Martina Melounová

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.

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.

Due to their nutritional value and sustainability, edible insect-based foods are gaining popularity in Europe. Their use is regulated by EU legislation, which defines authorised species and sets labelling requirements. Molecular tools are being developed to authenticate such products. In this study, yellow mealworm (Tenebrio molitor) larvae authorised for human consumption were fed wheat flour-based diets containing varying proportions of house cricket (Acheta domesticus) flour for 21 days. This was followed by a 48 h starvation period to assess the persistence of insect DNA in the digestive tract. Two novel, species-specific, single-copy markers were designed: ampd gene for the Acheta domesticus and MyD88 gene for the Tenebrio molitor. These were applied using qPCR and ddPCR. Both methods successfully detected cricket DNA in the guts of starved larvae. Linear regression analysis revealed a strong, statistically significant correlation between the proportion of Acheta domesticus flour in the diet and the normalised relative quantity of DNA. ddPCR proved to be more sensitive than qPCR, particularly in the detection of low DNA levels. These results suggest that the presence of DNA from undeclared insect species in edible insects may be indicative of their diet rather than contamination or adulteration. This highlights the importance of contextual interpretation in food authenticity testing.

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.

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.

Somatic hybridization has been frequently used to overcome sexual incompatibility between potato and its secondary germplasm. The primary objective of this study was to produce and evaluate somatic hybrids of Solanum tuberosum (Stub) and S. bulbocastanum (Sblb) for breeding purposes. In 2007, 23 somatic hybrids were produced using an electrofusion of mesophyll protoplasts of diploid (2n = 2x = 24) potato line StubDH165 and S. bulbocastanum PI24351 (Sblb66). Phenotype of somatic hybrids in field conditions were evaluated, together with constitution and stability of 30 nuclear (ncSSR) and 27 cytoplasmic (cpSSR) microsatellite markers and content of main glycoalkaloids. All somatic hybrids had very high field resistance against late blight, but the plants were infertile: the viability of pollen grains insignificantly varied between 0.58 and 8.97%. A significant somaclonal variation was observed in terms of the morphology of plants, the date of emergence, the quantity of harvested tubers, the content of glycoalkaloids in foliage, and nuclear microsatellite markers (ncSSR). The analysis of ncSSR identified five distinct genotypes of hybrids partly associated with phenotype variations. The process of somatic hybridization with regeneration of shoots was identified as the most likely source of somaclonal variation because the ncSSR genotypes of hybrids, which were maintained in vitro, remained stable for more than 10 years. The infertile somatic hybrids have no practical breeding potential, but they are considered very suitable for advanced studies of the differential expression of genes in the pathways linked to dormancy of tubers and synthesis of glycoalkaloids.

High-quality simple sequence repeat (SSR) markers are invaluable tools for revealing genetic variability which could be utilized for many purposes, such as breeding new varieties or the identifying current ones, among other applications. Based on the analysis of 3.7 million EST sequences and 15 genomic sequences from bacterial artificial chromosome (BAC) libraries, 200 trinucleotide genic (EST)-SSR and three genomic (gSSR) markers were tested, where 17 of them fulfilled all criteria for quality markers. Moreover, the reproducibility of these new markers was verified by two genetics laboratories, with a mean error rate per allele and per locus equal to 0.17%. These markers were tested on 38 accessions of Papaver somniferum and nine accessions of another five species of the Papaver and Argemone genera. In total, 118 alleles were detected for all accessions (median = 7; three to ten alleles per locus) and 88 alleles (median = 5; three to nine alleles per locus) within P. somniferum alone. Multivariate methods and identity analysis revealed high resolution capabilities of the new markers, where all but three pair accessions (41 out of 47) had a unique profile and opium poppy was distinguished from other species.