P. Sedlák

Comparative morphological and molecular identification of Haemonchus species in sheep

SummaryA combined approach in the determination of Haemonchus nematodes from sheep was applied in this trial. Using selected morphological characters 90.2 % females and 84.2 % males of Haemonchus contortus and 9.8 % females and 15.8 % males of Haemonchus placei were identified. Although cluster analysis based on morphological identification clearly separated two Haemonchus species, H. contortus was exclusively detected in all specimens using restriction cleavage of the ITS-2 region with FspBI endonuclease as well as through the sequencing analysis. Because sheep from both farms have never had contact with other ruminants, and the farmers apply only closed flock turnover, we assume that only H. contortus mono-infection occurred on both farms. This opinion is also supported by molecular data. The most striking result of our study was the finding which indicates that the discriminant function is not able to accurately identify Haemonchus males at the species level.

S-genotyping of 25 sweet cherry (Prunus avium L.) cultivars from the Czech Republic

ABSTRACT Sweet cherry (Prunus avium L.) is a self-incompatible species. Determination of the S-genotypes of cherry cultivars is crucial for breeding and to select appropriate cultivars for cross-fertilisation and fruit set. In this study, we characterised the S-genotypes of 25 sweet cherry cultivars, some of which had being bred at the Research and Breeding Institute of Pomology (RBIP), Holovousy, Czech Republic, and others were European cultivars in the RBIP collection. S-genotyping was carried out by polymerase chain reaction using consensus primers for the S-RNase and SFB genes, and capillary electrophoresis. Nine different known S-haplotypes (S1, S2, S3, S4, S5, S6, S9, S13, and S16) were identified and the cultivars were assigned to 12 incompatibility groups. One local cultivar, ‘Ptačka z Plzně’, originated from a wild forest seedling and used as a pollinator, was assigned to Group 0 of universal donors. The pedigree of some cultivars was confirmed by their S-genotype. This study represents the first comprehensive S-genotype screening of sweet cherry genetic resources in the Czech Republic and will be useful for the design of crossing programmes and orchard management of these sweet cherry cultivars.

Phenotype and molecular diversity evaluation of some wild 2n Solanum species (super series Rotata)

Rotatamarkers. Totally 63 alleles of 20 cpSSR loci were detected i.e. 3.15 alleles on average per one microsatellite locus. Alleles ranged from two to six per locus. The highest polymorphism was detected in the locus ntcp9 and lowest were recorded having by two alleles in seven of loci. The average value of observed heterozygosity (H

Sensitivity to Fungicides and Esential Oils in Czech Isolates of Phytophthora infestans

Abstract A total of 235 Phytophthora infestans isolates were collected from five regions of the Czech Republic during the growing seasons 2012–2014 and 2016 and examined using the in vitro amended agar method for their sensitivity to metalaxyl-M (MFX), propamocarb-HCl (PCH), and dimethomorph (DMM). A majority of the isolates (50%) were sensitive to MFX. Resistant isolates were found in all four years of the survey; they represented 30% of the samples. The EC50 values of PCH in inhibiting mycelial growth of 65% of the overall isolates were higher than 100 μg ml−1, which indicates the occurrence of insensitivity to PCH in the Czech P. infestans populations. DMM was very effective, and the mycelial growth of all isolates tested was completely suppressed at the concentration of 0.1 μg ml−1. Furthermore, the efficacy of 12 plant essential oils was tested against 20 isolates of P. infestans using the in vitro amended agar method. Essential oils of Cymbopogon winterianus, Litsea cubeba, Mentha spicata, Pelargonium graveolens, Syzygium aromaticum, and Thymus vulgaris were observed to have the highest antifungal activity against P. infestans, with minimal inhibitory concentrations less than or equal to 1 μl ml−1.

Innovative multiplex and its evaluation for effective genotyping of wild cherry

Trees from the family Rosaceae play an important role in forest and agricultural ecosystems. Therefore, they are often an object of interest for both forest and horticultural tree breeders. Here, we present the utilization of an effective microsatellite (SSRs) genotyping method for wild cherry (Prunus avium L.) and verified the discriminatory power of the presented multiplex by genotyping 48 genetically distinctive individuals (plus-trees). Concerned loci were previously proven to be cross-compatible among various cultivars of cherry, hence, the method could have a broader utilization beyond to the field of forestry. Our technique is based on post-PCR processing of 15 polymorphic SSRs loci amplified in three multiplex reactions with fluorescently labeled primers (6-FAM, VIC, PET and NED). All PCR products could be pooled and analyzed simultaneously (pseudo 15-plex). In order to make this approach feasible, we redefined sequences of several primers. Thus, utilizing modified primers provides non-overlapping amplicons of each fluorescent dye.

S-Genotype Diversity in Wild Cherry Populations in the Czech Republic

Abstract Wild cherry (Prunus avium L.) S-genotyping is aimed to uncover and thus make it possible to select appropriate genotypes applicable in establishing commercial plantations and advanced forest tree breeding activities. The general and long-term aim is to increase genetic gain in economically valuable traits while maintaining sufficient genetic variability (represented by diverse S-alleles in population). We genotyped 123 accessions from wild cherry growing areas in the Czech Republic using polymerase chain reaction based length polymorphisms detection of S-RNase and SFB genes. The studied plant material revealed 18 different S-haplotypes, 54 S-genotypes corresponded to 25 defined incompatibility groups of cultivated sweet cherry. Eighteen unique S-genotypes were designated to group ‘0’ as a universal pollinator. Eleven new incompatibility groups were found out, of which four were cross-compatible with sweet cherry cultivars. The most frequent was a new incompatibility group S14S21 followed by the group S12S14. The haplotypes S14 (13%) and S1 (10%) were the most frequent whereas S20 was less frequent in the wild populations of cherry. The present study of S-genotyping in the wild cherry population reveals the genetic diversity structure of natural populations and hopefully will help define the breeding strategy including more accurate planning activities such as the optimal seed design of orchards.

Virulence and Mating Type of Phytophthora infestans Isolates in the Czech Republic

Abstract To evaluate the frequency and stability of the occurrence of P. infestans races and mating types in the Czech Republic, 338 monosporic isolates were collected from 31 sites in different potato-growing areas from 2012 to 2014 and in 2016. In total, 142 isolates were evaluated for virulence and race structure using the detached leaflet assay on Black’s differential set, supplemented with cultivar Sarpo Mira and somatic hybrid REG 46F. With the exception of virulence for resistance genes R9 and Rpi-blb-1, all virulence genes were detected among isolates, with a predominance of genes R1, R3, R7, R10, and R11. Most isolates were virulent to five or more R-genes, with a mean virulence complexity of 7.1. Among the 38 races detected, the most commonly occurring races were 1.2.3.4.6.7.10.11 and 1.2.3.4.7.10.11. Of the 338 isolates tested by the pairing test and the cleaved amplified polymorphism sequence (CAPS) marker, 40% were of the A1 mating type and 60% were of the A2 mating type, with an A1 : A2 isolate ratio demonstrating the predominance of the A2 mating type each year of the survey.

Evaluation of variations in plastid DNA non-coding regions in selected species of the genus Solanum

Sedláková V., Sedlák P., Zeka D., Domkářová J., Doležal P., Vejl P. (2017): Evaluation of variations in plastid DNA noncoding regions in selected species of the genus Solanum. Czech J. Genet. Plant Breed., 53: 127−131. The diversity of three non-coding plastid DNA loci (trnL/trnF spacer, trnV/16SrRNA spacer, trnL/trnL intron) was assessed in 16 Solanum L. species (135 individuals). Polymorphisms were detected by denaturing gradient gel electrophoresis (DGGE) and verified by direct sequencing. No intraspecific diversity and only poor interspecific diversity was detected. Unique S. mochiquense Ochoa specific length polymorphism at the trnL/trnL locus represented by duplication of an 18 bp segment was discovered. The detected DGGE interspecific trnL/trnF locus polymorphism did not specifically associate with single point mutations in the sequence confirmed by sequencing. The DGGE method was found to be a simple and cheap pre-exploring tool for mutation detection in compared DNA regions. Some identified polymorphisms can be used in the management of genetic resources.