Inherited Retinal Degenerations (IRDs) are a heterogenous group of diseases which lead to vision impairment and can be found both in humans and in dogs. About 1 in 1,380 humans is estimated to suffer from an autosomal recessive IRD, which would be 5.5 million people worldwide, and many more are estimated to be unaffected carriers. This makes autosomal recessive IRDs likely the most common group of Mendelian diseases in humans. Today, about 300 genetic mutations have been connected to cause retinal diseases in humans. Whilst in dogs only 32 genes have been identified, numerous eye conditions have been described where the genetic cause has not yet been identified. This suggests that there are much more genetic causes to discover in the dog genome. Additionally, the dog serves well as a model organism to investigate IRDs as it is sharing morphological and genetic similarities with humans. For these reasons, proper software, a canine reference genome of high quality, and smart implementation of bioinformatic tools and methods are a big advantage to increase chances of finding new causative genetic variants and subsequently enable faster detection of possible preventions of the disease or at least alleviating its symptoms via early diagnosis. In this project, a pre-existing pipeline consisting of Bash scripts was stepwise improved with the goal to increase its efficiency. After controlling whether previous data could still be reproduced with the old pipeline in a first step, the software was exchanged to more updated versions in a second step. A main change was the replacement of the mapping tool Burrows-Wheeler Aligner (BWA) from bwa mem to bwa-mem2 mem, and the update of deprecated Genome Analysis Toolkit (GATK) 3.7 to version 4.3 or 4.4. Thirdly, the scripts were adapted from using the older canine reference genome CanFam3.1 to CanFam4. In a fourth step, for automatization and fastening the running time, the pipeline steps were implemented into the workflow management system Nextflow. Additionally, this step was partly aiming to make the pipeline in concordance with the FAIR-principles. All steps were tested on the same test data set, a Labrador retriever family trio, in which one genetic cause for a canine form of the IRD Stargardt disease in a previous study had been detected, namely an insertion in the ABCA4 gene. Lastly, the workflow was also tested on a second data set of a novel IRD of unknown genetic origin on two sibling pairs of Chinese Crested Dogs (CCR). The adjustment of the pipeline shows similar results regarding the change of mapping tool. Introducing the new reference genome revealed a drop of average coverage by one read average for when using CanFam4, while other results were similar. Using the new reference genome increased the number of unknown variants compared to findings with CanFam3.1. However, the known causative variant for the canine form of Stargardt disease, an insertion in ABCA4 gene, could be found in all cases. The run with Nextflow produced identical results to when the respective steps were run with Bash scripts, but it reduced the running time. Running the workflow on the new data set (CCR) and subsequent annotation and filtering indicate new candidates which could be further investigated as a potential cause for this currently unknown cause for an IRD.

The Scandinavian brown bear went through a severe bottleneck due to human actions in the early 1900’s that reduced the population to low numbers. After protective measures were taken, the population recovered and today it consists of around 3000 individuals. Such loss of genetic variation can have long-lasting effects on population viability even as populations recover, and is therefore important to consider in the management and conservation of species. Previous studies investigating the genetic effects of the bottleneck based on genetic markers, such as microsatellites and mitochondrial DNA, have rendered inconclusive results. Here, whole-genome sequencing of historical and contemporary bears was used to estimate heterozygosity, inbreeding and mutational load in the pre- and post- bottleneck population. Surprisingly, a significant increase in heterozygosity was found in the contemporary population and no significant increase in inbreeding over time was found. However, mutational load was higher in the contemporary subpopulation in the south of Sweden compared to the subpopulation in the north, and the southern subpopulation seems to have lost unique genetic variation after the bottleneck. The results indicate that although the population was negatively affected by the bottleneck, a following increase in gene flow could have contributed to successful recovery of the population.

Strains MS455 and MS389, endophytic bacteria, were isolated from healthy soybean plant growing adjacent to a patch of plants affected by charcoal rot disease, caused by the fungal pathogen Macrophomina phaseolina. The complete genomes of both strains were sequenced and identified as Burkholderia species Strain MS455 exhibits broad-spectrum antifungal activities against economically important pathogens, including Aspergillus flavus. Random and site-specific mutations were employed in discovery of the genes that share high homology to the ocf gene cluster of Burkholderia contaminans strain MS14, which is responsible for production of the antifungal compound occidiofungin. RNA-seq analysis demonstrated ORF1, a homolog to the ambR1 LuxR-type regulatory gene, not only regulates occidiofungin biosynthesis in MS455, but also involved in expression of multiple genes, especially those involved in ornibactin biosynthesis. Plate and corn kernel assays showed that growth of A. flavus and aflatoxin production were reduced significantly by MS455 as compared with buffer control and the ORF1 mutant. Strain MS389 shows significant antifungal and antibacterial activities as well. Mutagenesis study identified that the TatC gene, an important unit of twin-arginine translocase (Tat) secretion system, and the LysR-type transcriptional regulatory gene were essential for the antifungal activity of strain MS389. RNA-seq analysis implied that the pyrrolnitrin biosynthesis gene cluster and an uncharacterized NRPS / PKS gene cluster were involved in antifungal activity. By comparing several endophytic bacteria of Burkholderia, including MS455 and MS389, to pathogenic Burkholderia species, endophytic bacteria were observed to harbor multiple antimicrobial biosynthesis genes but lack certain pathogenic or virulence genes. The potential endophytic behavior related genes and characteristics related to antibiotic resistance, secretion system, and CRISPR-Cas profiles were well established. The research findings on strains MS455 and MS389 have provided important genetic clues for understanding their molecular mechanism of antimicrobial activities and exhibited their potential possibility as biocontrol agents.

付記する学位プログラム名: 霊長類学・ワイルドライフサイエンス・リーディング大学院 / 京都大学 / 新制・課程博士 / 博士(理学) / 甲第24471号 / 理博第4970号 / 新制||理||1709(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 村山 美穂, 教授 伊谷 原一, 教授 平田 聡 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Peregrine falcon subspecies taxonomy is widely debated due to uncertainty in their evolutionary history and unresolved phylogenetic reconstruction using both morphological and molecular data. Previous genetic work has shown limited support for subspecies taxonomy largely as a result of molecular markers used, potential contemporary gene flow, incomplete lineage sorting, and ancestral polymorphisms. With the advent of next-generation sequencing, the cost of generating large amounts of sequence data has dropped significantly, making whole genome re-sequencing (WGR) studies of non-model organisms more tangible. In this study, WGR methods have been utilized to investigate the phylogenetic relationships among all 20 currently recognized peregrine falcon subspecies. By generating whole-genome data for all 20 subspecies, subspecies specific diagnostic SNPs have been identified to aid in subspecies delimitation. Results of this study broadly support current subspecies, however, reveal that further study is needed to investigate regional relationships among subspecies in Asia, Australia, and western North America. With these results, conservation efforts can be further supported by allowing for accurate delimitation of local subspecies and subspecies boundaries.

In early 2020 the World Health Organization (WHO) declared a COVID-19 pandemic outbreak. As of March 2023, the Kingdom of Saudi Arabia has reported over 829 thousand cases and over 9 thousand deaths. Due to the public health emergency, the wild-type SARS-CoV-2 genome was fully sequenced early on the pandemic course. Afterward, different viral variants emerged around the world. The variant prevalence fluctuates over time, with some strains presenting increased transmissibility, changes in disease severity, and decreased efficacy of containment measures. In this respect, genomic surveillance and timely data sharing to public repositories represent a tool to track and detect emerging changes that require addressing with public health measurements. However, the Kingdom's genome contribution to the GISAID database represents merely 0.27% of the reported cases. To address this gap, we incorporated whole genome sequencing and the available metadata, to track the circulating viral lineages and identify the variants of concern (VOC) and variants of interest (VOI) over 14 months, from December 2021 to January 2023. We sequenced 581 genomes using the MinION MK1C platform of Oxford Nanopore Technologies (ONT) and assigned the viral lineages, the data primarily derived from COVID-19-positive patients from the city of Jeddah. The VOC Omicron was the principal circulating variant in the country (~99%), which aligns with the global trend. Our report included the sublineages BA.2.75, BQ.1, CH.1.1, XBB, and XBB.1.5, which are under WHO monitoring. We also reported the VOC Delta and the recombinant strains XF, XZ, and XPB. Finally, to validate the use of ONT for genomic surveillance, we compared the data quality of 93 samples sequenced in both ONT MinION and Illumina NaovaSeq platforms. There was 90% correspondence in the Nextstrain clade assignment (84/93) and 78% correspondence in the Pangolin sublineage assignment (73/93). MinION offered a shorter turnaround time, while Illumina produced a consistently higher breadth of genome coverage. In conclusion, the continued viral evolution patterns are reflected in the Kingdom's variant prevalence, where only Omicron circulated by January 2023. Likewise, current Variants Under Monitoring (VUM) were already identified in KSA, highlighting the need for increased genomic surveillance.

The objective of this thesis was to determine the viability of alternative genetic improvement strategies (GIS). Each alternative GIS combined the use of whole genome selection (WGS) with common reproductive methods (non-sexed semen artificial insemination (AI), sexed semen AI, embryo transfer utilizing non-sexed semen AI) that can be found on a commercial dairy operation. The viability of each GIS was determined using a discounted gene flow model, designed with parameters of a typical western dairy operation, to evaluate the following variables: reproductive method, selection intensity, accuracy of prediction and female age-class. Of the GIS investigated, a heifer-based strategy that used embryo transfer with 11% selection intensity and 85% accuracy was viable. This GIS generated 2.7 million dollars in present value of cumulative gross marginal returns. Despite such encouraging results, at the current prices for genotyping, reproductive methods and achievable prediction accuracy levels, all other GIS resulted in negative returns. Whole genome selection could be a powerful genetic improvement tool for the commercial dairy industry if high accuracy genotyping solutions and reproductive methods that allowed for high selection intensity were combined and priced less than $379.07 per individual.

No description available.

Swedish populations of Wels catfish (Silurus glanis) experienced severe declines during the 19th and 20th centuries. The main causes for the decline were loss of suitable spawning habitat and fragmentation of populations. Currently, three native and two reintroduced populations remain in Sweden. Thanks to national protection and progress in restoring habitats, population sizes have increased over the past three decades. Previous studies that used microsatellite loci have found that genetic diversity and effective population sizes in Sweden are low, while population differentiation is high. A study that used whole genome sequencing (WGS) confirmed these results for native Swedish populations (those found in the Båven, Emån, and Möckeln water systems). The current project uses the same WGS methods and expands on the previous study by considering samples from non-Swedish populations (river Garonne, France; river Ebro, Spain; hatchery, Czech Republic), as well as improving read-depth coverage and sampling from the introduced Swedish population in the Helge å water system. Both a genome-wide SNP-set and full mitochondrial sequences were used to assess genetic diversity within each population, and differentiation among them. Genetic diversity in Swedish populations is lower than in non-Swedish populations. Native Swedish populations are strongly differentiated from one another. The introduced Helge å population is strongly differentiated from Emån and Möckeln, but less so from Båven. Despite Helge å individuals having heritage that can be predominantly traced to Båven, there are clear signs of admixture with the other two native populations. Swedish populations are all strongly differentiated from the non-Swedish populations. Altogether, evidence of admixture and slightly greater genetic diversity than native Swedish populations in Helge å can at the surface be seen as promising signs. However, it remains uncertain whether these improvements are durable over multiple generations. Considering the poor genetic status of Sweden taken as a whole, and the questionable nature of the improvements seen in Helge å, the long-term viability of Swedish catfish populations remains uncertain.

Maternal binge alcohol consumption has been linked to congenital birth defects in the fetus. Said defects include abnormalities in heart development, a category of disease referred to as Congenital Heart Disease. Given the prevalence of Congenital Heart Disease, with a study showing around 49.9% of women having at least participated in binge alcohol consumption at least once during the early stages of their pregnancy and Congenital Heart Disease being linked to various complications in adulthood, this is a topic relevant to the clinical setting. Alcohol consumption has been linked to decreases in DNA methylation, which generally increases transcriptional expression of nearby genes. This thesis will focus on how alcohol affects the genomic-wide epigenetics of the embryonic heart with the aim of identifying specific genes and sites within those genes that are affected by alcohol exposure in utero. We hypothesize that embryonic mouse hearts exposed to ethanol will show a differential methylation pattern characteristic of hypomethylation versus control hearts not exposed to ethanol. To test this hypothesis, we used oral gavage to administer ethanol to pregnant mice at embryonic age E9.5 (a time associated with heart chamber formation). Maternal mice were sacrificed at E11.5, embryonic hearts were removed, and DNA was extracted for further experimentation with whole genome bisulfite sequencing. Analysis of whole genome bisulfite sequencing data showed a slight trend towards hypomethylation but suggested no significant changes in the overall methylation pattern in embryonic mouse hearts at the genomic level, but we have independently identified several genes whose expression is depressed in the embryonic mouse following a single maternal binge ethanol dose at E9.5, and thus we are investigating potential alcohol-induced DNA methylation alterations in specific target genes of interest. Future investigations into gene and site-specific DNA methylation profiles as well as other epigenetic modifications should prove useful in our quest to learn how maternal alcohol consumption causes cardiac malformations leading to congenital heart disease.