Development and applications of Plabsoft : a computer program for population genetic data analyses and simulations in plant breeding

Marker-assisted breeding approaches are promising tools for enhancement of the conventional plant breeding process. They have been successfully applied in many areas such as plant variety protection, classification of germplasm, assessment of genetic diversity, mapping of genes underlying important agronomic traits, and using the mapping information for selection decisions. Powerful and flexible bioinformatic tools are urgently required for a better integration of molecular marker applications and classical plant breeding methods. The objective of my thesis work was to develop and apply Plabsoft, a computer program for population genetic data analyses and simulations in plant breeding. The assumption of Hardy-Weinberg equilibrium is a cornerstone of many concepts in population and quantitative genetics. Therefore, tests for Hardy-Weinberg equilibrium are of crucial importance, but the assumptions underlying asymptotic chi-square tests are often not met in datasets from plant breeding programs. I developed and implemented in Plabsoft a new algorithm for exact tests of Hardy-Weinberg equilibrium with multiple alleles. The newly derived algorithm has considerable computational advantages over previously described algorithms and extends substantially the range of problems that can be tested. Knowledge about the amount and distribution of linkage disequilibrium (LD) in breeding populations is of fundamental importance to assess the prospects for gene mapping with whole-genome association studies. To analyze LD in breeding populations, I implemented various LD measures in Plabsoft and developed a new significance test for these LD measures. The routines were employed to analyze LD in 497 elite maize lines from a commercial hybrid breeding program, which were fingerprinted by 81 simple sequence repeat (SSR) markers covering the entire genome. Strong LD was detected and, therefore, whole-genome association studies were recommended as promising. However, LD between unlinked loci will most likely result in a high rate of false positives. The prediction of hybrid performance with DNA markers facilitates the identification of superior hybrids. The single marker models used so far do not take into account the correlation between allele frequencies at linked markers. To overcome this problem, the concept of haplotype blocks was proposed. I developed and implemented in Plabsoft three alternative algorithms for haplotype block detection suitable for plant breeding. The algorithms were applied for the haplotype-based prediction of the hybrid performance of 270 hybrids, the parents of which were fingerprinted with 20 amplified fragment length polymorphism (AFLP) primer combinations. Employing haplotypes resulted in an improved prediction of hybrid performance compared with single marker models. Consequently, haplotype-based prediction methods have a high potential to improve substantially the efficiency of hybrid breeding programs. Computer simulations can be employed to solve population genetic problems in plant breeding, for which the simplifying assumptions underlying the classical population genetic theory do not hold true. However, before the start of my thesis no flexible simulation software was available. I developed algorithms for simulation of single breeding steps and entire plant breeding programs and implemented these in Plabsoft. The routines allow the simulation of plant breeding programs as they are conducted in practice. The simulation routines of Plabsoft were validated by simulating two marker-assisted backcross programs in rice conducted by the International Rice Research Institute (IRRI). In the simulations, the frequency distributions of the proportion of recurrent parent genome in the backcross populations were assessed. The simulation results were in good agreement with the experimental data. Therefore, computer simulations are a useful tool for pre-test estimation of selection response in marker-assisted backcrossing. The application of Plabsoft was exemplified by two studies in maize. In the first study, the expected LD decay in the intermating generations of two recurrent selections programs was determined with simulations. This application demonstrates the use of Plabsoft to solve problems for which analytical results are not available. In the second study, the forces generating and maintaining LD in a hybrid maize breeding program were investigated with computer simulations. This application demonstrates the capability of modeling complex long-term breeding programs as performed in practice. The studies of my thesis provide an example for the broad range of possible applications of Plabsoft. In addition to the presented studies, Plabsoft has so far been employed in about 40 further studies, which corroborates the usefulness of Plabsoft for integrating new genomic tools in applied plant breeding programs.DNA Marker werden in der Pflanzenzüchtung zum Erkennen von Sortenplagiaten, zur Gruppierung von Zuchtmaterial, zur Überwachung der genetischen Diversität, zur Kartierung von Genen, die für die Ausprägung wichtiger agronomischer Merkmale verantwortlich sind, sowie zur marker-gestützten Selektion eingesetzt. Um die Markertechnologie in die Methodik der klassischen Pflanzenzüchtung zu integrieren, werden dringend flexible und leistungsfähige bioinformatische Konzepte und darauf basierende Computerprogramme benötigt. Das Ziel dieser Arbeit war es, Plabsoft, ein Computerprogramm zur populationsgenetischen Datenanalyse und Simulation von Pflanzenzüchtungsprogrammen, zu entwickeln und anzuwenden. Die Annahme, dass sich eine Population im Hardy-Weinberg Gleichgewicht befindet, liegt vielen Konzepten der Populationsgenetik und der quantitativen Genetik zugrunde. Deswegen sind statistische Tests auf Hardy-Weinberg Gleichgewicht von großer Bedeutung. In Datensätzen aus Pflanzenzüchtungsprogrammen treffen die statistischen Annahmen, welche den oft verwendeten Chi-Quadrat-Tests zugrunde liegen, häufig nicht zu. Aus diesem Grund wurde in dieser Arbeit ein neuer Algorithmus für einen exakten Test auf Hardy-Weinberg Gleichgewicht mit multiplen Allelen entwickelt und in Plabsoft umgesetzt. Der neu implementierte Algorithmus ist deutlich schneller als alle vorher beschriebenen Algorithmen und erlaubt somit eine bedeutende Erweiterung für den Anwendungsbereich exakter Hardy-Weinberg Tests. Die genaue Kenntnis der Höhe und Verteilung von Gametenphasenungleichgewicht (linkage disequilibrium, LD) in pflanzenzüchterischen Populationen ist von großer Bedeutung, um die Erfolgsaussichten genomweiter Assoziationsstudien abschätzen zu können. Zu diesem Zweck wurde die Berechnung der wichtigsten LD Maße in Plabsoft implementiert und ein neuer Signifikanztest für die LD Maße entwickelt. Die neu entwickelten Routinen wurden zur Analyse des LD in einem kommerziellen Hybridmaiszüchtungsprogramm verwendet. Hierzu wurden 497 Inzuchtlinien mit 81 SSR (simple sequence repeat, Mikrosatelliten) Markern genotypisiert und ein hohes Ausmaß an LD detektiert, so dass genomweite Assoziationskartierungsansätze vielversprechend erscheinen. Jedoch ist zu erwarten, dass aufgrund des hohen Ausmaßes an LD zwischen ungekoppelten Markerloci viele falsch positive Assoziationen beobachtet werden. Eine markergestützte Vorhersage der Hybridleistung vereinfacht die Identifizierung überlegener Kreuzungskombinationen. Bisher wurden hierfür nur Vorhersagemodelle verwendet, die auf einzelnen Markerloci basieren und die Korrelationsstruktur zwischen Allelen an benachbarten Markerloci nicht berücksichtigen. In der Humangenetik wurde vorgeschlagen, benachbarte Markerloci zu sogenannten Haploblöcken zusammenzufassen, um das Problem der Multikolinearität zu lösen. Im Rahmen dieser Arbeit wurden drei unterschiedliche Algorithmen zur Detektion von Haploblöcken im Zuchtmaterial erarbeitet und in Plabsoft umgesetzt. Die Routinen wurden für eine haplotyp-basierte Vorhersage der Leistung von 270 Hybriden verwendet, deren Eltern mit 20 AFLP (amplified fragment length polymorphism) Primerkombinationen untersucht wurden. Die Vorhersage der Hybridleistung konnte durch die Verwendung von Haploblöcken verbessert werden. Folglich haben haplotyp-basierte Vorhersagemethoden ein großes Potential, die Effizienz von Hybridzuchtprogrammen zu steigern. Computersimulationen können in der Pflanzenzüchtung zur Lösung populationsgenetischer Fragestellungen auch dann angewendet werden, wenn die Annahmen, welche der klassischen populationsgenetischen Theorie zugrunde liegen, nicht erfüllt sind. Vor Beginn dieser Arbeit stand jedoch keine Software zur Verfügung, welche auf flexible Art und Weise Simulationen pflanzenzüchterischer Fragestellungen ermöglicht hätte. Aus diesem Grund wurden Algorithmen entwickelt, die die Simulation einzelner Züchtungsschritte sowie kompletter Pflanzenzüchtungsprogramme ermöglichen. Die entwickelten Algorithmen wurden im Computerprogramm Plabsoft umgesetzt, so dass es jetzt möglich ist, komplexe Pflanzenzüchtungsprogramme praxisnah zu simulieren. Die Simulationsroutinen von Plabsoft wurden an einem experimentellen Datensatz zur markergestützten Introgression eines Überflutungstoleranzgens in Reis validiert. Hierzu wurde das gesamte Zuchtprogramm, wie es in der Praxis durchgeführt wurde, simuliert. In den Simulationen wurde die Häufigkeitsverteilung des rekurrenten Eltergenomanteils in den Rückkreuzungspopulationen erfasst. Die Simulationsergebnisse stimmten nahezu vollständig mit den experimentell beobachteten Daten überein. Dies belegt, dass Computersimulationen ein äußerst effektives Hilfsmittel sind, um den Selektionserfolg bei der markergestützten Rückkreuzung abzuschätzen. Die Anwendung der Simulations- und Analysesoftware Plabsoft wurde exemplarisch an zwei Studien dargestellt. In der ersten Studie wurde mit Hilfe von Simulationen der zu erwartende Abfall an LD in den Durchkreuzungsgenerationen bei zwei rekurrenten Selektionsprogrammen in Mais bestimmt. Diese Studie demonstriert die Anwendung von Plabsoft zur Lösung von Fragestellungen, für welche keine analytische Lösungen zur Verfügung stehen. In der zweiten Studie wurden mit Hilfe von Computersimulationen die Ursachen untersucht, welche in einem Hybridmaiszuchtprogramm LD generieren und aufrecht erhalten. Hiermit wurde gezeigt, dass mit Plabsoft komplexe praktische Zuchtprogramme modelliert werden können. Die Studien dieser Arbeit geben einen Überblick über das breite Anwendungsspektrum der entwickelten Simulations- und Analysesoftware Plabsoft. Darüber hinaus wurde Plabsoft bis jetzt in vierzig weiteren Studien verwendet, womit die Nützlichkeit von Plabsoft für die Integration neuer genomischer Werkzeuge in die angewandte Züchtungsforschung zweifelsfrei belegt wird

Geometry and chronology of growth and drowning of Middle Triassic carbonate platforms (Cernera and Bivera/Clapsavon) in the Southern Alps (northern Italy)

Abstract.: The depositional architecture and the geometric relationships between platform-slope deposits and basinal sediments along with paleontological evidence indicate the time interval of the younger Anisian Reitziites reitzi ammonoid zone to largely represent the main stage of platform aggradation at the Cernera and Bivera/Clapsavon carbonate platforms. Published and new U-Pb age data of zircons from volcaniclastic layers bracketing the stratigraphic interval of platform growth constrain the duration of platform evolution to a time span shorter than 1.8±0.7m.y., probably in the order of 0.5-1m.y., reflecting fast rates of vertical platform aggradation exceeding 500 m/m.y. In the range of growth potentials for shallow-water carbonate systems estimated in relation to the time span of observation, this high rate is in agreement with values for short intervals of 105-106yrs (e.g., Schlager 1999). After drowning, the platforms at Cernera and Bivera/Clapsavon were blanketed by thin pelagic carbonates. On the former platform flanks the draping sediments in places comprise red nodular pelagic limestones (Clapsavon Limestone) similar in facies to the Han Bulog Limestones occurring elsewhere in Middle Triassic successions of the Mediterranean Tethys. The drowning of vast areas of former carbonate platforms possibly triggered the onset of bottom-water circulation in adjacent basins as suggested by the abrupt transition from laminated to bioturbated pelagic nodular limestones in the Buchenstein Formation which occurred close to the time of initial platform submergence. During the Late Ladinian the topographic features of the drowned platforms were onlapped by rapidly deposited, predominantly clastic successions including coarse breccias and volcanic rocks sealing and preserving the peculiar stratigraphic settin

Time- and temperature-dependent postmortem concentration changes of the (synthetic) cannabinoids JWH-210, RCS-4, as well as ∆9-tetrahydrocannabinol following pulmonary administration to pigs

In forensic toxicology, interpretation of postmortem (PM) drug concentrations might be complicated due to the lack of data concerning drug stability or PM redistribution (PMR). Regarding synthetic cannabinoids (SC), only sparse data are available, which derived from single case reports without any knowledge of dose and time of consumption. Thus, a controlled pig toxicokinetic study allowing for examination of PMR of SC was performed. Twelve pigs received a pulmonary dose of 200 µg/kg BW each of 4-ethylnaphthalene-1-yl-(1-pentylindole-3-yl)methanone (JWH-210), 2-(4-methoxyphenyl)-1-(1-pentyl-indole-3-yl)methanone (RCS-4), and Δ9-tetrahydrocannabinol via an ultrasonic nebulizer. Eight hours after, the pigs were put to death with T61 and specimens of relevant tissues and body fluids were collected. Subsequently, the animals were stored at room temperature (n = 6) or 4 °C (n = 6) and further samples were collected after 24, 48, and 72 h each. Concentrations were determined following enzymatic cleavage and solid-phase extraction by liquid-chromatography tandem mass spectrometry applying the standard addition approach. High concentrations of the parent compounds were observed in lung, liver, kidney and bile fluid/duodenum content as well as brain. HO-RCS-4 was the most prevalent metabolite detected in PM specimens. In general, changes of PM concentrations were found in every tissue and body fluid depending on the PM interval as well as storage temperature

Know Your Epidemic, Know Your Response: COVID-19 in the United States

We document that during the week of March 10-16, the Covid-19 pandemic fundamentally affected the perceptions of U.S. residents about the health risks and socioeconomic consequences entailed by the pandemic. During this week, it seems, everything changed. Not only did the pandemic progress rapidly across the United States, but U.S. residents started to realize that the threat was real: increasing Covid-19 caseloads heightened perceptions of infection risks and excess mortality risks, concerns about the economic implications increased substantially, and behavioral responses became widespread as the pandemic expanded rapidly in the U.S. In early to mid-March 2020, average perceptions about the coronavirus infection risks are broadly consistent with projections about the pandemic, while expectations about dying conditional on infection and expectations about Covid-19-related excess mortality during the next months are possibly too pessimistic. However, some aspects of Covid-19 perceptions are disconcerting from the perspective of implementing and sustaining an effective societal response to the pandemic. For instance, the education gradient in expected infection risks entails the possibility of having different perceptions of the reality of the pandemic between people with and without a college education, potentially resulting in two different levels of behavioral and policy-responses across individuals and regions. Unless addressed by effective health communication that reaches individuals across all social strata, some of the misperceptions about Covid-19 epidemic raise concerns about the ability of the United States to implement and sustain the widespread and harsh policies that are required to curtail the pandemic. Our analyses also reveal perceptions of becoming infected with the virus, and dying from Covid-19, were driven upwards by a rapidly increasing national caseload, and perceptions of the economic consequences and the adaptation of social distancing were affected by both national and state-level cases

Is adipose tissue suitable for detection of (synthetic) cannabinoids? A comparative study analyzing antemortem and postmortem specimens following pulmonary administration of JWH-210, RCS-4, as well as ∆9-tetrahydrocannabinol to pigs

Examining fatal poisonings, chronic exposure may be refected by the concentration in tissues known for long-term storage of drugs. Δ9-tetrahydrocannabinol (THC) persists in adipose tissue (AT), but sparse data on synthetic cannabinoids (SC) are available. Thus, a controlled pig study evaluating antemortem (AM) disposition and postmortem (PM) concentration changes of the SC 4-ethylnaphthalene-1-yl-(1-pentylindole-3-yl)methanone (JWH-210) and 2-(4-methoxyphenyl)-1-(1-pentyl-indole3-yl)methanone (RCS-4) as well as THC in AT was performed. The drugs were administered pulmonarily (200 µg/kg body weight) to twelve pigs. Subcutaneous (s.c.) AT specimens were collected after 15 and 30 min and then hourly up to 8 h. At the end, pigs were sacrifced and s.c., perirenal, and dorsal AT specimens were collected. The carcasses were stored at room temperature (RT; n=6) or 4 °C (n=6) and specimens were collected after 24, 48, and 72 h. After homogenization in acetonitrile and standard addition, LC–MS/MS was performed. Maximum concentrations were reached 0.5–2 h after administration amounting to 21±13 ng/g (JWH-210), 24±13 ng/g (RCS-4), and 22±20 ng/g (THC) and stayed at a plateau level. Regarding the metabolites, very low concentrations of N-hydroxypentyl-RCS-4 (HO-RCS-4) were detected from 0.5 to 8 h. PM concentrations of parent compounds did not change signifcantly (p>0.05) over time under both storage conditions. Concentrations of HO-RCS-4 signifcantly (p<0.05) increased in perirenal AT during storage at RT. These results suggest a rapid distribution and persistence in s.c. AT. Furthermore, AT might be resistant to PM redistribution of parent compounds. However, signifcant PM increases of metabolite concentrations might be considered in perirenal AT

A novel histopathological scoring system to distinguish urticarial vasculitis from chronic spontaneous urticaria

Background: Urticarial vasculitis (UV) is defined by long-lasting urticarial lesions combined with the histopathologic findings of leukocytoclastic vasculitis. As one of the major unmet needs in UV, diagnostic criteria are rather vague and not standardized. Moreover, there seems to be considerable overlap with chronic spontaneous urticaria (CSU), particularly for the normocomplementemic variant of UV. Therefore, this study aimed to develop a diagnostic scoring system that improves the histopathologic discrimination between UV and CSU. Methods: Lesional skin sections of patients with clinical and histopathologic diagnosis of UV (n = 46) and CSU (n = 51) were analyzed (blinded to the diagnosis) for the following pre-defined criteria: presence of leukocytoclasia, erythrocyte extravasation, fibrin deposits, endothelial cell swelling, ectatic vessels, blurred vessel borders, dermal edema, intravascular neutrophil, and eosinophil numbers and numbers of dermal neutrophils, macrophages and mast cells. Results: The greatest differences between UV and CSU samples were observed for leukocytoclasia (present in 76% of UV vs. 3.9% of CSU samples; p < 0.0001), erythrocyte extravasation (present in 41.3% of UV vs. 2.0% of CSU samples; p < 0.0001), and fibrin deposits (present in 27.9% of UV vessels vs. 9.7% of CSU vessels; p < 0.0001). Based on these findings, we developed a diagnostic score, the urticarial vasculitis score (UVS), which correctly assigned 37 of 46 cases of UV and 49 of 51 cases of CSU to the previously established diagnosis. Conclusion: Our results suggest that the UVS, a combined quantitative assessment of the three criteria leukocytoclasia, fibrin deposits and extravasated erythrocytes, distinguishes UV from CSU in skin histopathology. The UVS, if validated in larger patient samples, may help to improve the diagnostic approach to UV

Optimum allocation of resources for QTL detection using a nested association mapping strategy in maize

In quantitative trait locus (QTL) mapping studies, it is mandatory that the available financial resources are spent in such a way that the power for detection of QTL is maximized. The objective of this study was to optimize for three different fixed budgets the power of QTL detection 1 − β* in recombinant inbred line (RIL) populations derived from a nested design by varying (1) the genetic complexity of the trait, (2) the costs for developing, genotyping, and phenotyping RILs, (3) the total number of RILs, and (4) the number of environments and replications per environment used for phenotyping. Our computer simulations were based on empirical data of 653 single nucleotide polymorphism markers of 26 diverse maize inbred lines which were selected on the basis of 100 simple sequence repeat markers out of a worldwide sample of 260 maize inbreds to capture the maximum genetic diversity. For the standard scenario of costs, the optimum number of test environments (Eopt) ranged across the examined total budgets from 7 to 19 in the scenarios with 25 QTL. In comparison, the Eopt values observed for the scenarios with 50 and 100 QTL were slightly higher. Our finding of differences in 1 − β* estimates between experiments with optimally and sub-optimally allocated resources illustrated the potential to improve the power for QTL detection without increasing the total resources necessary for a QTL mapping experiment. Furthermore, the results of our study indicated that also in studies using the latest genomics tools to dissect quantitative traits, it is required to evaluate the individuals of the mapping population in a high number of environments with a high number of replications per environment

Tracking a Tuberculosis Outbreak Over 21 Years: Strain-Specific Single-Nucleotide Polymorphism Typing Combined With Targeted Whole-Genome Sequencing

Background. Whole-genome sequencing (WGS) is increasingly used in molecular-epidemiological investigations of bacterial pathogens, despite cost- and time-intensive analyses. We combined strain-specific single-nucleotide polymorphism (SNP) typing and targeted WGS to investigate a tuberculosis cluster spanning 21 years in Bern, Switzerland. Methods. On the basis of genome sequences of 3 historical outbreak Mycobacterium tuberculosis isolates, we developed a strain-specific SNP-typing assay to identify further cases. We screened 1642 patient isolates and performed WGS on all identified cluster isolates. We extracted SNPs to construct genomic networks. Clinical and social data were retrospectively collected. Results. We identified 68 patients associated with the outbreak strain. Most received a tuberculosis diagnosis in 1991-1995, but cases were observed until 2011. Two thirds were homeless and/or substance abusers. Targeted WGS revealed 133 variable SNP positions among outbreak isolates. Genomic network analyses suggested a single origin of the outbreak, with subsequent division into 3 subclusters. Isolates from patients with confirmed epidemiological links differed by 0-11 SNPs. Conclusions. Strain-specific SNP genotyping allowed rapid and inexpensive identification of M. tuberculosis outbreak isolates in a population-based strain collection. Subsequent targeted WGS provided detailed insights into transmission dynamics. This combined approach could be applied to track bacterial pathogens in real time and at high resolutio

IgE Mediated Autoallergy against Thyroid Peroxidase – A Novel Pathomechanism of Chronic Spontaneous Urticaria?

Chronic spontaneous urticaria (csU), which is characterized by recurrent episodes of mast cell-driven wheal and flare-type skin reactions, is often associated with elevated total IgE levels and thyroid autoimmunity. We speculate that some csU patients express IgE autoantibodies against thyroid antigens such as thyroid peroxidase (TPO), which could bind to skin mast cells and induce their activation.We developed and used a site-directed human IgE capture ELISA to quantify IgE-anti-TPO. We used this assay and investigated csU patients (n = 478) and healthy control subjects (n = 127) for IgE-anti-TPO and then assessed IgE-anti-TPO-positive and -negative csU patients for clinical and serological differences. ( = 61%, IgE-anti-TPO: median 6.67, interquartile range 5.39–8.24). IgE-anti-TPO-positive and -negative csU patients had very similar distributions of age and gender as well as disease activity and duration. IgE-anti-TPO-positive csU patients exhibited significantly higher IgG-anti-TPO levels and lymphocyte counts as well as decreased C4 complement levels.Our findings show that a sizeable subgroup of csU patients expresses IgE antibodies against thyroid peroxidase. These autoantibodies could cause “autoallergic” mast cell activation, a novel pathomechanism of chronic spontaneous urticaria

Assessing How the Aluminum-Resistance Traits in Wheat and Rye Transfer to Hexaploid and Octoploid Triticale

The mechanisms of aluminum (Al) resistance in wheat and rye involve the release of citrate and malate anions from the root apices. Many of the genes controlling these processes have been identified and their responses to Al treatment described in detail. This study investigated how the major Al resistance traits of wheat and rye are transferred to triticale (x Tritosecale Wittmack) which is a hybrid between wheat and rye. We generated octoploid and hexaploid triticale lines and compared them with the parental lines for their relative resistance to Al, organic anion efflux and expression of some of the genes encoding the transporters involved. We report that the strong Al resistance of rye was incompletely transferred to octoploid and hexaploid triticale. The wheat and rye parents contributed to the Al-resistance of octoploid triticale but the phenotypes were not additive. The Al resistance genes of hexaploid wheat, TaALMT1, and TaMATE1B, were more successfully expressed in octoploid triticale than the Al resistance genes in rye tested, ScALMT1 and ScFRDL2. This study demonstrates that an important stress-tolerance trait derived from hexaploid wheat was expressed in octoploid triticale. Since most commercial triticale lines are largely hexaploid types it would be beneficial to develop techniques to generate genetically-stable octoploid triticale material. This would enable other useful traits that are present in hexaploid but not tetraploid wheat, to be transferred to triticale