Research and Breeding Institute of Pomology Holovousy

The European plum (Prunus domestica L.) is a hexaploid species that is grown worldwide for its tasty fruits. Many pomological forms and varieties exist, and thus it is important for genebank curators, breeders, growers, and/or control authorities to distinguish them with certainty. The purpose of this study was to select and verify a set of simple sequence repeat (SSR) markers for reliable genotyping, and to optimize their use in a one-reaction format for easy routine practice. After testing 78 SSR markers from different diploid Prunus species, 8 SSR markers were selected, multiplexed, and successfully verified as being able to distinguish all 242 unique genotypes tested. The selected markers were relatively easily scored and highly heterogenic, giving more than 35 alleles/genotype on average. The allele atlas was created to become a valuable tool for allele calling that should lead to standardized and reliable genotyping results between laboratories. The population analysis confirmed high diversity of the Czech germplasm collection used. The kit was also successfully tested for diploid "plums" of various origins and interspecies hybrids, as these are sometimes phenotypically indistinguishable from hexaploid European plums. The one-tube approach substantially simplified the plum genotyping laboratory workflow, minimizes errors, and saves labor, time, and money.

Abstract Spiders are generalist predators able to respond very well to environmental conditions. Therefore, they are good bioindicators for forest management practices. The vertical distribution of spiders in forest stands has very rarely been studied. Thus, we investigated patterns in species richness, diversity, life‐history traits and functional diversity of spiders within the aboveground vertical stratification of oak dominated forests and their response to the canopy cover gradient. There were three localities sampled during four growing seasons (2008–2011, Southern Moravia, Czech Republic). Spiders were sampled monthly by flight intercept traps, which were placed in three stratigraphical levels of oak stands. Altogether, 3592 spiders, representing 18 families, and 112 distinguishable species were collected during the study. We found that species richness, total abundance the abundance of ambush hunters, other hunters and scarce, rare and very rare species of spiders increased from ground level to the canopy, and that species composition changed across the vertical gradient. Some of these characteristics were significantly influenced by interaction with canopy cover. Less common ballooning species preferred the ground part of forest stratification. Canopy cover evaluation showed that the abundance of orb web weavers and less common ballooning species was highest in the more closed canopies. The opposite trend was found for sheet web weavers, very abundant species and open habitat species. Space web weavers, other hunters and very common ballooning species showed increased abundance in the higher layers under conditions of more open canopy cover. Our results suggest that sampling spiders across the vertical gradient of forest stands might be important for a proper biodiversity and management practice evaluation, although most recent assessments were conducted on ground‐dwelling spiders obtained from pitfall traps.

Ants are key ecosystem service providers and can serve as important biological control agents in pest management. However, the effects of insecticides on common farmland ant species are poorly understood. We tested the effects of three commonly used insecticides on ants (Hymenoptera, Formicidae). The tested insecticides were acetamiprid (neonicotinoid; formulated as Mospilan 20 SP), deltamethrin (pyrethroid; formulated as Sanium Ultra), and sulfoxaflor (sulfilimine; formulated as Gondola). We tested two ant (Hymenoptera: Formicidae) species with different colony founding strategies, Lasius niger (Linnaeus, 1758) and Myrmica rubra (Linnaeus, 1758). We sprayed their queens with insecticides at concentrations recommended for use in foliar applications in agriculture, i.e., at 1.25 g L−1 (acetamiprid), 0.6 g L−1 (sulfoxaflor), and 0.875 g L−1 (deltamethrin). Further, we diluted the compounds in distilled water and tested them at 10%, 1%, and 0.1% of the field-recommended concentrations, and used distilled water as a control. We monitored the survival of the queens and the number of eggs laid. All three tested insecticides caused severe lethal and sublethal concentration-dependent effects. Even at concentrations three orders of magnitudes lower than recommended for field applications, significantly lower numbers of eggs were found in the queens’ nests. The extent of the sublethal effects of acetamiprid and sulfoxaflor was concentration-dependent and differed between the two ant species. Besides bees and bumblebees, ants represent an important group of hymenopterans that are severely affected even by low concentrations of the tested compounds and therefore should be included in risk assessment schemes.

The total aboveground biomass production, nutritional status, bearing and branching behaviors of the central leader and one year old shoots of young apple trees have been analyzed. The shoots were further characterized according to the length, shoot demography, and the production of terminal and lateral flowers. All the characteristics are described in connection with nitrogen supply and cultivar. Nitrogen represents one of the major macronutrients involved in the growth and development of the fruit trees. The understanding of the effect of nitrogen supply for flower bud formation can be further improved by detailed analyses of tree architecture. While the biomass production was cultivar specific, the trees within particular cultivar were characterized by almost similar growth with respect to the nitrogen supply. Cultivar ´Rubinola´ exhibited similar branching pattern, but higher vigor than ´Topaz´. As a result of higher apical dominance, ´Rubinola´ produced higher proportion of long shoots, but a lower quality of short shoots than ´Topaz´. Consequently, cultivar ´Rubinola´ produced only few terminal flowers on short shoots and lateral flowers dominantly in the distal zone, while ´Topaz´ was characterized by intensive terminal flowering, but the lateral flowers were more abundant in the median zone. Even a lower dose of spring nitrogen improved the flower bud formation on both terminal and lateral positions extending the flowering zone on one-year-old shoots. This further changed the branching and bearing behavior of the apple trees, which particularly allows to optimize their fertilization management. However, this effect appears to be further regulated by mechanism connected with apical dominance.

In sweet cherry (Prunus avium L.), quantitative trait loci have been identified for fruit maturity, colour, firmness, and size to develop markers for marker-assisted selection. However, resolution is usually too low in those analyses to directly target candidate genes, and some associations are missed. In contrast, genome-wide association studies are performed on broad collections of accessions, and assemblies of reference sequences from Tieton and Satonishiki cultivars enable identification of single nucleotide polymorphisms after whole-genome sequencing, providing high marker density. Two hundred and thirty-five sweet cherry accessions were sequenced and phenotyped for harvest time and fruit colour, firmness, and size. Genome-wide association studies were used to identify single nucleotide polymorphisms associated with each trait, which were verified in breeding material consisting of 64 additional accessions. A total of 1 767 106 single nucleotide polymorphisms were identified. At that density, significant single nucleotide polymorphisms could be linked to co-inherited haplotype blocks (median size ~10kb). Thus, markers were tightly associated with respective phenotypes, and individual allelic combinations of particular single nucleotide polymorphisms provided links to distinct phenotypes. In addition, yellow-fruit accessions were sequenced, and a ~ 90-kb-deletion on chromosome 3 that included five MYB10 transcription factors was associated with the phenotype. Overall, the study confirmed numerous quantitative trait loci from bi-parental populations using high-diversity accession populations, identified novel associations, and genome-wide association studies reduced the size of trait-associated loci from megabases to kilobases and to a few candidate genes per locus. Thus, a framework is provided to develop molecular markers and evaluate and characterize genes underlying important agronomic traits.

‘Candidatus Phytoplasma spp.’ are pathogenic bacteria that infect many plant species. ‘Candidatus Phytoplasma pyri’, one of the members of the 16SrX group causes pear decline disease that adversely affects pear crops. To describe the prevalence of ‘Ca. P. pyri’ genotypes in the Czech Republic, 143 pear samples were collected from 41 locations including commercial orchards as well as trees along roads. Phytoplasma was detected by PCR in 115 samples, and it was possible to determine imp gene genotype in 84 samples. The most frequent genotypes were A1, B1, and C, which were identified in 71% of phytoplasma positive samples. ‘Ca. P. pyri’ was present either alone or as a mix of two populations in 88% of genotyped samples, and in another 6% of samples it was found in a mixed infection with ‘Ca. Phytoplasma mali’. A sole infection with ‘Ca. Phytoplasma mali’ was observed in 6% of samples. As for symptoms, 19% of symptomatic samples were found to be phytoplasma negative, and 74% of asymptomatic samples proved to be phytoplasma positive; leaf roll was more often observed in phytoplasma positive samples, while leaf narrowing rather indicated the absence of phytoplasma. The mildest symptoms were observed in samples infected with ‘Ca. P. pyri’ of the A1 imp genotype.

Non-invasive flesh firmness prediction using near infrared spectroscopy has been perfected and validated on three apple varieties. Three novel calibration models were developed following three year's of repeated large-scale sampling of stored commercial apple varieties ‘Gala’, ‘Red Jonaprince’ and ‘Jonagored’. The spectroscopic results were compared directly with those obtained using the invasive method. Increased accuracy of calibration models was achieved with the newly established data collection model. The results exhibited coefficient of determination for calibration, R2, and ratio of prediction to deviation (RPD) in excess of 0.91 and 2.3, respectively, thus enabling excellent prediction of flesh firmness via a non-invasive and fast spectroscopic approach. The highest R2 obtained was 0.94, RPD 2.6, root mean square error of calibration 5.87 N, and root mean square error of cross-validation (internal) 6.75 N for variety ‘Red Jonaprince’. Our complex long-term study provided excellent external validated calibration models and the approach can help developing calibration models for commercial sorting lines using near infrared spectroscopy.

Drought stress is a serious threat. Therefore, improvements in crop productivity under conditions of limited water availability are vital to keep global food security. Apples and cherries belong to the most produced fruit worldwide. Thus, searching for their tolerant or resistant cultivars is beneficial for crop breeders to produce more resistant plants. We studied five apple (“Malinové holovouské”, “Fragrance”, “Rubinstep”, “Idared”, “Car Alexander”) and five cherry (“Regina”, “Napoleonova”, “Kaštánka”, “Sunburst”, “P-HL-C”) cultivars for their adaptation in response to progressive drought stress. The reaction of an in vitro culture to osmotic stress simulated by increasing polyethylene glycol (PEG) concentration in medium was evaluated through the morphological (fresh and dry weight, water content, leaf area), physiological (chlorophyll and carotenoids content), and biochemical (reactive oxygen species and malondialdehyde content) parameters. Drought-like stress negatively affected the water content, leaf areas, and chlorophyll content in both fruit species. Oxidative status and membrane damage of plants under water deficiency conditions occurred to be important indicators of stress tolerance mechanism. Cherries exhibited higher hydrogen peroxide levels compared to apples, whereas their malondialdehyde values were generally lower. The overall results indicated wide tolerance range to water deficit among apple and cherry in vitro culture as well as among cultivars within single plant species.