Browsing by Subject "Sequence analysis"

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Detection and molecular characterization of canine babesiosis causative agent Babesia canis in naturally infected dogs in the Dobrogea area (Southeastern Romania)
(2023) Ionita, Mariana; Leica, Laurentiu; Wassermann, Marion; Mitrea, Emanuel; Nicorescu, Isabela Madalina; Mitrea, Ioan Liviu
Canine babesiosis is an emerging tick-borne disease of major veterinary concern in Europe. Its prevalence has increased in the last two decades and is spreading rapidly toward the north. The aim of this study was to investigate the genetic diversity of Babesia spp. strains isolated from naturally infected dogs in a tick-endemic area (Dobrogea) in southeastern Romania. For this purpose, a total of twenty-three samples from dogs diagnosed with various clinical forms of babesiosis, evaluated by means of clinical history, physical examination, and hematological tests, were subjected to a molecular investigation using PCR, sequencing analysis, and genetic characterization. A microscopic examination of thin Diff-quick-stained blood smears revealed large intra-erythrocytic Babesia piroplasms in all dogs. The PCR and sequencing analysis results indicated the presence of Babesia canis in 22 dogs (95.7%) and Babesia vogeli in 1 dog (4.3%). Among the B. canis isolates, two genotypes were distinguished based on two nucleotide substitutions (GA→AG) observed in the 18S rRNA gene sequences (at positions 609 and 610), with the AG genotype predominating (54.5% of samples), while the GA variant was identified in 9.1% of samples. In the remaining isolates (36.4%), both variants were identified. The B. vogeli-positive dog also tested positive for antibodies against Ehrlichia canis and displayed severe disease. This study reports, for the first time, the presence of genetically heterogenic B. canis strains in dogs with clinical babesiosis in Romania. These findings provide a basis for future studies on the relationship between the genetic structure of the causative agents of canine babesiosis in Romania and the course of the disease.
Population genomics of herbicide resistance in Alopecurus myosuroides
(2022) Kersten, Sonja; Schmid, Karl J.
Over the past 50 years, herbicides have often replaced mechanical and manual human weed control, thus representing a major factor in yield productivity in modern agriculture. Herbicide applications, however, exert strong selection pressures on weeds. As a consequence, these species have developed herbicide resistance through adaptive, beneficial alleles that increase in number to ensure the persistence of the populations, a phenomenon known as evolutionary rescue. A major research question is whether herbicide resistance adaptation is more likely to arise from standing genetic variation that was present before the onset of herbicide selection or from de novo mutations that arose after herbicide selection began. To address this question, I focused on target-site resistance (TSR) point mutations, which cause a lower binding affinity to the target protein of the respective herbicides. I first investigated the diversity of TSR haplotypes in populations of the grass species Alopecurus myosuroides (common name: blackgrass), and compared it with the TSR diversity outcome of simulated populations under both evolutionary scenarios. I first conducted a population genetics study of A. myosuroides, which is the most problematic weed in winter cereals across the European continent due to rapid resistance evolution. To obtain genome-wide polymorphic markers, I adapted a restriction site-associated DNA sequencing protocol to this species. I began by analyzing the diversity and population structure in a smaller local South German collection. The fact that I could differentiate populations on a local scale motivated me to extend the study to a European-wide collection, in which I found clear population structure, albeit with low differentiation and some evidence for admixture across Europe. In addition, I generated highly accurate long-read amplicons from single individuals of two loci, ACETYL-COA CARBOXYLASE (ACCase) and ACETOLACTATE SYNTHASE (ALS), which are the targets of the two main herbicide modes of action used in European cereal crops. I obtained completely phased haplotype information, supporting the analysis of haplotype diversity on a population level. I found a remarkable diversity of beneficial TSR mutations at the field level arising from multiple haplotypes of independent origin, so called soft sweeps. I used this information to perform forward simulations to investigate the evolutionary origin of these mutations. I found evidence that a majority of resistance mutations originated from standing genetic variation. While this at first may appear surprising, it is consistent with very large census and effective population sizes in blackgrass. Since long-read amplicon sequencing of single individuals could be costly and time consuming, I extended the analysis to pools of 150 to 200 individuals from Germany, Belgium, France, the Netherlands and the United Kingdom. By combining the power of a more stringent accuracy criterion in our long-reads and a novel clustering software (PacBio amplicon analysis), I was able to preserve individual haplotype information in pooled samples. Furthermore, in a proof of concept experiment, I was able to recover in our pools most haplotypes previously sequenced in individuals. The amplicon study provides a versatile workflow that can be easily adapted to any gene of interest in different species. In conclusion, I found that many A. myosuroides populations likely already have the genetic prerequisites not only for rapid evolution of resistance to currently used herbicides, but also to herbicides that have not yet been brought to market.