Petr Sedlák

High-throughput molecular technology DArTseq generates markers for potential use in molecular breeding of crops. Using DArTseq, we analysed a comprehensive set of 333 European potato varieties reflecting the outcomes of long-term breeding hist...

The most important potato viruses, PVY and PLRV, are transmitted by vector aphids, particularly Myzus persicae under the climatic conditions of Czechia. The integrated seed-potato protection requires detailed information on virus transmission potential in the potato fields. We developed a new system for simultaneous detection of PVY and PLRV in aphid vectors using two pairs of virus-specific primers targeting conserved genomic regions. The primers function reliably in duplex SYBR Green-based one-step qPCR combined with melting analysis, enabling virus identification by distinct amplicon melting temperatures. The method was validated using diverse virus strains from gene bank collections, artificially infected aphids and aphids collected during field monitoring. The presence of PLRV and PVY was assessed in 446 aphids, including Myzus persicae , Aphis nasturtii and Phorodon humuli , captured using yellow pan traps during 2024. Of these, 42% were PVY-positive, 2.6% PLRV-positive and 1.2% carried mixed infection. The frequency of PVY- and PLRV-positive aphids corresponded with viruses occurrence in postharvest tubers testing. This methodology is suitable for detection of PVY and PLRV in potato plants, rapid evaluation of aphid infection potential in potato fields and studies of viruses transmissibility by other aphid species.

The genus Solanum comprises numerous wild and cultivated species that are important for potato breeding. This pilot-scale study aimed to evaluate the genetic diversity in 44 accessions from Solanum sect. Petota, comprising wild species, Andean...

Potato leaf diseases are the greatest threat to potato yields worldwide. A novel system for detection of spores was designed, combining a rotation spore trap with droplet digital PCR (ddPCR). This novel system was then evaluated for disease signalling and to study the dynamics of leaf pathogens. The spore traps were placed in six representative potato fields within Czechia’s primary potato growing regions. Up to 24 spores’ specimens were collected per locality in three-day intervals from May to September 2024. The DNA extraction procedures were optimised and new species-specific combinations of primers and TaqMan probes were designed, verified, and multiplexed to detect Alternaria alternata and A. solani based on polymorphisms in beta-tubulin gene, and P. infestans based on unique sequences in Avr4 effector protein gene. The optimised ddPCR protocol worked consistently in both singleplex and multiplex. Reliable detection limits were measured to be approximately one copy of target DNA per µL of PCR mix. Field testing detected the presence of Alternaria spp. spores only. Although Phytophthora infestans were present, its sporangia levels remained below the detection threshold; likely due to the prevailing climatic conditions that year. Single Alternaria species generally differed by the dynamics of seasonal conditions, and these differences were consistent amongst all localities. The progression pattern of A. alternata strongly correlated with five-day moving sums of temperatures exceeding 25 °C (growing degree hours - GDH), suggesting a suitable indicator for signalling plant protection responses.

The complex life cycle, high reproductive potential and ability to quickly develop resistance to insecticides are key factors contributing to the destructiveness of the peach–potato aphid (Myzus persicae Sulzer) among pest species. Monitoring its population dynamics at a large scale allows us to better understand M. persicae biology and take relevant measures for pest management. For this purpose, reliable molecular tools are needed. Based on the analysis of 128,362 microsatellite loci, we developed four multiplex assays including 49 comprehensively characterised SSR markers. Internal validation confirmed the species specificity and low genotyping error (e_a = 0.8%, e_l = 0.99%, e_obs = 22.7%) of the assays. A total of 194 alleles were identified (mean = 4 alleles per locus, range = 2–8 alleles per locus) within a group of 365 aphid accessions collected in the Vysočina region (Czechia). The studied aphid population showed the typical characteristics expected of the species with clonal or partially clonal reproduction (heterozygote excess, negative F_IS, moderate-to-high linkage disequilibrium (LD), and distortion of the H-W equilibrium for most of the loci), and did not exhibit any stratification on a spatiotemporal level. Owing to the high discriminatory power of the markers, we discovered that the population sample was founded upon a small number of fundatrices, as only five dominating lineages comprising over 70% of all accessions were identified. In conclusion, this study identified a significant number of new high-quality markers with the high discriminatory power necessary for revealing the population structure and dynamics of M. persicae, which holds considerable potential in both general biological and agricultural research.

The Colorado potato beetle (CPB; Leptinotarsa decemlineata) is an important potato pest with known resistance to pyrethroids and organophosphates in Czechia. Decreased efficacy of neonicotinoids has been observed in last decade. After the restriction of using chlorpyrifos, thiacloprid and thiamethoxam by EU regulation, growers seek for information about the resistance of CPB to used insecticides and recommended antiresistant strategies. The development of CPB resistance to selected insecticides was evaluated in bioassays in 69 local populations from Czechia in 2017–2022 and in 2007–2022 in small plot experiments in Zabcice in South Moravia. The mortality in each subpopulation in the bioassays was evaluated at the field-recommended rates of insecticides to estimate the 50% and 90% lethal concentrations (LC₅₀ and LC₉₀, respectively). High levels of CPB resistance to lambda-cyhalothrin and chlorpyrifos were demonstrated throughout Czechia, without significant changes between years and regions. The average mortality after application of the field-recommended rate of lambda-cyhalothrin was influenced by temperature before larvae were sampled for bioassays and decreased with increasing temperature in June. Downwards trends in the LC₉₀ values of chlorpyrifos and the average mortality after application of the field-recommended rate of acetamiprid in the bioassay were recorded over a 6-year period. The baseline LC₅₀ value (with 95% confidence limit) of 0.04 mg/L of chlorantraniliprole was established for Czech populations of CPBs for the purpose of resistance monitoring in the next years. Widespread resistance to pyrethroids, organophosphates and neonicotinoids was demonstrated, and changes in anti-resistant strategies to control CPBs were discussed.

A panel of X-linked microsatellite markers was newly designed using the data from a previous sequencing project available in NCBI and used for a study of the Colorado potato beetle (CPB, Leptinotarsa decemlineata) X-haplotype variability. The analysis of scaffolds 49 and 61 (newly identified as fragments of CPB chromosome X) found ten high-quality markers, which were arranged in two PCR multiplexes and evaluated in both 420 CPB adults, collected from 14 localities of Czechia and Slovakia, and 866 larvae from five single-female families from two more Czech localities. Length polymorphisms found in 6 loci have predicted 192 potential X-haplotypes, however, only 36 combinations were detected in the adult males (N = 189), and seven additional ones in the larvae. The X-haplotypes were also generally unevenly distributed; five of the most frequent haplotypes were detected in 55% of males, 19 repeating up to ten-times in 38.7% of males and the remained 12 occurred uniquely in 6.3% of males. Bulk analysis of X-haplotypes dissimilarity indicated seven haplotype groups diversified by mutations and recombinations. Two haplotypes showed a distinctive regional distribution, which indicates an east–west disruption of CPB migration probably caused by different environments of localities in the South Bohemia region and Vysocina region. On the contrary, the results indicate a south–north migration corridor alongside the Vltava River. In the single-female families, from 6 to 13 distinct paternal haplotypes were detected, which proved and quantified a frequented polyandry in CPB.

Proof of concept salt tolerance and plasticity. Wild germplasm may represent a precious source of genetic variability for salt tolerance. This study evaluated the morphological changes occurring under controlled and saline conditions in tuber-bearing S. bulbocastanum, S. commersonii, S. chomatophyllum, S. multidissectum, S. pinnactisectum, S. phureja, and cultivated S. tuberosum. An in vitro screening method was employed. Significant phenotypic variations were observed for all phenotypic traits analyzed at all NaCl levels (0, 40, 60, and 120 mM). In addition, a significant correlation between root plasticity and salt tolerance was found. Further, changes in proline and total phenolic content were assessed to envisage the metabolic adjustments of tolerant clones towards salinity. The most promising sources of tolerance were identified in S. commersonii and S. multidissectum and information obtained is discussed from a breeding perspective.

The Colorado potato beetle (CPB,Leptinotarsa decemlineataSlechtd.) is an invasive pest with economic importance worldwide. Sex ratios during egg-hatching and a frequency of polyandry in single-female families were analysed to clarify the reproduction strategy of CPB, which was still known only in fragments. 1296 just hatching 1st instar CPB larvae were collected from 19 single-female families, of which 13 were random families collected from potato fields and 6 were families produced by laboratory farming of naturally fertilised females. All larvae were analysed to detect a sex using a qPCR-based method and to detect polymorphisms in genotypes of 9 microsatellite (SSR) markers. The bias in sex ratio in favour of females was confirmed using linear mixed-effects model in both experimental groups of families: field collections (F = 36.39; P = 0.0001) and laboratory farming (F = 13.74;P = 0.0139). The analysis of diversity in microsatellites proved the polyandry in all progenies as 73% of analysed segregation patterns did not match with the patterns expected for full-sib progenies; on average per locus, 46% of allelic and 49.7% of genotype ratios showed irregular segregation. Both findings contribute toward understanding CPB success rate as an invasive species, as the preferential bearing of females with polyandry has a great potential to keep fitness of progenies, to maintain and operate population diversity, and to accelerate the reproduction of the pest.

The identification of sex in larvae of insects is usually challenging or even impossible, while in adults the sexual dimorphism is usually evident. Here, we used copy number analysis to develop a method of sex detection in Colorado potato beetle (Leptinotarsa decemlineata), which has an X0 sex determination system. The X linked geneLdVsscand autosomal geneLdUBE3Bwere identified as appropriate target and reference loci, respectively. The copy numbers (CNV) ofLdVsscin males and females were estimated using standard droplet digital PCR (ddPCR) and real-time PCR (qPCR). With both methods, CNVs were bimodally distributed (BA_ddPCR = 0.709 and BA_qPCR = 0.683) with 100% ability to distinguish females from males. The use of qPCR-based sex detection in a broad collection of 448 random CPB adults showed a perfect association (Phi = 1.0,p < 0.05) with the true sexes of adults, with mean CNV in females of 2.032 (SD = 0.227) and 0.989 in males (SD = 0.147). In the collection of 50 random 4th instar larvae, 27 females and 23 males were identified, consistent with the expected 1:1 sex ratio (p = 0.689). The method is suitable for sexing in all stages of ontogenesis. The optimal cost-effective application of the method in large populations requires the DNA extraction using CTAB, the qPCR assay in one biological replicate and three technical replicates of each marker, and the use of one randomly chosen male per run to calibrate calculation of CNV.