间长短无关。     

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.     

Agrobacterium-mediated transformation of apricot (Prunus armeniaca L.) leaf explants

A protocol for Agrobacterium-mediated stable transformation for scored, whole leaf explants of the apricot (Prunus armeniaca) cultivar Helena was developed. Regenerated shoots were selected using a two-step increased concentrations of paromomycin sulphate. Different factors affecting survival of transformed buds, including possible toxicity of green fluorescent protein (GFP) and time of exposure to high cytokine concentration in the regeneration medium, were examined. Transformation efficiency, based on PCR analysis of individual putative transformed shoots from independent lines was 5.6%, when optimal conditions for bud survival were provided. Southern blot analysis on four randomly chosen PCR-positive shoots confirmed the presence of the nptII transgene. This is the first time that stable transformation of an apricot cultivar is reported and constitutes also one of the few reports on the transformation of Prunus cultivars.     

Genomic and cDNA microsatellites from apricot (Prunus armeniaca L.)

We developed primers for the amplification of 24 polymorphic nuclear microsatellites in apricot (Prunus armeniaca L.). Thirteen loci originated from three genomic libraries enriched for TC, TG and AAG motifs. Eight loci were developed from three fruit EST (Expressed‐Sequence‐Tag) libraries and three from a leaf cDNA microsatellite‐enriched library. There were up to nine alleles per polymorphic locus in 12 different cultivars. No difference in allele numbers were shown between cDNA and genomic‐source loci. Mean expected heterozygosity was 0.65 (range: 0.15–0.87). Mendelian segregation was confirmed for all loci. These markers should be helpful for diversity studies, genome mapping and cultivar identification in apricot and related species.     

Non-TIR-NBS-LRR resistance gene analogs in apricot (Prunus armeniaca L.)

Genes encoding for proteins with nucleotide-binding site and leucine-rich repeat motifs (NBS-LRR) have been suggested to play a general role in plant defence mechanism. In Prunus species, many TIR (Toll / Interleukin-1 Receptor), and only very few non-TIR sequences were identified, which was explained either by the unequal distribution of TIR/non-TIR sequences in the Prunus genome or by the incapability of primers in the amplification of non-TIR RGAs. The objective of this work was to check whether a new semi-nested PCR strategy can be developed for the targeted isolation of non-TIR-NBS-LRR Resistance Gene Analog (RGA) sequences from apricot. Three primers (CUB-P-loop F, CUB-Kin2 F and CUB-HD R) were designed, from which CUB-Kin2 F and CUB-HD R were constructed to anneal selectively to the non-TIR sequences. A colony Polymerase Chain Reaction (PCR) indicated that out of the 96 clones tested 28 showed amplification using the newly developed primers, while no amplification occurred when using the formerly described primers. Half of the 28 positive clones were sequenced and they turned out to represent 11 different non-TIR RGA sequences. A phylogenetic analysis was carried out based on an alignment containing 293 Rosaceae and 21 non-Rosaceaa sequences. A significantly higher ratio (91%) of non-TIR sequences were arranged in multi-genera clades than that of (57%) the TIR groups confirming that non-TIR sequences might be of more ancient origin than TIR sequences.     

Effective pollination period in apricot (Prunus armeniaca L.) varieties

A study of the effective pollination period (E.P.P.) of six varieties of apricot has shown that in the warm climate of the South Mediterranean this parameter is extremely short. The percentage of fruit set of emasculated flowers diminishes rapidly with time. Temperature plays an important role in this process and is responsible for the rapid degradation of the stigma, which hinders pollen germination.     

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).     

Self-compatibility and incompatibility in tetraploid sour cherry (Prunus cerasus L.)

. Gametophytic self-incompatibility (GSI) typically "breaks down" due to polyploidy in many Solanaceous species, resulting in self-compatible (SC) tetraploid individuals. However, sour cherry (Prunus cerasus L.), a tetraploid species resulting from hybridization of the diploid sweet cherry (P. avium L.) and the tetraploid ground cherry (P. fruticosa Pall.), is an exception, consisting of both self-incompatible (SI) and SC individuals. Since sweet cherry exhibits GSI with 13 S-ribonucleases (S-RNases) identified as the stylar S-locus product, the objectives were to compare sweet and sour cherry S-allele function, S-RNase sequences and linkage map location as initial steps towards understanding the genetic basis of SI and SC in sour cherry. S-RNases from two sour cherry cultivars that were the parents of a linkage mapping population were cloned and sequenced. The sequences of two S-RNases were identical to those of sweet cherry S-RNases, whereas three other S-RNases had unique sequences. One of the S-RNases mapped to the Prunus linkage group 6, similar to its location in sweet cherry and almond, whereas two other S-RNases were linked to each other but were unlinked to any other markers. Interspecific crosses between sweet and sour cherry demonstrated that GSI exists in sour cherry and that the recognition of common S-alleles has been maintained in spite of polyploidization. It is hypothesized that self-compatibility in sour cherry is caused by the existence of non-functional S-RNases and pollen S-genes that may have arisen from natural mutations.     

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.     

Antioxidant properties of prunes (Prunus domestica L.) and their constituents

Prunes contain large amounts of phenolics and show high antioxidant activity. The aim of this study is to clarify the contents of caffeoylquinic acid (CQA) isomers, and to estimate the contribution of these isomers to the antioxidant activity of prunes. Furthermore, structural elucidation and evaluation of antioxidant activity of prune components were also performed. CQA isomers in prunes were quantified by HPLC analysis, and it has become apparent that prunes contain relatively high amount of 4-O-caffeoylquinic acid. The contribution of CQA isomers to the antioxidant activity of prunes was revealed to be 28.4% on the basis of oxygen radical absorbance capacity (ORAC); hence, it was indicated that residual ORAC is dependent on unknown antioxidant components. Total 28 compounds were isolated and their structures were elucidated by NMR and MS analyses. Four abscisic acid related compounds, a chromanon, and a bipyrrole were novel. Each CQA isomer in prunes showed high antioxidant activities when measured by the oil stability index (OSI) method, O2- scavenging activity, and ORAC. Other isolated compounds such as hydroxycinnamic acids, benzoic acids, coumarins, lignans, and flavonoid showed high ORAC values. Furthermore, a novel chromanon indicated a remarkable synergistic effect on ORAC of CQA isomers.     

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.     

甜樱桃(Prunus avium L.)品种S基因型鉴定

根据蔷薇科S-RNase基因(S基因)高度保守区C2和RC4区设计一对特异引物PruC2和PruC4R,对甜樱桃品种的基因组DNA进行S基因特异PCR扩增。克隆S基因的扩增片段,核酸序列在GenBank上搜索,确定了4种S基因的核酸序列和大小。结果表明,在琼脂糖凝胶上位置相同的扩增带其核酸序列相同,是同一种S基因。4种S基因扩增片段的大小分别是：S1为677bp,S3为762bp,S4为945bp,S6为456bp。参试的自交不亲和品种的S基因型分别是：红灯、红艳、早红宝石和先锋相同,为S1S3;抉择、红丰和那翁相同,为S3S4;大紫为S1S6;长把红为S1S4;养老为S2S6;自交亲和品种外引7号和斯太拉为S3S4。     

The effects of external magnesium concentration on the growth and magnesium inflow rates of micropropagated cherry rootstocks 'F.12/1' (Prunus avium L.) and 'Colt' (Prunus avium L. × Prunus pseudocerasus L.)

The cherry rootstock 'F.12/1' is more susceptible to Mg deficiency than the cherry rootstock 'Colt'. The effects of different external concentrations (3000, 500, 50 and 10 µM) of Mg on the growth of micropropagated plants of 'F.12/1' and 'Colt' were investigated in a flowing solution culture system. The relative growth rates (RGR) and total dry weight of both cultivars decreased similarly with the reduction in the external concentrations of Mg. The decreases were caused by a lower net assimilation rate (unit leaf rate). 'F.12/1' had a greater RGR than 'Colt' at all external concentrations of Mg and this is ascribed to its greater leaf weight ratio (leafiness). 'Colt' partitions more dry matter to roots than 'F.12/1', resulting in a smaller shoot: root dry weight ratio. 'F.12/1' required a greater inflow rate of Mg than 'Colt' to maintain its maximum growth rate. When the external concentration of Mg fell below 500 µM the concentration of Mg in the leaves of 'F.12/1' fell well below the critical concentration whereas for 'Colt' this did not occur until the concentration fell below 50 µM.     

Strains of Prunus necrotic ringspot virus in hop (Humulus lupulus L.)

Purified preparations of Prunus necrotic ringspot virus (NRSV) from hop plants formed two light-scattering zones when centrifuged in sucrose density gradients; the upper and lower zones contained particles 25 mμ and 31 mμ in diameter respectively whose sedimentation coefficients were 79 S and 107 S. NSRV isolates from hop were of two distinct serological types: ‘A’ strains, serologically very closely related to NRSV isolates from apple; and ‘C’ strains more nearly related to NRSV from cherry. The variety Fuggle is tolerant to hop mosaic (not related to NRSV) and different selections of apparently healthy female plants usually contained A strains; but C strains were usually isolated from nettlehead-diseased plants. Either A or C strains occurred in male plants grown with the hop-mosaic tolerant varieties. In mosaic-sensitive varieties (Goldings and Bramlings) apparently healthy female plants tested were usually infected with C strains; either A or C types occurred in mosaic-sensitive male plants. NRSV was not detected in the seventy-four hop seedlings obtained from virus-infected plants. Some varieties developed nettlehead when infected with NRSV (A) or (C) + the hop form of arabis mosaic virus, but not with NRSV (A) or (C) alone. Others developed nettlehead when infected with arabis mosaic virus + NRSV (C) but not with arabis mosaic + NRSV (A). A and C strains can multiply together in the same hop plant. There is evidence of partial antagonism, however, and the fluctuating behaviour of the nettlehead syndrome probably reflects changes in the relative concentration of the two serotypes.