Estimation of genetic diversity in some Iranian wild Prunus subgenus Cerasus accessions using inter-simple sequence repeat (ISSR) markers

As Iran is one of the main origins of Prunus germplasm. In this study, ISSR markers were used for genetic diversity evaluation of 39 accessions of subgenus Cerasus belonging to six species i.e. Prunus avium L., Prunus cerasus L., Prunus mahaleb L., Prunus incana Pall., Prunus microcarpa Boiss., and Prunus brachypetala Boiss.. With 12 ISSR primers, 151 polymorphic bands were detected with polymorphism ratio range of 81.8%–100%. The lowest similarity (0.04) was found between P. avium and P. microcarpa genotypes and the mean of similarity between all genotypes was 0.28. Cluster analysis separated improved cultivars from wild accessions. Improved cherry cultivars and rootstocks were placed closer to the P. avium than the other species. The principal coordinate analysis (PCoA) supported the cluster analysis results. The wild accessions were separated according to their species and collection sites. ISSR markers are useful techniques for genetic diversity evaluation in Prunus subgenus Cerasus.     

Genetic diversity of some Iranian sweet cherry (<Emphasis Type="Italic">Prunus avium</Emphasis>) cultivars using microsatellite markers and morphological traits

The aim of this study was to characterize 23 important Iranian sweet cherry (Prunus avium) cultivars collected from different provinces of Iran and 1 foreign cultivar, which was used as control, considered for breeding programs by using 21 microsatellite markers and 27 morphological traits. In sweet cherry (Prunus avium) accessions, leaf, fruit, and stone morphological characters were evaluated during two consecutive years. The study revealed a high variability in the set of evaluated sweet cherry accessions. The majority of important correlations were determined among variables representing fruit and leaf size and variables related to color. Cluster analysis distinguished sweet cherry accessions into two distinct groups. Principal component analysis (PCA) of qualitative and quantitative morphological parameters explained over 86.59% of total variability in the first seven axes. In PCA, leaf traits such as leaf length and width, and fruit traits such as length, width, and weight, and fruit flesh and juice color were predominant in the first two components, indicating that they were useful for the assessment of sweet cherry germplasm characterization. Out of 21 SSR markers, 16 were polymorphic, producing 177 alleles that varied from 4 to 16 alleles (9.35 on average) with a mean heterozygosity value of 0.82 that produced successful amplifications and revealed DNA polymorphisms. Allele size varied from 95 to 290 bp. Cluster analyses showed that the studied sweet cherry genotypes were classified into five main groups based mainly on their species characteristics and SSR data. In general, our results did not show a clear structuring of genetic variability within the Iranian diffusion area of sweet cherry, so it was not possible to draw any indications on regions of provenance delimitation. The results of this study contribute to a better understanding of sweet cherry genetic variations in Iran, thus making for more efficient programs aimed at preserving biodiversity and more rational planning of the management of reproductive material.     

Development and Evaluation of a Genome-Wide 6K SNP Array for Diploid Sweet Cherry and Tetraploid Sour Cherry

High-throughput genome scans are important tools for genetic studies and breeding applications. Here, a 6K SNP array for use with the Illumina Infinium® system was developed for diploid sweet cherry (Prunus avium) and allotetraploid sour cherry (P. cerasus). This effort was led by RosBREED, a community initiative to enable marker-assisted breeding for rosaceous crops. Next-generation sequencing in diverse breeding germplasm provided 25 billion basepairs (Gb) of cherry DNA sequence from which were identified genome-wide SNPs for sweet cherry and for the two sour cherry subgenomes derived from sweet cherry (avium subgenome) and P. fruticosa (fruticosa subgenome). Anchoring to the peach genome sequence, recently released by the International Peach Genome Initiative, predicted relative physical locations of the 1.9 million putative SNPs detected, preliminarily filtered to 368,943 SNPs. Further filtering was guided by results of a 144-SNP subset examined with the Illumina GoldenGate® assay on 160 accessions. A 6K Infinium® II array was designed with SNPs evenly spaced genetically across the sweet and sour cherry genomes. SNPs were developed for each sour cherry subgenome by using minor allele frequency in the sour cherry detection panel to enrich for subgenome-specific SNPs followed by targeting to either subgenome according to alleles observed in sweet cherry. The array was evaluated using panels of sweet (n = 269) and sour (n = 330) cherry breeding germplasm. Approximately one third of array SNPs were informative for each crop. A total of 1825 polymorphic SNPs were verified in sweet cherry, 13% of these originally developed for sour cherry. Allele dosage was resolved for 2058 polymorphic SNPs in sour cherry, one third of these being originally developed for sweet cherry. This publicly available genomics resource represents a significant advance in cherry genome-scanning capability that will accelerate marker-locus-trait association discovery, genome structure investigation, and genetic diversity assessment in this diploid-tetraploid crop group.     

The occurrence of nitrate reductase in leaves of prunus species

Nitrate reductase was found in leaves of apricot Prunus armeniaca, sour cherry P. cerasus, sweet cherry P. avium, and plum P. domestica, but not in peach P. persica, from trees grown in sand culture receiving a nitrate containing nutrient solution. Nitrate was found in the leaves of all species. Nitrate and nitrate reductase were found in leaves of field-grown apricot, sour cherry, and plum trees. The enzyme-extracting medium contained insoluble polyvinylpyrrolidone, and including dithiothreitol or mercaptobenzothiazole did not improve enzyme recovery. Inclusion of cherry leaf extract diminished, and peach leaf extract abolished, recovery of nitrate reductase from oat tissue. Low molecular weight phenols liberated during extraction were probably responsible for inactivation of the enzyme. The enzyme from apricot was two to three times as active as from the other species. Both nicotine adenine diphosphopyridine nucleotide and flavin mononucleotide were effective electron donors. The enzyme was readily induced in apricot leaves by 10 mm nitrate supplied through the leaf petiole.     

Hybridization success is largely limited to homoploid <Emphasis Type="Italic">Prunus</Emphasis> hybrids: a multidisciplinary approach

Prunus fruticosa is a rare shrub occurring in Eurasian thermophilous forest-steppe alliances. The species frequently hybridizes with cultivated Prunus species in Europe (allochthonous tetraploid P. cerasus and partly indigenous diploid P. avium). Propidium iodide flow cytometry, distance-based morphometrics, elliptic Fourier analysis and embryology were employed to evaluate the extent of hybridization in six Slovak populations. Flow cytometric analyses revealed three ploidy levels: diploid (P. avium), triploid (P. × mohacsyana) and tetraploid (P. fruticosa, P. × eminens and P. cerasus). In addition, P. fruticosa and P. cerasus, at the tetraploid level, were found to differ in absolute genome size. An embryological evaluation suggested the existence of a triploid block in P. × mohacsyana and significant potential for hybridization among tetraploid taxa (indicated also by a continuous distribution of genome size data and further mirrored by morphometrics). Although hybrids significantly differ in ploidy level and embryological characteristics, they are almost indistinguishable using morphological characters. Hybridization with P. cerasus thus turns out to be a significant threat to wild populations of P. fruticosa compared to the relatively weak influence of P. avium.     

Genetic relatedness among some wild cherry (<Emphasis Type="Italic">Prunus</Emphasis> subgenus <Emphasis Type="Italic">Cerasus</Emphasis>) genotypes native to Iran assayed by morphological traits and random amplified polymorphic DNA analysis

Subgenus Cerasus species are useful genetic resources for cherry breeding programs. A total of 17 morphological traits together with 19 random amplified polymorphic DNA (RAPD) primers were used to study 39 accessions including 34 wild Cerasus subgenus genotypes belonging to Prunus avium L., P. cerasus L., P. mahaleb L., P. microcarpa Boiss., P. incana Pall., and P. brachypetala Boiss. species, along with an unknown wild Cerasus sample, two advanced cherry cultivars (‘Lambert’ and ‘Bulgar’), and two rootstocks (‘Colt’ and ‘Gisela 6’). Genotypes were separated into different groups according to their species and collection sites using cluster analysis performed by Ward’s clustering method based on morphological data. Nineteen RAPD primers from 60 screened produced 304 polymorphic reproducible bands (98.15% polymorphism). According to the similarity matrix, the lowest similarity was obtained between P. avium and P. microcarpa samples. A dendrogram was prepared by the unweighted pair-group method with arithmetic average (UPGMA), and the accessions were separated according to their species and geographic origin. In both morphological and molecular results, the advanced cultivars and rootstocks were separated from wild genotypes, and the unknown genotype was grouped with P. mahaleb accessions. Grouping by morphological characteristics was compared with the results of RAPD analysis, with no significant correlations between morphological and molecular data being found. This is the first report of molecular (RAPD) genetic diversity study in wild Cerasus subgenus genotypes from Iran, and the results demonstrate the high potential of RAPD analysis for discrimination of Cerasus subgenus genotypes.     

Intra-allelic variation in introns of the <Emphasis Type="Italic">S</Emphasis><Subscript><Emphasis Type="BoldItalic">13</Emphasis></Subscript><Emphasis Type="Italic">-RNase</Emphasis> allele distinguishes sweet,wild and sour cherries

The cherry (Prunus avium), a self-incompatible diploid species, and the sour cherry (Prunus cerasus), a self-incompatible or self-compatible allotetraploid species derived from P. avium and Prunus fruticosa, share several S-RNase alleles, including S ₁₃ . An inactive form, S ₁₃ °, is found in some sour cherries. Two (AT) microsatellites are associated with allele S ₁₃ -RNase, one in the first intron and one in the second. Their length polymorphisms were studied in 14 sweet and 17 wild cherries (both P. avium) and in 42 sour cherries. Fluorescent primers amplifying each microsatellite were designed and amplification products sized on an automated sequencer. Variants ranged from 247 to 273 bp for the first intron microsatellite and from 308 to 322 bp for the second. There were 34 combinations and, surprisingly, the lengths of the two microsatellites were correlated. Generally, the sweet, wild and sour cherries had different combinations, and the four examples of S ₁₃ °-RNase were associated with three different combinations. Certain sequences associated with the microsatellites match footprints of transposons. The distribution of combinations indicated little overlap between the three populations analysed and provided useful insights into relationships of some of the accessions allowing some parentages to be checked. In the diploid sweet and wild cherries, S ₁₃ variants presumably resulted from slippage during replication, but in the tetraploid sour cherries, which can have more than one copy of S ₁₃ or S ₁₃ °, intra-allelic crossing over may have generated new variants. The possible involvement of transposable elements in the origin of these microsatellites is considered.     

A study of ethylene in apple, red raspberry, and cherry

High ethylene levels were associated with flower abscission in apple (Malus sylvestris) and cherry (Prunus avium and Prunus cerasus), “June drop” of immature cherries, and harvest drop of apple and red raspberry (Rubus idaeus). However, an increase in ethylene content was not associated with June drop of apples and harvest drop of cherries. During the period of fruit ripening on the plant, the largest increases in ethylene occurred in apple flesh and red raspberry receptacular tissue. Ethylene remained low throughout the period of sweet and tart cherry ripening. The data obtained indicated marked ethylene gradients between adjacent tissues. Increases of ethylene in some tissues may have resulted from ethylene diffusion from adjacent tissues containing high levels of ethylene.     

Isozyme diversity in sour,sweet, and ground cherry

Thirty-six sour (Prunus cerasus L.), sweet (P. avium L.), and ground cherry (P. fruticosa Pall.) selections were evaluated for seven enzyme systems and principal coordinate analysis was used to examine isozyme divergence among these cherry species. The enzyme systems studied were phosphoglucose isomerase (PGI), isocitrate dehydrogenase (IDH), phosphoglucomutase (PGM), 6-phosphogluconate dehydrogenase (6-PGD), leucine aminopeptidase (LAP), shikimate dehydrogenase (SKDH), and malate dehydrogenase (MDH). The first principal coordinate, which accounted for 41% of the total variation, separated the diploid sweet cherry selections from the sour, ground, and sour x ground cherry tetraploids. An additional 86 selections were evaluated for up to six of the enzyme systems to determine the polymorphisms at the enzyme loci and the level of heterozygosity between the diploid sweet cherry and the tetraploid species and interspecific hybrids. 6-PGD was the most polymorphic enzyme exhibiting 16 patterns. The tetraploid cherry species were more heterozygous than the diploid sweet cherry with an average heterozygosity of 78% compared to 19% for the diploids.     

Phenotypic and genotypic variation in Iranian sour and duke cherries

Phenotypic and genotypic variation and structure of 29 sour cherry (P. cerasus) and duke cherry (P. x gondouinii) genotypes from different regions of Iran were identified using random amplified polymorphic DNA (RAPD) markers and morphological characters. Furthermore, one Prunus mahaleb genotype was used as an outgroup for molecular analysis. For morphological analysis, 23 variables were recorded to detect similarities between and among studied sour and duke cherries. Most studied characteristics were showing a high degree of variability. Principal component analysis showed that the first three components explained a total of 73.87 % of the whole phenotypic variability. Based on the morphological cluster analysis, studied sour and duke cherry genotypes were placed into three main clusters. The first main cluster included 16 sour cherry genotypes. The second main cluster contained all duke cherry genotypes and eight sour cherry genotypes, while, only one sour cherry genotype was placed in third main cluster. For RAPD analysis, 17 primers generated a total of 233 discernible and reproducible bands across genotypes analyzed, out of which 214 (91.51 %) were polymorphic with varied band size from 300 to 3000 bp. According to the similarity matrix, the lowest similarity was obtained between P. mahaleb, as an outgroup, and sour cherry. Dendrogram based on molecular data separated genotypes according to their species and geographic origin. Low correlation was observed between the similarity matrices obtained based on morphological and RAPD data. The information obtained here could be valuable for devising strategies for conservation of Iranian sour and duke cherries.     

Assessing past agrobiodiversity of Prunus avium L. (Rosaceae): a morphometric approach focussed on the stones from the archaeological site H?tel-Dieu (16th century, Tours, France)

Abundant and diverse Prunus fruitstone remains from cherries, plums, sloes, peaches, etc. are frequently recovered from archaeological waterlogged contexts such as wells, latrines, lake dwellings etc. in Europe. The distinction between most of the Prunus species, based on traditional morphological characters of the fruit stones, is usually not problematic. However the discrimination between P. avium L., P. cerasus L. and related cherry species, based on classical criteria alone, often turns out to be ambiguous because of the increasing number of varieties which have been bred since Roman times. By combining geometric and traditional morphometrical approaches, the overall variation in shape and size of stones from French and Swiss excavations dating from the 1st century to the 16th century a.d. were assessed. Among these important archaeobotanical data, the detailed examination of 100 waterlogged stones from the 16th century H?tel-Dieu cesspit at Tours, France, revealed that the morphological diversity is structured into two distinct morphotypes which diverge mainly according to geometrical features. Finally, the comparison between morphological features of these well-preserved archaeological stones and modern reference material including P. avium, P. cerasus and P. × gondouinii, suggests that these two morphotypes, which have been initially attributed to P. avium (long stones) and P. avium/cerasus (rounded stones) according to traditional morphological parameters, would correspond to two different cultivated varieties, both belonging to Prunus avium. Results presented in this work constitute new and preliminary data obtained during the development of this project that throw light on morphological variability and biosystematic aspects.     

Abscisic Acid in Prunus Trees: Isolation and the Effect on Growth of Excised Shoot Tissue

The abscisic acid content of the mature shoot tissue of vigorous Prunus avium, medium sized Prunus cerasus and dwarfing Prunus cerasus self (S) was estimated by spectropolarimetry and gas chromatography. There is no correlation between the levels of ABA found in the tissue and the dwarfing potential of the trees investigated. However, Prunus arium stem tissue shows the lowest sensitivity to ABA, whereas the explants of normal and dwarfing Prunus cerasus exhibit a stronger inhibition.     

Cell number regulator genes in Prunus provide candidate genes for the control of fruit size in sweet and sour cherry

Striking increases in fruit size distinguish cultivated descendants from small-fruited wild progenitors for fleshy fruited species such as Solanum lycopersicum (tomato) and Prunus spp. (peach, cherry, plum, and apricot). The first fruit weight gene identified as a result of domestication and selection was the tomato FW2.2 gene. Members of the FW2.2 gene family in corn (Zea mays) have been named CNR (Cell Number Regulator) and two of them exert their effect on organ size by modulating cell number. Due to the critical roles of FW2.2/CNR genes in regulating cell number and organ size, this family provides an excellent source of candidates for fruit size genes in other domesticated species, such as those found in the Prunus genus. A total of 23 FW2.2/CNR family members were identified in the peach genome, spanning the eight Prunus chromosomes. Two of these CNRs were located within confidence intervals of major quantitative trait loci (QTL) previously discovered on linkage groups 2 and 6 in sweet cherry (Prunus avium), named PavCNR12 and PavCNR20, respectively. An analysis of haplotype, sequence, segregation and association with fruit size strongly supports a role of PavCNR12 in the sweet cherry linkage group 2 fruit size QTL, and this QTL is also likely present in sour cherry (P. cerasus). The finding that the increase in fleshy fruit size in both tomato and cherry associated with domestication may be due to changes in members of a common ancestral gene family supports the notion that similar phenotypic changes exhibited by independently domesticated taxa may have a common genetic basis.     

A draft genome of sweet cherry (Prunus avium L.) reveals genome‐wide and local effects of domestication

Sweet cherry (Prunus avium L.) trees are both economically important fruit crops but also important components of natural forest ecosystems in Europe, Asia and Africa. Wild and domesticated trees currently coexist in the same geographic areas with important questions arising on their historical relationships. Little is known about the effects of the domestication process on the evolution of the sweet cherry genome. We assembled and annotated the genome of the cultivated variety “Big Star*” and assessed the genetic diversity among 97 sweet cherry accessions representing three different stages in the domestication and breeding process (wild trees, landraces and modern varieties). The genetic diversity analysis revealed significant genome‐wide losses of variation among the three stages and supports a clear distinction between wild and domesticated trees, with only limited gene flow being detected between wild trees and domesticated landraces. We identified 11 domestication sweeps and five breeding sweeps covering, respectively, 11.0 and 2.4 Mb of the P. avium genome. A considerable fraction of the domestication sweeps overlaps with those detected in the related species, Prunus persica (peach), indicating that artificial selection during domestication may have acted independently on the same regions and genes in the two species. We detected 104 candidate genes in sweep regions involved in different processes, such as the determination of fruit texture, the regulation of flowering and fruit ripening and the resistance to pathogens. The signatures of selection identified will enable future evolutionary studies and provide a valuable resource for genetic improvement and conservation programs in sweet cherry.     

Cherry leaf spot resistance in cherry (Prunus) is associated with a quantitative trait locus on linkage group 4 inherited from P. canescens

Cherry leaf spot (CLS), caused by the fungal pathogen Blumeriella jaapii (Rehm) Arx (telomorph Phloeosporella padi [Lib.] Arx), is a major disease in all humid cherry-growing regions worldwide causing leaf yellowing and defoliation. The diploid Prunus species, P. canescens, had previously been identified as a source of CLS resistance. Therefore, the objective of this study was to identify quantitative trait loci (QTL) for CLS resistance derived from P. canescens in both diploid sweet cherry (P. avium) and tetraploid sour cherry (P. cerasus). Because of the simpler genetics of diploid cherry, the initial investigation was done with P. canescens-derived materials from crosses with sweet cherry, followed by validation using P. canescens-derived plant materials from sour cherry. A major QTL controlling P. canescens-derived CLS resistance, named CLSR_G4, was identified on linkage group 4 in sweet cherry and validated in sour cherry. All CLS-resistant individuals had one P. canescens-derived allele for CLSR_G4. A second QTL may be necessary for CLS resistance as one-fifth–one-third of the progeny individuals with the P. canescens-derived allele for CLSR_G4 were susceptible.     

Indole-3-butyric acid and myo-inositol impacts on in vitro rooting of the cherry rootstocks CAB-6P and Gisela 6

The present study investigates the effects of indole-3-butyric acid (IBA) alone and in combination with myo-inositol on in vitro rooting and biochemical responses in the cherry rootstocks CAB-6P (Prunus cerasus L.) and Gisela 6 (Prunus cerasus × Prunus canescens). For the CAB-6P rootstock, the best results for root number (6.31), fresh mass (FM), dry mass (DM), and rooting percentage (100 %) were obtained on Murashige and Skoog (MS) medium with 2 mg dm^?3 IBA and maximum root length (30.57 mm) was obtained at 1 mg dm^?3 IBA. Myo-inositol suppressed the positive effects of IBA on root length. In the Gisela 6 explants, the inclusion of 2 mg dm^?3 IBA together with 0.5 mg dm^?3 of myo-inositol in the culture medium significantly enhanced root number (9.91) and root FM and DM. The root length was maximum in the combination of the lowest IBA and myo-inositol concentrations (0.5 mg dm^?3). The rooting percentage was the greatest (100 %) with the application of 1 mg dm^?3 IBA alone. In both explants, the application of IBA alone or in combination with myo-inositol resulted in a lower leaf proline content in comparison with the control (without growth regulators). The maximum leaf chlorophyll content was at 1 mg dm^?3 IBA in the CAB-6P whereas at 2 mg dm^?3 IBA and 1 mg dm^?3 myo-inositol in Gisela 6. Addition of myo-inositol mostly increased sugar content in comparison with control or IBA alone in both rootstocks.     

Diurnal Expansion and Contraction of Leaves and Fruits of English Morello Cherry

Changes in leaf thickness and fruit diameter were measured ingrowth chambers under varying conditions of light, humidity,temperature, and soil moisture for potted trees of English Morellocherry (Prunus cerasus L. grafted on Prunus mahaleb root stock).Decrease in leaf thickness of well-watered plants was inducedby illumination following a period of darkness. In the light,leaf thickness decreased when vapour-pressure deficit (VPD)was rising or high. The magnitude of diurnal change in leafthickness was related to amount of change in VPD of the air.Leaf thickness increased in the dark when stomata were closed.When changes in VPD were minimized, leaves under 8-h days and16-h nights still decreased in thickness during the day andexpanded at night, but the degree of change was not as greatas when VPD also fluctuated diurnally. When severe internal water deficits developed in trees duringprolonged droughts the correlations of changes in leaf thicknesswith VPD and light intensity were low. Marked decline in leafthickness resulted with little re-expansion in the dark at lowVPD. Soil irrigation resulted in rapid hydration and expansionof leaves. Changes in fruit diameter in contrast were positivelycorrelated with VPD fluctuations and were not influenced byphotoperiod.     

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.     

Genetic variation in wild Prunus L. subgen. Cerasus germplasm from Iran characterized by nuclear and chloroplast SSR markers

Key message

The selected material of Cerasus subgen. will be useful for conservation and management and important for Prunus breeding programs.

Abstract

Knowledge of relationships among the cultivated and wild species of Cerasus is important for recognizing gene pools in germplasm and developing effective conservation and management strategies. In this study, genetic and phylogenetic relationships of wild Cerasus subgenus species naturally growing in Iran, including P. avium (mazzard), P. mahaleb, P. brachypetala, P. incana, P. yazdiana, P. microcarpa subsp. microcarpa, P. microcarpa subsp. diffusa and P. pseudoprostrata and three commercial species, sweet cherry (P. avium), sour cherry (P. cerasus) and duke cherry (P. x gondouinii) was investigated based on 16 nuclear SSR and five chloroplast SSR. Very high level of polymorphism was detected among the studied species based these molecular markers, indicating high inter and intraspecific genetic variation. Inter and intraspecific genetic similarity coefficients varied from 0.00 to 1.00, indicating high genetic variation in studied germplasm. These two molecular markers types could distinguish differences between all species so that accessions of each species were placed into a single group. Based on molecular markers, a close correlation was observed between intraspecific variation and geographical distribution. Furthermore, based on nuSSR primers, most wild species showed 2–4 alleles and may be tetraploid. In conclusion, the conservation of these highly diverse native populations of Iranian wild Cerasus germplasm is recommended for future breeding activity.