Browsing by Subject "SNP"

Now showing 1 - 7 of 7
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    Characterization of genetic variation among Ethiopian barley (Hoerdeum vulgare L.) genotypes
    (2019) Abtew, Wosene Gebreselassie; Knierim, Andrea
    Barley (Hordeum vulgare L.) is a major cereal crop in Ethiopia and accounts for 8% of the total cereal production based on cultivation area. Farmers may face unpredictable rainfall and drought stress patterns such as terminal drought where rainfall ends before crops have completed their physiological maturity, which then poses a challenge to crop production. The absence of efficient weather forecasts and a lack of efficient communication channels for resource-poor farmers ask for the development of varieties that are robust to such irregularities. A goal of plant breeding for areas with variable climate and limited resources for agricultural inputs is to produce stable varieties with higher average yield across diverse environments and growing conditions. Genotype by environment (G x E) interactions, however, frequently interfere with the selection of widely adapted genotypes. Knowledge about the yield stability of existing Ethiopian barley varieties and landraces under changing environmental variables is important for the future development of barley varieties with high and stable yields. In addition, yield components are quantitative with substantial influence of environment. Yield components also compensate each other in trait correlation dynamics. Since grain yield is a more complex trait than its components, environmental effects and genotype-by-environment (G x E) interactions for grain yield are stronger than for its components. Therefore, indirect selection of yield components may be more efficient than selection on grain yield per se to obtain higher yielding and stable cultivars. A study, therefore, was initiated to 1) characterize the response of a diverse set of barley genotypes to different locations and variable planting dates and identify genotypes with wide adaptation and stable performance and/or genotypes with specific altitude and planting date 2) determine traits that contribute to high and stable yields across a range of different environments and planting dates 3) determine the pattern of population structure and genetic parameters among genotypes conserved in Ethiopian and German gene banks in for different period of time as well as currently growing in farmers’ field. In order to meet the objectives 18 genotypes were tested at four different sowing dates with 15 days interval in different locations (Ambo and Jimma) and years (2012 and 2013). The tested genotypes revealed a wide variation for both static and dynamic yield stability measures. Compared to improved cultivars, farmers landraces displayed higher average static stability and similar superiority indices (dynamic stability). These landraces are therefore a source of germplasm for breeding resilient barley cultivars. Staggered planting proved to be a useful method for evaluating genotype stability across environmental factors beyond location and season. In addition, we also noticed that compensatory relationship between kernels per spike and thousand kernel weight in landraces. Kernels per spike and number of fertile tillers can be proposed as robust traits in barley breeding for a wider adaptation as they had significant and consistent positive total effects on grain yield. In order to determine the pattern of population structure and genetic parameters among genotypes of different origin and gene banks, DNA samples were subject to double-digest by ApeK1 and Hind III enzymes. After sequencing, raw read was checked for major quality parameters. Sequence reads were then filtered for sequencing artifacts and low quality reads (preprocessing). The pre-processed reads were aligned to genome of barley cultivar Morex to call SNPs. Values of observed heterozygosity (Ho) ranged from 0.250 to 0.337 and were higher than the expected heterozygosity (He) that varied from 0.180 to 0.242 in genotypes of all origins. The inbreeding coefficient (FIS) values that ranged between -0.240 and -0.639 across the regions were also higher and negative suggesting existence of excess outcrossing than expected. Based on the inferred clusters by the ADMIXTURE, high Fst values were observed between clusters suggesting high genetic differentiation among the genotypes tested though differentiation was not based on location. In addition, genetic differentiation computed based on the predetermined location, altitude and source of genotypes suggested weak differentiation among the groups. These results indicate that, in Ethiopia, barley genetic variation between regions and altitudes were less pronounced than within region and altitude variations. This calls for the germplasm collection strategies to be cautious in considering location and altitude as a main factor of variation thus strategies should focus on exploiting the within region variation also for better germplasm conservation and utilization. The static yield stability of landrace has to be utilized by breeders for their wider recommendations for those farmers who cannot afford use of farm inputs and specific cultivars. In addition, the relative robustness as well as plasticity of traits sorted by the current study can be incorporated in the breeding strategy of barley in Ethiopia.
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    Comparison of omics technologies for hybrid prediction
    (2019) Westhues, Matthias; Melchinger, Albrecht E.
    One of the great challenges for plant breeders is dealing with the vast number of putative candidates, which cannot be tested exhaustively in multi-environment field trials. Using pedigree records helped breeders narrowing down the number of candidates substantially. With pedigree information, only a subset of candidates need to be subjected to exhaustive tests of their phenotype whereas the phenotype of the majority of untested relatives is inferred from their common pedigree. A caveat of pedigree information is its inability to capture Mendelian sampling and to accurately reflect relationships among individuals. This shortcoming was mitigated with the advent of marker assays covering regions harboring causal quantitative trait loci. Today, the prediction of untested candidates using information from genomic markers, called genomic prediction, is a routine procedure in larger plant breeding companies. Genomic prediction has revolutionized the prediction of traits with complex genetic architecture but, just as pedigree, cannot properly capture physiological epistasis, referring to complex interactions among genes and endophenotypes, such as RNA, proteins and metabolites. Given their intermediate position in the genotype-phenotype cascade, endophenotypes are expected to represent some of the information missing from the genome, thereby potentially improving predictive abilities. In a first study we explored the ability of several predictor types to forecast genetic values for complex agronomic traits recorded on maize hybrids. Pedigree and genomic information were included as the benchmark for evaluating the merit of metabolites and gene expression data in genetic value prediction. Metabolites, sampled from maize plants grown in field trials, were poor predictors for all traits. Conversely, root-metabolites, grown under controlled conditions, were moderate to competitive predictors for the traits fat as well as dry matter yield. Gene expression data outperformed other individual predictors for the prediction of genetic values for protein and the economically most relevant trait dry matter yield. A genome-wide association study suggested that gene expression data integrated SNP interactions. This might explain the superior performance of this predictor type in the prediction of protein and dry matter yield. Small RNAs were probed for their potential as predictors, given their involvement in transcriptional, post-transcriptional and post-translational regulation. Regardless of the trait, small RNAs could not outperform other predictors. Combinations of predictors did not considerably improve the predictive ability of the best single predictor for any trait but improved the stability of their performance across traits. By assigning different weights to each predictor, we evaluated each predictors optimal contribution for attaining maximum predictive ability. This approach revealed that pedigree, genomic information and gene expression data contribute equally when maximizing predictive ability for grain dry matter content. When attempting to maximize predictive ability for grain yield, pedigree information was superfluous. For genotypes having only genomic information, gene expression data were imputed by using genotypes having both, genomic as well as gene expression data. Previously, this single-step prediction framework was only used for qualitative predictors. Our study revealed that this framework can be employed for improving the cost-effectiveness of quantitative endophenotypes in hybrid prediction. We hope that these studies will further promote exploring endophenotypes as additional predictor types in breeding.
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    Identification and analysis of a transcriptome of Douglas-fir (Pseudotsuga menziesii) and population structure inference using different next-generation sequencing techniques
    (2015) Müller, Thomas; Schmid, Karl J.
    Predictions assume severe changes in the climatic conditions in Central Europe in the coming decades. Longer periods of drought and less precipitation during summer are expected. Plants cannot change their habitat and have to adapt to the new conditions or their offspring has to colonize new ecological niches. Due to the long generation times in trees it is important to know if and how trees can cope with the expected climatic conditions. Forest managers already give thought to the composition of future forests, because they have to choose species and populations which have no or only few problems with the changed climate. Douglas-fir (Pseudotsuga menziesii) is a promising tree species for this purpose, because it is adapted to different habitats and climate zones in its natural distribution range in North America. The two main varieties, coastal and interior Douglas-fir, differ genotypically and phenotypically, e.g. in drought tolerance. Douglas-fir trees, mainly of the coastal variety, showed good growth performances in field trials in Germany. Hence, a research project called "DougAdapt" was designed to analyze and to link genotypic and phenotypic differences in several coastal and interior Douglas-fir provenances. In this project, trees from field trials and from greenhouse experiments were sampled. To analyze the genetic diversity of the provenances we first generated reference sequences. Even with modern and cost-efficient next-generation sequencing technologies it would be very expensive to decipher the ~ 19 gigabases of the Douglas-fir genome completely. An alternative to whole genome sequencing is transcriptome sequencing, in which only the coding regions of a genome are sequenced. The transcriptome sequencing, which was performed for the first time in Douglas-fir, resulted in a large number of putative unique transcripts (PUTs). Comparisons with published transcriptomes of other plant species showed that the PUTs represented the transcriptome of Douglas-fir comprehensively. As the sampled seedlings were part of a drought stress experiment and grew under controlled conditions, we were able to identify drought related candidate PUTs, which may be part of the trees response to drought. Furthermore, more than 27,000 previously unknown single nucleotide polymorphisms (SNPs) in Douglas-fir could be identified. SNPs can influence the phenotype of individuals, and they can be used for instance as markers or to analyze genetic diversity. The analysis of genetic diversity of Douglas-fir provenances and the search for genes which may be part of local adaptation were performed with a sequence capture experiment. In sequence capture only predefined regions of a genome are sequenced. We showed that sequence capture based on PUTs as target regions is applicable in species with large and mostly unknown genomes. The polymorphic drought related candidate PUTs showed higher genetic differentiation than the remaining genes. Nevertheless, none of them was among the candidate PUTs for positive selection, which in turn are probably part of the local adaptation of the trees. Despite a high level of gene flow between coastal and interior provenances, the SNP data showed genetic differentiation between both varieties but only very low differentiation between the coastal provenances. We also investigated if genotyping-by-sequencing (GBS) is a suitable method to detect polymorphisms in Douglas-fir and compared the results of two GBS experiments with the sequence capture. The genome is digested with one or several restriction enzymes in GBS. Afterwards, only fragments with a specific length are sequenced, which considerably reduces the part of the genome that is sequenced as well as the costs. The advantage compared to sequence capture is the possibility to sample more individuals at the same time with less effort and costs. We showed that a digestion with two restriction enzymes results in more SNPs with less missing data, compared to using only one restriction enzyme. Both GBS methods returned considerably less SNPs than the sequence capture. Nevertheless, it was possible to distinguish between southern interior, northern interior, and coastal provenances using SNP data of the GBS experiments. GBS, especially with two restriction enzymes, seems to be a promising approach to genotype a large number of Douglas-fir trees and to obtain SNPs at low costs, which can be used in several tasks like genome-wide association studies. A large amount of sequence data and SNPs were analyzed in this thesis. Together with phenotypic information, these data will be crucial for the analysis of useful traits in Douglas-fir, like drought tolerance. Furthermore, the results concerning the Douglas-fir genome and the genetic diversity of different provenances will be beneficial in breeding programs and association studies, which in turn can be helpful to choose the optimal provenances for a given location.
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    Investigations on herbicide resistance in Apera spica-venti populations
    (2011) Massa, Dario; Gerhards, Roland
    Weeds belong to the main biotic yield-limiting factors in agricultural fields worldwide. Since the introduction of herbicides in the global market more than six decades ago, agro-ecosystems have been characterized by a strong reliance on herbicides for weed management. However, the selection pressure exerted on weed populations by long-term application of herbicides with the same mode of action has imposed selection for resistance within several weed species. Apera-spica-venti (L.) Beauv. represents one of the most abundant annual grass weeds in autumn-sown crops of Central and Eastern European arable lands. Since the first report on herbicide-resistant A. spica-venti populations in 1994, several cases have been documented. Therefore, it is assumed that this species is evolving resistance to herbicides. The main objectives of the present work were: 1. The development of reliable testing procedures under greenhouse conditions for the verification of herbicide resistance in A. spica-venti populations; 2. The quantification of resistance in A. spica-venti at different herbicide doses using dose-response assays and digital image analysis; 3. The elucidation of resistance mechanisms in A. spica-venti populations at the molecular genetic level through laboratory experiments; 4. The evaluation of the influence of farm management factors on the occurrence and spread of herbicide resistance in A. spica-venti populations through the use of statistical modeling; 5. The development of a geo-referenced database for documenting the spatial and temporal distribution of herbicide-resistant A. spica-venti populations in Europe; 6. The verification of the results obtained from the greenhouse assays under realistic field conditions and the detection of the introgression of herbicide resistance traits into the sensitive population after two generations. The main resistance testing procedure consisted of growing plants in the greenhouse from seeds collected in the suspect fields and spraying them with herbicides. Applications were carried out with an automated precision sprayer either at a single dose or at a range of doses. Herbicide efficacy was then assessed at 15 and 30 days after treatment by direct comparison with untreated controls. Results from the greenhouse assays showed that most of the screened popula¬tions (~70%) have evolved resistance to herbicides, particularly acetolactate synthase (ALS)-inhibitors. Dose-response assays revealed resistance factors at the ED90 (ED90 resistant / ED90 sensitive) of up to 140 after treatment with flupyrsulfuron-methyl, thus indicating that A. spica-venti is a resistance-prone grass weed. A novel method for quantifying resistance based on the assessment of percent canopy cover using digital image analysis has been developed in the greenhouse to provide a potential alternative to the labour-intensive and time-consuming dry weight assessments. Laboratory experiments conducted on over 70 ALS-resistant populations revealed that target site mutations were responsible for the observed resistance. Cleaved Amplified Polymorphic Sequences (CAPS) marker analysis and sequencing of the amplified 3? and 5? ends of the ALS gene by Rapid Amplification of cDNA Ends (RACE) revealed the presence of previously known as well as novel mutations endowing resis¬tance to ALS-inhibitors. The development of a risk assessment model allowed the evaluation of the influence of farm management factors on the probability of resistance occurrence in A. spica-venti populations. Results showed that a high percentage of winter crops in the rotation (>75%), together with reduced or no-till practices, early sowing and high population density significantly increase the risk of resistance emergence. An internet-based geo-referenced database was developed to document the spatial and temporal distribution of herbicide-resistant A. spica-venti populations in Europe. Finally, field experiments were carried out over the course of two years (2008/2010) by sow-ing ALS-resistant and susceptible A. spica-venti populations in winter wheat plots. Resistant plants survived herbicide application, completed their life cycle and set vital seeds, which showed a significantly higher germination rate compared to the sensitive population. However, maximum yield losses of only ~10% could be ob¬served at A. spica-venti panicle densities of >400 m-2. Greenhouse bioassays conducted at the end of the first year with plants from seeds collected in the sensitive plots showed an increase in tolerance to ALS-inhibitors of ~20%, thus suggesting introgression of herbicide resis¬tance traits into the sensitive population already after one year. The outcomes of this work add knowledge to the understanding of the mechanisms underlying resistance to herbicides in A. spica-venti populations and provide weed scientists and consultants with useful tools for the reliable diagnosis and prevention of herbicide resistance in weed populations.
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    Investigations on herbicide resistant grass weeds
    (2009) Balgheim, Natalie; Balgheim, Natalie
    Weeds are one of the most troublesome threats for farmers, causing high yield losses and serving as hosts for pathogens and insect pests. Since the introduction of chemical weed control agricultural production systems have changed. During the last years the number of herbicide resistant grass weeds is steadily increasing especially in cereal monocultures. These monocultures are characterised by the repeated use of herbicides with the same modes of action and minimum-tillage practices. All these factors can one by one or all together lead to the development of herbicide resistant grass weeds. In general herbicide resistance is the result of heritable changes to biochemical processes that enable plant survival when treated with herbicides. Two different mechanisms are commonly known to confer resistance: target-site resistance and non-target-site resistance. First is the result of an altered target enzyme, where a single point mutation is changing the amino acid structure and exclude herbicide from effectively binding to the target enzyme. The second one, non-target-site resistance, can be summarised as the mechanisms which includes all other mechanisms than target-site resistance, for example rapid metabolic degradation or translocation of herbicides. In Germany, the most trouble causing weeds associated with target-site resistance are the grass weeds Alopecurus myosuroides Huds. and A. spica-venti L. Beauv.. All investigations carried out during this thesis are dealing with those two weed species. Therefore the main objectives of this thesis are the following: To characterise the resistance levels and patterns of both species. To identify the underlying resistance mechanisms. To develop molecular markers for rapid detection of target-site based resistance. To get an idea of the spatial and temporal distribution of herbicide resistant grass weeds in arable fields. Both investigated species are highly adapted to cereals and developed resistance against ACCase and ALS inhibiting herbicides. So they are an increasing problem for German farmers and in consideration of the fact, that both weeds have developed multiple resistances, detecting and management strategies for controlling and preventing of these weeds are absolutely necessary. Carried out dose response relationships proved strong resistance of the A. myosuroides biotype BR(R) against cycloxydim and fenoxaprop, where low resistance was expressed against clethodim. However, biotype BL(R) showed resistance to fenoxaprop and clodinafop only. Dose response experiments carried out with the A. spica-venti biotype showed resistance to sulfosulfuron and iodo-/mesosulfuron. No cross resistances could be detected in both species. The carried out DNA analysis revealed target-site resistance as the underlying resistance mechanism. BR(R) and BL(R) showed well known substitutions: an amino acid change on position 1781 with in the CT domain result in a change of Leu to Ile which confers resistance to APPs and CHDs in the biotype BR(R). The mutation of Gly to Ala on position 2096 within the CT domain causes resistance to APPs only. Also in the A. spica-venti biotype a amino acid change is the responsible resistance mechanism: a change of Pro to Thr at position 197. These sequencing results serve as basis for the development molecular markers. Designed markers based on dCAPS technology. Such markers were developed to detect SNPs which can cause amino acid changes on the constitutive enzymes. Developed markers can rather differentiate between heterozygous and homozygous resistant alleles. Their technology is based on the fact that restriction endonucleases can cut DNA strands on specific recognition sites. This fact can be used for developing markers which are cutting the DNA in a previously generated PCR fragment on the mutation or wild-type sites, respectively. If there is no recognition site, it can be implemented by specific primers during the PCR. By these markers suspicious samples can be analysed and the results give an advice for management strategies, because target- and non-target-site resistance need different controlling strategies. Investigations on the spatial and temporal distribution of weed populations where carried out on an arable field, invested with herbicide resistant A. myosuroides. Collected and analysed leave samples give information about the spatial dynamics of homozygous, heterozygous and sensitive plants in the field. Results show that the distribution of resistant plants depends on the weed density. Besides the weeds are distributed heterogeneous on the field and occur in patches that are persistent over several years. This example revealed that herbicide resistance is rather associated with crop cultivation measurements. Changes in herbicidal and cultivation measurements shall be practiced to control and to prevent the occurrence of herbicide resistant grass weeds.
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    Molecular mapping of resistance and aggressiveness in the cereal/Fusarium head blight pathosystem
    (2016) Kalih, Rasha; Miedaner, Thomas
    Fusarium head blight (FHB) is one of the most destructive fungal diseases in small-grain cereals worldwide causing significant yield losses and contamination of grain with mycotoxins e.g., deoxynivalenol (DON). This renders the grain unsuitable for human consumption and animal feeding. Exploring the genetic mechanism of FHB resistance is considered the key tool for modern cereal breeding activities. Triticale, the intergeneric hybrid between wheat and rye, is an important cereal crop in Poland and Germany. Resistance breeding using genetic mapping to identify quantitative-trait loci (QTL) associated with FHB resistance represents the best strategy for controlling the disease. In parallel, understanding the mechanism of aggressiveness and DON production of F. graminearum will be a significant contribution to improve FHB management. The objectives of the present work were (1) identification of QTL related to FHB resistance in triticale, together with the analysis of the correlation of FHB severity with other related traits such as plant height and heading stage, (2) correlation between DON production and FHB severity, (3) mapping of dwarfing gene Ddw1 in triticale and studying its effect on FHB resistance, plant height and heading stage, (4) detection of SNPs in candidate genes associated with aggressiveness and DON production of a large Fusarium graminearum population in bread wheat. To study the genetic architecture of FHB resistance in triticale, five doubled-haploid (DH) triticale populations with 120 to 200 progenies were successfully tested under field conditions by inoculation with Fusarium culmorum (FC46) in multiple environments. All genotypes were evaluated for FHB resistance, plant height and heading stage. DArT markers were used to genotype triticale populations. Significant genotypic variances (P<0.001) were observed for FHB severity in all populations combined with high heritability. Twenty-two QTLs for FHB resistance in triticale were reported with two to five QTL per population, thus confirming the quantitative inheritance of FHB resistance in triticale. The most prominent (R2 ≥ 35%) QTLs were located on chromosomes 6A, 3B, 4R, and 5R. QTLs for plant height and heading stage were also detected in our work, some of them were overlapping with QTLs for FHB resistance. Correlation between FHB severity, DON content and Fusarium damaged kernels (FDK) in triticale was studied in the population Lasko x Alamo. Significant genotypic variance was detected for all traits. However, low correlation between FHB severity and DON content (r=0.31) was found. Interestingly, correlation between FHB severity and FDK rating was considerably higher (r=0.57). For FHB severity, two QTLs were detected in this population. A QTL located on chromosome 2A with minor effect for FHB severity was also a common QTL for DON content and FDK rating and explained ≥34% of genotypic variance for these two traits. A second QTL on chromosome 5R was a major QTL but it has no effect on DON content or FDK rating. For analyzing the rye dwarfing gene Ddw1 derived from the father Pigmej, 199 (DH) progenies were genotyped with DArT markers and in addition with conserved ortholog set (COS) markers linked to the Ddw1 locus in rye. QTL analyses detected three, four, and six QTLs for FHB severity, plant height and heading stage, respectively. Two specific markers tightly linked with Ddw1 on rye chromosome 5R explained 48, 77, and 71 % of genotypic variation for FHB severity, plant height, and heading stage, respectively. This is strong evidence, that we indeed detected the rye gene Ddw1 in this triticale population. Another objective was to highlight the association between quantitative variation of aggressiveness and DON production of 152 F. graminearum isolates with single nucleotide polymorphism (SNP) markers in seven candidate genes. One to three significant SNPs (P < 0.01 using cross-validation) were associated to FHB severity in four genes (i.e., Gmpk1, Mgv1, TRI6, and Erf2). For DON content, just one significant SNP was detected in the gene Mgv1 explaining 6.5% of the total genotypic variance. In conclusion, wide genetic variation in FHB resistance in triticale has been observed in five populations. QTL mapping analyses revealed twenty-two QTLs for FHB resistance derived from wheat and rye genomes. QTLs located on the rye genome were reported here for the first time and they are a new source for FHB resistance in triticale. In parallel, analysis of the diversity of four pathogenicity genes in F. graminearum is an important first step in inferring the genetic network of pathogenicity in this fungal pathogen.
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    Untersuchung der spatio-temporalen Verbreitung von Brucellose-Ausbruchsstämmen in Ägypten mittels cgSNP-Analyse und Multi Lokus VNTR-Analyse
    (2022) Holzer, Katharina; Beyer, Wolfgang
    Brucellosis is a worldwide zoonosis caused by the genus Brucella, which includes 12 species, and it is of great importance for the public health sector. In animals, the disease can lead to abortions, which entails high economic losses. The disease can be transmitted from animals to humans directly by penetrating through mucous membranes and wounds in the skin, or indirectly by consuming unpasteurized milk, milk products made from it, or meat that has not been sufficiently heated. The bacteria can also be transmitted via aerosols, which among other things categorizes these pathogens in risk class three. Contact with infected animals, especially their reproductive organs, aborted fetuses and discharges therefore represent a risk factor. The disease in humans is serious, an antibiotic treatment lasting several weeks is necessary. Although many countries are considered brucellosis-free, African countries, for example, are still badly affected. This work focuses on Brucella from Egypt. Although brucellosis is endemic in Egypt, there is just one publication with very little data about outbreak analysis of the species Brucella (B.) melitensis and B. abortus based on whole genome sequencing. Some other earlier publications are based on the so-called Multiple Locus VNTR analysis (MLVA), which in this present work turned out to be unsuitable for epidemiological analysis or outbreak analysis. VNTR means variable number of sequence repetitions in the DNA. In order to make concrete statements on outbreak analysis, a so-called single nucleotide polymorphism analysis of the core genome (cgSNP analysis) was carried out based on whole genome sequencing. While the MLVA includes highly mutable DNA regions, in the cgSNP analysis they are excluded. Therefore, the cgSNP analysis enables a more precise classification of the genotypes and thus also the classification of the outbreak strains, while the MLVA can just be used for the differentiation of isolates due to the use of highly mutable sequence regions. This became clear through the direct comparison of both methods including the metadata, so that previously published data that described the outbreak analysis for Brucella using the MLVA cannot be used. Over and above that in contrast to an in silico MLVA based on genome sequencing, a laboratory-based MLVA is prone to errors. In a direct comparison, the in silico MLVA showed reliable results in 100% of the cases and should therefore replace the laboratory-based MLVA. For the cgSNP analysis a total of 185 isolates from animals and humans were available. Out of a total of 185 samples, 137 were classified as B. melitensis and 49 as B. abortus, suggesting that B. melitensis infections are more common than B. abortus infections in Egypt. Furthermore, conserved outbreak strains in Egypt, detected by the cgSNP analysis and genetically differing only minimally, have persisted for several years and have spread or are still spreading, as well as constantly newly introduced outbreak strains or infections. Among the B. abortus isolates, B. abortus RB51 vaccine strains from aborted animals could be detected, confirming that the RB51 vaccine strain also causes abortions. While most B. melitensis strains are assigned to West Mediterranean origin, one isolate each is assigned to American and East Mediterranean origin. Such a classification by a so-called canonical SNP assay (canSNP assay), as was carried out for the B. melitensis isolates, is not possible for B. abortus for methodological reasons. A further cgSNP analysis was used to check existing public database entries from other countries for possible sources of introduction of the detected Egyptian outbreak strains. Italian origin is likely for most B. melitensis isolates, while UK origin is likely for a certain group of B. abortus isolates. The remaining B. abortus isolates could not be assigned unequivocally, but would possibly be close to the isolates from the USA. In order to confirm or refute possible relatives, much more isolates from different regions are necessary for a comparison. Due to the large number of different outbreak strains in Egypt, the origin of these outbreak strains should be outside the country, so that importations can be assumed.