Molecular characterization and genetic diversity of Prunus rootstocks

Twenty microsatellite primer pairs, previously developed in peach, were used to characterize and to explore genetic relationships among 44 clones, representing three groups of rootstocks defined as: (1) Peach-based rootstocks (Prunus dulcis × P. persica, P. persica × P. davidiana); (2) Myrobalan-Marianna plums (P. cerasifera and interspecific hybrids having P. cerasifera as a parent); and (3) Slow growing plums (P. insititia, P. domestica, and P. domestica × P. spinosa). Eighteen SSR markers, from the 20 initially used, were able to amplify polymorphic products for the Peach-based rootstocks and 13 common markers gave also polymorphism for the Myrobalan-Marianna and Slow growing plums groups. The Dice coefficient of similarity was calculated between all pairs of accessions and their genetic similarity represented by a principal coordinate analysis. The genetic diversity detected among the 44 clones studied divided them in three groups, which are in agreement with their current taxonomic classification and their morphological characteristics. A set of three microsatellites (BPPCT001, CPPCT022 and UDP98-407) can distinguish between all the clones analyzed. The analysis within groups reveal another two sets of three SSR to distinguish between the clones from the Peach based rootstocks and the Myrobalan-Marianna plums, respectively, and only a single SSR is needed to distinguish within the clones from the Slow growing plums group. These results demonstrate the high potential of the SSR analysis for peach rootstock identification and studies of diversity in Prunus species.     

Cross-transferable polymorphic SSR loci in Prunus species

This work reports the transferability and polymorphism of previously reported SSRs in 10 Prunus species. The availability of a large number of SSRs in the genus Prunus makes marker choice random, while preventing comparison of results in fingerprinting studies. The availability of SSR markers, polymorphic in a wide sample of Prunus species, would facilitate marker choice, while allowing the comparison of results. In this work, microsatellite markers useful for analyzing 10 different Prunus species (P. persica, P. dulcis, P. armeniaca, P. domestica, P. insititia, P. salicina, P. cerasifera, P. avium, P. cersus and P. mahaleb) were searched through screening SSRs previously reported to be conserved and/or polymorphic in more than one Prunus species. A selected group of 13 SSRs, transferable to the 10 species, was analyzed in terms of their usefulness for analyzing these species. The amplification range, polymorphism and variability detected by these loci are reported. The information provided will be useful for Prunus genetic studies as well as conservation and management of Prunus germplasm resources.     

Assessment of genetic diversity of Latvian and Swedish sweet cherry (Prunus avium L.) genetic resources collections by using SSR (microsatellite) markers

Three previously described highly polymorphic SSR (microsatellite) primer pairs were tested on 126 sweet cherry (Prunus avium L.) accessions to adapt a fast, reliable method for preliminary screening of sweet cherry germplasm collections and to compare two sweet cherry germplasm collections: at the Latvia State Institute of Fruit-Growing, Dobele (LIFG-Dobele) and at the Division of Horticultural Genetics and Plant Breeding at Balsgård, Department of Crop Sciences, Swedish University of Agricultural Sciences (SLU-Balsgård). The SSR loci were highly polymorphic with 4–10 different alleles and 5–18 genotypes. Heterozygosity values ranged from 0.431 to 0.809, gene diversity (PIC) values ranged from 0.400 to 0.753, and the discriminating power of each locus varied from 0.631 to 0.894. The combined discriminating power of all loci was highly effective (0.996). Sixteen identical accession groups with the same allele profile were discovered in both collections. This study demonstrated that SSR fingerprinting with the three primer pairs tested, can be used for preliminary characterization of sweet cherry germplasm collections.     

Genetic diversity and phylogenetic relationships of Prunus microcarpa C.A. Mey. subsp. tortusa analyzed by simple sequence repeats (SSRs)

Prunus microcarpa C.A. Mey. subsp. tortusa is a deciduous shrub well adapted to severe winter and dry-hot summer conditions. As the first step to explore the genetic and horticultural potential of P. microcarpa C.A. Mey. subsp. tortusa, we used SSRs to elucidate the genetic variation within its populations dispersed in upper Mesopotamia. We also investigated its phylogenetic relationship with economically important Prunus species; almond, apricot, sweet cherry, peach and plums. Using 47 amplifying SSR primer pairs, 63 P. microcarpa C.A. Mey. subsp. tortusa genotypes sampled from five locations and 15 cultivars belonging to other Prunus species were assayed. The cross-species transportability of SSRs was 96% indicating a high degree of homology between P. microcarpa C.A. Mey. subsp. tortusa and the other Prunus species. The genetic distance between P. microcarpa C.A. Mey. subsp. tortusa genotypes belonging to a particular geographic site was lower than that between genotypes of different geographic origins. Cluster analysis differentiated P. microcarpa C.A. Mey. subsp. tortusa genotypes according to their geographic sites and separated them from the other Prunus species. P. microcarpa C.A. Mey. subsp. tortusa and sweet cherry, the subgenus Cerasus, were located in the same major cluster, the other Prunus species, belonging to the subgenera Amygdalus and Prunus, were located in another one. The analysis of molecular variance (AMOVA) revealed that genetic variation among individuals within populations (59.10%) was much higher than among Prunus groups (29.28%) and among P. microcarpa C.A. Mey. subsp. tortusa populations of different geographic sites (11.61%). The results indicate a substantial genetic diversity in P. microcarpa C.A. Mey. subsp. tortusa and the need of exploring a wider area to increase the chance of finding a particular genotype.     

Microsatellite fingerprinting of homonymous grapevine (Vitis vinifera L.) varieties in neighboring regions of South-East Turkey

Genotyping of Turkish grapevine (Vitis vinifera L.) germplasm was characterized by use of six highly polymorphic microsatellite loci (VVS2, VVMD5, VVMD7, VVMD27, VrZAG62, VrZAG79). In this study we aimed to clarify the relationships between homonymous varieties coming from different regions. Our results showed a large degree of genetic variability among most of the homonymous cultivars. The number of alleles per locus ranged from 10 to 21, and gene diversity (expected heterozygosity) values ranged from 0.85 to 0.93. Cultivars presenting the same names of Sergi karas? (sampled from ?anl?urfa and Gaziantep), Yediveren (sampled from ?anl?urfa, Gaziantep, and National Germplasm Repository Vineyard in Tekirda?) and Serpenek?ran (sampled from ?anl?urfa and Gaziantep) were clustered together, or very close to each other, in a phenogram. Moreover, the alleles at the six microsatellite loci analyzed were found to be similar in terms of base pairs within each of these three closely positioned varieties. However, all the other cultivars failed to show a suitable clustering pattern when comparing their DNA profiles and names. Similarly named cultivars were not generally grouped together in the phenogram. On the other hand, we detected a tendency for differently named homonymous grape cultivars to cluster together.     

Molecular characterization and genetic relationship among almond cultivars assessed by RAPD and SSR markers

RAPDs and SSRs were used to study the genetic diversity of Iranian almond cultivars and their relationship to important foreign cultivars and three related species. Eight unidentified almond Shahrodi cultivars and three wild almonds (Prunus communis, Prunus orientalis and Prunus scoparia) were also included. Of the primers tested, 42 (out of 80) RAPD and 18 (out of 26) SSR primers were selected for their reproducibility and high polymorphism. A total of 664 polymorphic RAPD bands were detected out of 729 bands. The number of presumed alleles revealed by the SSR analysis ranged from 3 to 10 alleles per locus with a mean value of 6.64 alleles per locus. Both techniques discriminated the genotypes very effectively, but only RAPDs were able to discriminate the cultivars Monagha and Sefied. Results demonstrated an extensive genetic variability within the tested cultivars as well as the value of SSR markers developed in peach for characterization of almond and related species of Prunus. Dice similarity coefficient was calculated for all pair wise comparisons and was used to construct a UPGMA dendrogram. For both markers a high similarity in dendrogram topologies was obtained although some differences were observed. All dendrograms, including that obtained by the combined use of both the marker data, depicted the phenetic relationships among the cultivars and species, depending upon their geographic region and/or pedigree information. Almond cultivars clustered with accession of P. communis showing their close relationship. P. orientalis and P. scoparia were clustered out of the rest of P. dulcis.     

The Use of Dinitrocresol-Mineral Oil Sprays for the Control of Prolonged Rest in Apple Orchards

Summary

Pluots are putative hybrids between plums (Prunus salicina Lindl.) and apricots (P. armeniaca L.). The capability to distinguish among plum and pluot cultivars is important in breeding and cultivation. We investigated the genetic diversity among 14 plums, 6 pluots and one plumcot representing commercial cultivars in California, with 28 microsatellite markers. We also tested seven apricot cultivars as a reference to ®nd evidence of apricot in the ancestry of pluots and plumcot. The parental material used in the original cross that produced the pluot and plumcot was not available. Of the 28 SSR markers, 25 were from sweet cherry (Prunus avium L.) and three from peach (Prunus persica L.). Approximately 80% of the cherry primers generated ampli®cation products in plum and pluots, showing transportability between these Prunus species. One to eight putative alleles per locus were displayed by the tested SSRs in plums and pluots. In plum and pluot samples a total of 100 alleles were identi®ed with an average of 4.3 alleles per primer combination. The SSR markers were successfully used for the discrimination of all tested cultivars. In pluots, 76 alleles were found in which 63 (83%) were speci®cally coming from plum, 9 (12%) were common in plum, pluots and apricot while no allele in the pluots was observed that was contributed from apricot. In plumcot, 49 alleles were observed in which 25 (51%) were from plum, 18 (36%) were speci®cally from apricot and 6 (12%) were common in plum, plumcot and apricot. Relationships among the 28 plum, pluot and apricot cultivars were represented by a dendrogram, constructed on the basis of 168 SSR markers. The dendrogram showed the plums and pluots form a cluster distinct from the apricots, with pluot cultivars interspersed among plum cultivars and more closely related to plum than to apricot. Plumcot made a separate branch and was placed between the plum and apricot cluster. These results suggest that the SSR markers are valuable tools for identi®cation of cultivars and diversity analyses in plum.     

S-RNase based S-genotyping of Japanese plum (Prunus salicina Lindl.) and its implication on the assortment of cultivar-couples in the orchard

Determination of the genetic compatibility between self-incompatible cultivars is crucial in agriculture. The Rosaceae family carries the S-RNase-mediated gametophytic self-incompatibility (GSI) system. Each haplotype is conferred by an S-locus. The S-locus contains two highly polymorphic genes, S-RNase and SFB, which are characteristic of each haplotype and therefore these genes are ideal markers for molecular S-genotyping. In this study 43 Japanese plum cultivars grown in Israel were S-genotyped based on their S-RNase gene sequences. Four alleles, S^b, S^c, S^e and S^h are widespread and together are responsible for 87% of the S-haplotypes therefore many of the cultivar combinations are semi-compatible. In Israel semi-compatibility was shown to correlate with low yield. However, two cultivars, ‘Wickson’ S^fS^k and ‘Shiro’ S^fS^g carry rare S-haplotypes and, therefore, are fully compatible with most of the analyzed cultivars.     

Early detection of graft incompatibility in apricot (Prunus armeniaca L.) using phenol analyses

The study investigated the role of phenols in apricot graft incompatibility. Assays of phloem with cambium from 1-year-old apricot trees of cultivars Marlen, Leskora and Betinka which were grafted on the rootstocks of different genetic origin: M-LE-1, Lesiberian, MY-KL-A, Tetra, Penta, Green Gage, Julior, MRS 2/5 and Isthara were analysed with HPLC (together 23 scion/stock combinations). The phloroglucinol, catechin, p-coumaric acid and further non-identified phenols with the retention time 23–25 and 30 min were determined. The content of individual phenol compounds was related to specific cultivar/rootstock combination. The minimum number of statistical significant differences in the phenol content between tissues above and below graft union was established in homospecific combinations (P. armeniaca/P. armeniaca). Cultivars Marlen, Leskora and Betinka differ in the degree of compatibility or incompatibility with rootstocks. The pattern of non-identified phenol 23 in different graft combinations is similar to catechin and p-coumaric acid.     

Droplet-vitrification of apical shoot tips of Rubus fruticosus L. and Prunus cerasifera Ehrh.

Apical shoot tips excised from in vitro plantlets of blackberry (Rubus fruticosus L. ‘?a?anska Bestrna’) and cherry plum (Prunus cerasifera Ehrh.) were tested for recovery after cryopreservation using the droplet-vitrification technique. Following treatment for 30 min with a loading solution comprising 1.9 M glycerol and 0.5 M sucrose, explants were dehydrated with a highly concentrated cryoprotectant solution, so called vitrification solution. Shoot tips were dehydrated for 10, 20 and 30 min at room temperature with a solution derived from the original PVS2 solution (containing 37.5% (w/v) glycerol, 15% (w/v) dimethylsulfoxide, 15% (w/v) ethylene glycol and 22.5% (w/v) sucrose) and for 60, 90 and 120 min using the PVS3 solution (containing 50% (w/v) glycerol and 50% (w/v) sucrose). Explants were cooled by direct immersion in LN in 10 μl droplets of vitrification solution placed on aluminium foil strips. Rewarming was done by direct plunging of foil strips in a preheated (37 °C) unloading solution (0.8 M sucrose) for 30 s, after which an equal volume of unloading solution (at room temperature) was added for further incubation for 30 min. As for regrowth of blackberry, PVS3 proved more effective than the modified PVS2, but the difference was significant (P < 0.05) only for the shortest treatment duration. The duration of PVS3 treatment had no significant effect on regrowth of cryopreserved shoot tips (45.8–70%). By contrast, a 30-min treatment with modified PVS2 solution resulted in a significant increase in regeneration percentage (30%), as compared with a 10-min treatment with the same solution (5%). Cherry plum shoot tips were very sensitive to both vitrification solutions and growth recovery of cryopreserved samples was generally lower (5–20%) than that of blackberry explants. No significant influence of PVS treatment (both type of solution and treatment duration) on regrowth of cryopreserved shoot tips was observed with cherry plum shoot tips. Experiments performed in France and in Serbia produced similar results, thereby showing the robustness and reproducibility of the protocols developed.     

Comparison of the use of morphological,protein and DNA markers in the genetic characterization of Iranian wild Prunus species

In this study, the genetic diversity of four Iranian wild Prunus species including Prunus eleagnifolia, Prunus hauskenchtii, Prunus scoparia and Prunus lycioides were investigated using morphological, protein and DNA markers. DNA markers included nuclear and chloroplast SSRs and self-incompatibility (S) allele amplification. At the morphological level, leaf width showed significant differences between the four wild Prunus species. Concerning fruit and kernel characters, their values are relatively similar indicating the high degree of homoplasy described in Prunus. Nuclear SSR markers have been the most abundant markers with a higher polymorphism in comparison with morphological, protein and chloroplast SSR markers. Results also indicated the high variability present in the S locus. On the other hand, the correlation between the clustering based on DNA markers and protein were in general low. Dendogram performed using nuclear and chloroplast SSR indicated a more diffuse clustering between the wild almond species probably due to the natural introgression of genes observed in these wild almond species. Data from the analysis of the total protein seems to be more accurate to establish taxonomy relationships in these very close wild species.     

Effect of synthetic auxins on fruit size of five cultivars of Japanese plum (Prunus salicina Lindl.)

Most of the Japanese plum (Prunus salicina) cultivars grown in Israel produce relatively small fruit. Application of 2 l solution tree⁻¹ of 25 mg l⁻¹ 2,4-dichlorophenoxypropionic acid (2,4-DP) as butoxyethyl ester (Power™), 15 mg l⁻¹ 3,5,6-trichloro-2-pyridyloxyacetic acid (3,5,6-TPA) as free acid (Maxim^®), or 25 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) + 30 mg l⁻¹ naphthaleneacetic acid (NAA) (0.3% Amigo™) at the beginning of pit-hardening, when fruitlet diameter was ca. 22 mm, caused an appreciable and significant increase in fruit size. The yield of large fruit per cv.: ‘Kesselmen’ (100% increase), ‘Songold’ (100%), ‘Black Diamond’ (800%), ‘Royal Diamond’ (160%) and ‘Royal Zee’ (100%). As a result, the total yield of all five cultivars was also increased dramatically. Anatomical studies with ‘Songold’ revealed that the main effect of these synthetic auxins was via direct stimulation of fruit cell enlargement. The above auxins had no negative effect either on fruit quality at harvest (and after 1 week in shelf-life), or on return yield in the following year.     

Rose (Rosa hybrida L.) EST-derived microsatellite markers and their transferability to strawberry (Fragaria spp.)

The ‘Genome database for Rosaceae (GDR)’ provides a large collection of expressed sequence tags (ESTs) harboring simple sequence repeats (SSRs) from several Rosaceae genera, including Rosa (rose). Primer pairs flanking SSR were designed for 312 unique Rosa ESTs based on GDR database. Eight rose (Rosa hybrida L.) genotypes were tested for PCR amplification, and 287 (92%) of the primer pairs generated allele-specific PCR bands that were readily scored. From 183 (63.7%) primer pairs that evidenced polymorphic alleles among the eight rose cultivars, 20 pairs evidencing EST sequence homology to known gene functions and high levels of polymorphism were selected and utilized for DNA fingerprinting and genetic diversity assessments of 47 rose hybrids. A total of 202 polymorphic bands were scored and generated unique fingerprints for each rose hybrid. The Nei–Li genetic similarity coefficients among 1081 pair-wise comparisons of 47 cultivars exhibited a broad range of genetic variations from 0.30 (‘Grand King’ and ‘Carnival’) to 0.99 (‘First Red’ and ‘Red Champ’). UPGMA cluster analysis divided 47 hybrids into five major groups and two sub-groups. The cross-species transferability of 273 EST-SSR primer pairs was evaluated using four genotypes of the strawberry, a genus member of the Rosaceae family. PCRs on the DNA samples of strawberry were successful for 165 primer pairs; among these, 123 pairs amplified 243 polymorphic bands. As surrogates of the marker transfer, the phenetic relationship among the four strawberry genotypes was evaluated. Genetic similarity coefficients varied from 0.78 (‘Maehyang’ and ‘Janghyee’) to 0.64 (‘Janghyee’ and ‘Pragana’). The results of cluster analysis showed that the three octaploid strawberry cultivars were quite similar, whereas the diploid ‘Pragana’ was related distantly at the genomic DNA level. The EST-SSR markers developed in the present study can be efficiently utilized for genetic diversity studies in Rosaceae.     

Phylogenetic analysis in some Diospyros spp. (Ebenaceae) and Japanese persimmon using chloroplast DNA PCR-RFLP markers

Ten universal primer pairs of chloroplast genome were used to amplify non-coding regions of chloroplast DNA (cpDNA) in 7 Diospyros L. species including 29 genotypes and approximately 20.4 kb, 12.6% of the chloroplast genome were analyzed. The amplified products were digested by seven restriction enzymes. The results showed that there were abundant polymorphisms in inter-specific cpDNA within the genus Diospyros. However, it was not observed intra-species variations in 22 tested genotypes of Diospyros kaki (Japanese persimmon), except for ‘Male strain No. 9’. Persimmon had close relationship with Diospyros lotus and Diospyros glaucifolia, but distantly with Diospyros virginiana and Diospyros rhombifolia in diagram based on principal coordinates analysis and Wagner parsimony method. The discrepancy of digesting pattern in cpDNA suggested that the genotype Jinzaoshi was distinct with the remaining Diospyros spp., which revealed that Jinzaoshi may be a new species of the genus Diospyros.     

A SCAR marker linked to the N gene for resistance to root knot nematodes (Meloidogyne spp.) in pepper (Capsicum annuum L.)

The root-knot nematodes (Meloidogyne spp.) are important nematode pests and cause serious diseases in pepper in the world. No molecular markers linked to the nematodes resistance N gene have been reported. In this paper, ‘Carolina Wonder’ (Capsicum annuum L.), a sweet pepper line resistant to root-knot nematode with N gene, ‘20080-5-29’ (C. annuum L.), an inbred line susceptible to root-knot nematode with good horticultural characteristics, and their F₂ progeny with 320 individuals were used as materials. Evaluation of resistance and susceptibility of parental lines, F₁ and F₂ progeny inoculated with root-knot nematodes (Meloidogyne incognita) were carried out. ‘Bulked segregant analysis’ method was used to search for polymorphic markers from 512 pairs of AFLP primers. Based on the assessment of resistance and susceptibility and polymorphism of the AFLP marker in F₂ population, the genetic linkage distance between the AFLP marker and the N gene was estimated. One AFLP marker E39/M41-339 was obtained and transferred to a SCAR marker amplifying a 315 bp DNA fragment linked to the N resistant allele and a 331 bp fragment linked to the N⁺ susceptible allele. The distance between the molecular marker and the nematodes resistance N gene is 6.3 cM. This research delivered a valuable tool for the marker assisted selection of nematodes resistance in pepper.     

Direct and indirect effects of seed related characters on number of seed in guava (Psidium guajava L.) fruits

Dessert quality of guava fruit is considerably reduced by high seed content. Number of seeds in guava is associated with different seed and fruit characters. Influence of different seed characters on number of seeds per fruit (NSPF) was studied by analysing character association and direct and indirect effects on NSPF and seed related traits for 68 genotypes of guava collected from diverse sources and conserved at National Active Germplasm Site, CISH, Lucknow. Seed related characters were studied at the colour break stage of the fruits using image analysis software for counting number of seeds. The data was subjected to path analysis to find out direct and indirect effects of different characters on number of seeds in the fruits. At genotypic and phenotypic levels, NSPF was significantly and positively associated with seed weight per fruit (SWPF), number of seeds 100 g⁻¹ pulp (NSPHP) and fruit weight (FW). The genotypic (0.0029) and phenotypic (0.0563) residual values were fairly very low, which revealed that variables included in this study had significant contribution in determining NSPF. The NSPHP exhibited high positive direct effect on NSPF. The maximum direct response (P = 0.737, G = 1.004) of this component was attributed to the indirect positive effects of the SWPF and fruit:seed weight proportion (FSWP). SWPF also had a very high positive direct effect (P = 0.521, G = 0.694) on the NSPF. Correlation and path coefficient analysis revealed that SWPF, NSPHP and 100-seed weight (100SW) were deciding factors for realizing improvement for NSPF. The importance of small seeded genotypes in selection of less seeded varieties was emphasized.     

DNA content,morphometric and molecular marker analyses of Citrus limonimedica, C. limon and C. medica for the determination of their variability and genetic relationships within the genus Citrus

This work investigated the fingerprinting and phenotyping of Citrus germplasm; species selected were of historical importance belonging to Citrus limonimedica Lush. and its supposed ancestors, along with some other species of the Citrus genus. An integrated approach based on the exploitation of nuclear DNA content, morphological traits and molecular markers, such as RAPD fingerprints and ITS-based SNPs, was employed. We studied a core collection of 54 distinct accessions, including 43 genotypes of the Citrus species (18 species or supposed species) and 11 genotypes of the Poncirus genus, which was used as the reference outgroup. Morphological trait analysis and statistical analysis of DNA content and markers were useful for reconstructing a Citrus phylogeny. In particular, our experiments aimed at estimating the genetic variation within and the genetic relatedness among C limon (L.) Burm., C. limonimedica and C. medica L. to shed light on the hybrid origin hypothesis of C. limonimedica. The results of the multidisciplinary analyses allowed us to confirm a remarkable differentiation between Poncirus and Citrus genera and to highlight a close relationship among the three investigated Citrus species but a distinct difference between these three species and other species in the Citrus genus. RAPD fingerprints and ITS polymorphisms enabled us to point out a variation gradient between lemon and citron, with C. limonimedica as a possible intermediate species. Some accessions of C. medica and C. limonimedica that deviate from such a trend suggest recurrent introgression and/or hybridisation with other species of Citrus.     

Variation in total phenolics and antioxidant capacity among European plum genotypes

Our objective was to determine the amount of variation in total phenolics and antioxidants present in European plum fruits, so that it can be utilized in breeding programs to enhance the health benefits. Total antioxidant capacity and total phenolic content of the fruit, and the fruit skin color were determined in 20 genotypes, comprising of released varieties and advanced selections of European plums. Among the 20 genotypes, the total antioxidant capacity ranged from 105 to 424 mg ascorbic acid equivalents (AAE)/100 g fresh weight (FW) while the total phenolic content was 86–413 mg gallic acid equivalents (GAE)/100 g FW. The two parameters had a strong correlation of r² = 0.96. A direct correlation between skin color intensity and total phenolic content could also be observed. This study demonstrates that there is adequate variation in total phenolic compounds and antioxidants within European plums and hence there is potential for improvement towards enhancing these health-promoting phytochemicals in this fruit.