cor1 Gene: A Suitable Marker for Identification of Opium Poppy (Papaver somniferum L.).

This paper discusses the development of rapid, reliable, and accurate polymerase chain reaction (PCR) assays for detecting opium poppy (Papaver somniferum L.) in food. Endpoint, quantitative, and digital PCRs were compared based on the amplification of a newly developed DNA marker targeting the NADPH-dependent codeinone reductase (COR) gene. Designed assays were shown to be highly specific and sensitive in discriminating opium poppy from other plant species, even in heat-treated and food samples. Digital PCR was the most sensitive, with a detection limit of up to 5 copies, i.e., approximately 14 pg of target DNA per reaction. Quantitative and digital PCR further allowed the quantification of opium poppy in up to 1.5 ng and 42 pg (15 copies) of target DNA in a sample, respectively. In addition, two duplex PCRs have been developed for the simultaneous detection of opium poppy DNA and representatives of (i) the Papaveraceae family or (ii) the Plantae kingdom. Finally, all designed assays were successfully applied for analysis of 15 commercial foodstuffs; two were suspected of being adulterated. The study results have an important impact on addressing food fraud and ensuring the safety and authenticity of food products. Beyond food adulteration, the study may also have significant implications for forensics and law enforcement.

Bias in sex ratios and polyandry rate in reproduction of Leptinotarsa decemlineata.

The Colorado potato beetle (CPB, Leptinotarsa decemlineata Slechtd.) 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.

Phenotypic, molecular and biochemical evaluation of somatic hybrids between Solanum tuberosum and S. bulbocastanum.

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.

Detection of sex in adults and larvae of Leptinotarsa decemlineata on principle of copy number variation.

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 gene LdVssc and autosomal gene LdUBE3B were identified as appropriate target and reference loci, respectively. The copy numbers (CNV) of LdVssc in males and females were estimated using standard droplet digital PCR (ddPCR) and real-time PCR (qPCR). With both methods, CNVs were bimodally distributed (BAddPCR = 0.709 and BAqPCR = 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.

Czechoslovakian Wolfdog Genomic Divergence from Its Ancestors Canis lupus, German Shepherd Dog, and Different Sheepdogs of European Origin.

This study focused on the genomic differences between the Czechoslovakian wolfdog (CWD) and its ancestors, the Grey wolf (GW) and German Shepherd dog. The Saarloos wolfdog and Belgian Shepherd dog were also included to study the level of GW genetics retained in the genome of domesticated breeds. The dataset consisted of 131 animals and 143,593 single nucleotide polymorphisms (SNPs). The effects of demographic history on the overall genome structure were determined by screening the distribution of the homozygous segments. The genetic variance distributed within and between groups was quantified by genetic distances, the FST index, and discriminant analysis of principal components. Fine-scale population stratification due to specific morphological and behavioural traits was assessed by principal component and factorial analyses. In the CWD, a demographic history effect was manifested mainly in a high genome-wide proportion of short homozygous segments corresponding to a historical load of inbreeding derived from founders. The observed proportion of long homozygous segments indicated that the inbreeding events shaped the CWD genome relatively recently compared to other groups. Even if there was a significant increase in genetic similarity among wolf-like breeds, they were genetically separated from each other. Moreover, this study showed that the CWD genome carries private alleles that are not found in either wolves or other dog breeds analysed in this study.

OpiumPlex is a novel microsatellite system for profiling opium poppy (Papaver somniferum L.).

Opium poppy (Papaver somniferum L.) is a versatile plant exploited by the pharmaceutical and food industries. Unfortunately, it is also infamously known as a source of highly addictive narcotics, primarily heroin. Drug abuse has devastating consequences for users and also has many direct or indirect negative impacts on human society as a whole. Therefore, developing a molecular genetic tool for the individualization of opium poppy, raw opium or heroin samples could help in the fight against the drug trade by retrieving more information about the source of narcotics and linking isolated criminal cases. Bioinformatic analysis provided insight into the distribution, density and other characteristics of roughly 150 thousand microsatellite loci within the poppy genome and indicated underrepresentation of microsatellites with the desired attributes. Despite this fact, 27 polymorphic STR markers, divided into three multiplexed assays, were developed in this work. Internal validation confirmed species-specific amplification, showed that the optimal amount of DNA is within the range of 0.625-1.25 ng per reaction, and indicate relatively well balanced assays according to the metrics used. Moreover, the stutter ratio (mean + 3 SD 2.28-15.59%) and allele-specific stutters were described. The analysis of 187 individual samples led to the identification of 158 alleles in total, with a mean of 5.85 alleles and a range of 3-14 alleles per locus. Most of the alleles (151) were sequenced by the Sanger method, which enabled us to propose standardized nomenclature and create three allelic ladders. The OpiumPlex system discriminates most of the varieties from each other and pharmaceutical varieties from the others (culinary, dual and ornamental).

New EST-SSR Markers for Individual Genotyping of Opium Poppy Cultivars (Papaver somniferum L.).

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.

Long-term occurrence of Trichuris species in wild ruminants in the Czech Republic.

The aim of this study was to identify Trichuris species in wild ruminants from 32 localities in the Czech Republic using morphological and molecular methods (ITS1-5.8S RNA-ITS2 region polymorphisms). Trichurids were obtained from 176 wild ruminants (roe deer, sika deer, red deer, fallow deer and mouflons) that were culled between 2009 and 2017. Trichuris discolor is the predominant trichurid of all of the above-mentioned wild ruminants, whereas Trichuris ovis was identified less frequently in roe deer, fallow deer, sika deer and mouflons. Red deer were parasitised exclusively by T. discolor. Young hosts under 1 year of age were more intensively infected by trichurids than were adults (χ2 = 32.02, p = 0.00). Trichurid prevalence results obtained through coprological methods and those based on parasitological dissections differed significantly (χ2 = 16.26, p = 0.00). The regression analysis indicated that the eggs per gram (EPG) threshold (20 EPG) was exceeded only if the host was parasitised by more than 7 trichurid females. Full concordance between the positive results obtained by the coprological methods and those obtained via direct dissections was achieved when the number of trichurid females per host exceeded 51.

Dealing with AFLP genotyping errors to reveal genetic structure in Plukenetia volubilis (Euphorbiaceae) in the Peruvian Amazon.

An analysis of the population structure and genetic diversity for any organism often depends on one or more molecular marker techniques. Nonetheless, these techniques are not absolutely reliable because of various sources of errors arising during the genotyping process. Thus, a complex analysis of genotyping error was carried out with the AFLP method in 169 samples of the oil seed plant Plukenetia volubilis L. from small isolated subpopulations in the Peruvian Amazon. Samples were collected in nine localities from the region of San Martin. Analysis was done in eight datasets with a genotyping error from 0 to 5%. Using eleven primer combinations, 102 to 275 markers were obtained according to the dataset. It was found that it is only possible to obtain the most reliable and robust results through a multiple-level filtering process. Genotyping error and software set up influence both the estimation of population structure and genetic diversity, where in our case population number (K) varied between 2-9 depending on the dataset and statistical method used. Surprisingly, discrepancies in K number were caused more by statistical approaches than by genotyping errors themselves. However, for estimation of genetic diversity, the degree of genotyping error was critical because descriptive parameters (He, FST, PLP 5%) varied substantially (by at least 25%). Due to low gene flow, P. volubilis mostly consists of small isolated subpopulations (ΦPT = 0.252-0.323) with some degree of admixture given by socio-economic connectivity among the sites; a direct link between the genetic and geographic distances was not confirmed. The study illustrates the successful application of AFLP to infer genetic structure in non-model plants.

Reliable molecular differentiation of Trichuris ovis and Trichuris discolor from sheep (Ovis orientalis aries) and roe deer (Capreolus capreolus) and morphological characterisation of their females: morphology does not work sufficiently.

The main aim of the study was to evaluate associations between morphological variability of Trichuris females from sheep and roe deer and their rDNA polymorphism in whipworm populations from the Czech Republic. The results introduced the use of new molecular markers based on the internal transcribed spacer (ITS)1-5.8S RNA-ITS2 region polymorphisms, as useful tools for the unambiguous differentiation of congeners Trichuris ovis and Trichuris discolor. These markers revealed both parasites in roe deer and in sheep; however, T. ovis females predominated in sheep while T. discolor females occurred mostly in roe deer. Additional analysis of ITS1-5.8 rRNA-ITS2 discovered the genetic uniformity of the analysed T. discolor but high haplotype variation of T. ovis. Simultaneously, molecularly designated female individuals of both species were categorised into four morphotypes (MT) on the basis of morphology of genital pore area. MT1 and MT4 (vulvar opening on everted vaginal appendage/on visible cuticular bulge) occurred only in T. ovis, MT2 (uneverted vagina-vulvar opening without any elevation) was identified only in T. discolor and MT3 (transient type of vulvar opening on a small swelling) was observed in both species. Statistical analysis of biometric data confirmed that morphology of vulva is not a reliable marker for the species determination. On the basis of the ITS1-5.8S RNA-ITS2 region variability, we carried out a phylogenetic analysis (maximum likelihood method, Hasegawa-Kishino-Yano model) which showed that T. ovis haplotypes from the Czech Republic and Ireland and T. discolor haplotypes from the Czech Republic, Spain, Iran and Japan are sister OTUs.

Proteomic and physiological approach reveals drought-induced changes in rapeseeds: Water-saver and water-spender strategy.

The cultivar-dependent differences in Brassica napus L. seed yield are significantly affected by drought stress. Here, the response of leaf proteome to long-term drought (28days) was studied in cultivars (cvs): Californium (C), Cadeli (D), Navajo (N), and Viking (V). Analysis of twenty-four 2-D DIGE gels revealed 134 spots quantitatively changed at least 2-fold; from these, 79 proteins were significantly identified by MALDI-TOF/TOF. According to the differences in water use, the cultivars may be assigned to two categories: water-savers or water-spenders. In the water-savers group (cvs C+D), proteins related to nitrogen assimilation, ATP and redox homeostasis were increased under stress, while in the water-spenders category (cvs N+V), carbohydrate/energy, photosynthesis, stress related and rRNA processing proteins were increased upon stress. Taking all data together, we indicated cv C as a drought-adaptable water-saver, cv D as a medium-adaptable water-saver, cv N as a drought-adaptable water-spender, and cv V as a low-adaptable drought sensitive water-spender rapeseed. Proteomic data help to evaluate the impact of drought and the extent of genotype-based adaptability and contribute to the understanding of their plasticity. These results provide new insights into the provenience-based drought acclimation/adaptation strategy of contrasting winter rapeseeds and link data at gasometric, biochemical, and proteome level. SIGNIFICANCE: Soil moisture deficit is a real problem for every crop. The data in this study demonstrates for the first time that in stem-prolongation phase cultivars respond to progressive drought in different ways and at different levels. Analysis of physiological and proteomic data showed two different water regime-related strategies: water-savers and spenders. However, not only water uptake rate itself, but also individual protein abundances, gasometric and biochemical parameters together with final biomass accumulation after stress explained genotype-based responses. Interestingly, under a mixed climate profile, both water-use patterns (savers or spenders) can be appropriate for drought adaptation. These data suggest, than complete "acclimation image" of rapeseeds in stem-prolongation phase under drought could be reached only if these characteristics are taken, explained and understood together.

Significant relationships among frost tolerance and net photosynthetic rate, water use efficiency and dehydrin accumulation in cold-treated winter oilseed rapes.

Five winter oilseed rape cultivars (Benefit, Californium, Cortes, Ladoga, Navajo) were subjected to 30 days of cold treatment (4 °C) to examine the effect of cold on acquired frost tolerance (FT), dehydrin (DHN) content, and photosynthesis-related parameters. The main aim of this study was to determine whether there are relationships between FT (expressed as LT50 values) and the other parameters measured in the cultivars. While the cultivar Benefit accumulated two types of DHNs (D45 and D35), the other cultivars accumulated three additional DHNs (D97, D47, and D37). The similar-sized DHNs (D45 and D47) were the most abundant; the others exhibited significantly lower accumulations. The highest correlations were detected between LT50 and DHN accumulation (r=-0.815), intrinsic water use efficiency (WUEi; r=-0.643), net photosynthetic rate (r=-0.628), stomatal conductance (r=0.511), and intracellular/intercellular CO2 concentration (r=0.505). Those cultivars that exhibited higher Pn rate in cold (and further a significant increase in WUEi) had higher levels of DHNs and also higher FT. No significant correlation was observed between LT50 and E, PRI, or NDVI. Overall, we have shown the selected physiological parameters to be able to distinguish different FT cultivars of winter oilseed rape.