The genome-wide dynamics of purging during selfing in maize

Self-fertilization (also known as selfing) is an important reproductive strategy in plants and a widely applied tool for plant genetics and plant breeding. Selfing can lead to inbreeding depression by uncovering recessive deleterious variants, unless these variants are purged by selection. Here we investigated the dynamics of purging in a set of eleven maize lines that were selfed for six generations. We show that heterozygous, putatively deleterious single nucleotide polymorphisms are preferentially lost from the genome during selfing. Deleterious single nucleotide polymorphisms were lost more rapidly in regions of high recombination, presumably because recombination increases the efficacy of selection by uncoupling linked variants. Overall, heterozygosity decreased more slowly than expected, by an estimated 35% to 40% per generation instead of the expected 50%, perhaps reflecting pervasive associative overdominance. Finally, three lines exhibited marked decreases in genome size due to the purging of transposable elements. Genome loss was more likely to occur for lineages that began with larger genomes with more transposable elements and chromosomal knobs. These three lines purged an average of 398 Mb from their genomes, an amount equivalent to three Arabidopsis thaliana genomes per lineage, in only a few generations

Self-hybridization in Leishmania major

Genetic exchange between differen

Life finds a way: the recovery of frog populations from a chytridiomycosis outbreak

Emerging infectious diseases are a serious threat to wildlife, but not all populations or species have the same response to outbreaks. In some cases, diseases shift from being epizootic to enzootic, allowing populations to recover, but both the causes of recoveries and the long-term consequences of disease outbreaks remain poorly understood. My PhD aimed to further our knowledge of these important topics by using a frog assemblage in the Australian Wet Tropics as a model system for understanding recoveries from disease outbreaks. This region was impacted by an outbreak of the fungal disease chytridiomycosis (caused by the pathogen Batrachochytrium dendrobatidis [Bd]) in the late 1980s and early 1990s, during which high elevation populations of several frog species declined or disappeared, while low elevation populations remained stable. Following the outbreak, some species recovered at upland sites, but the patterns of both declines and recoveries vary among species. Litoria dayi disappeared from upland sites and has never recovered. Litoria nannotis disappeared from upland sites and has largely recovered. Litoria serrata declined at upland sites and has recovered, and Litoria wilcoxii did not decline substantially at any elevation. These different histories with the disease presented a great opportunity for studying the factors that allowed some species to recover, while apparently precluding recovery in others, and my thesis examined both population genetics and microbiomes of frogs in this system. My primary goals were to examine the long-term consequences of the outbreak (e.g., fragmentation, inbreeding, loss of diversity) and test several hypotheses for the differences in the history of declines and recoveries among species (e.g., differences in dispersal abilities, a lack of adaptive potential due to lost diversity, differences in microbiomes). I used single nucleotide polymorphisms to examine connectivity patterns, test for a loss of diversity, and test for Bd-driven selection. I examined low elevation populations of L. nannotis, L. serrata, and L. dayi that survived the outbreak, and compared them to recovered upland populations of L. nannotis and L. serrata. I sampled L. dayi at three national parks and L. nannotis and L. serrata at two national parks. All three species showed high levels of connectivity within a given park, and there was no structuring along streams, suggesting that all three species have good dispersal abilities. No inbreeding was present in any species, and all species showed high genetic diversity levels north of Paluma Range National Park. At Paluma, however, both L. nannotis and L. serrata had reduced genetic diversity, and diversity levels followed a west–east pattern, with higher diversity on the western half of the park (L. dayi does not occur at Paluma). These diversity patterns matched habitat patterns, with higher diversity in wetter areas with larger sections of rainforest, suggesting that the size and quality of refuge habitat may play an important role in the retention of genetic diversity during a disease outbreak. I did not find consistent evidence of selection in L. nannotis, but there was consistency among outlier testes for L. dayi. These tests could not conclusively demonstrate that L. dayi was undergoing diseaseinduced selection, but they were suggestive. Prior to analysing the microbiomes of the frog species, it was necessary to test or develop several microbiome methodologies. First, microbiome data often need to be normalized prior to analysis, and many methods are available, but several of the most popular methods use variance standardizing techniques that can distort ecological data. Therefore, I compared six methods (rarefaction, proportions, upper quartile, CSS, edgeRTMM, and DESeq-VS) using both a published data set and simulations. My results showed that upper quartile, CSS, edgeR-TMM, and DESeq-VS failed to fully standardize reads, and inflated minor differences among rare micro-organisms while suppressing large differences among common micro-organisms, thus distorting community comparisons. In contrast, using proportions or rarefaction produced accurate results, with proportions outperforming rarefaction. Another common issue with microbiome studies is the ubiquitous presence of bacterial contamination. This problem has been widely documented, but no method of accurately removing contaminate reads exists. Therefore, I developed an algorithm for identifying and removing contaminate reads, wrote an R package (microDecon) to implement it, and tested it using two large simulations, a published data set, and a sequencing experiment. All tests showed that microDecon was highly accurate and improved the results in 98.1% of cases. Having tested and developed these methods, I was able to apply them to the microbiomes of frog populations. Multiple laboratory studies have documented beneficial effects of bacteria for amphibian hosts during Bd infections, and several field studies have suggested that microbiomes may play important roles in infection dynamics. Nearly all of this research has focused on bacteria, while the fungal microbiomes of amphibians remain largely unexplored. Therefore, I examined both the fungal and bacterial microbiomes of L. dayi, L. nannotis, L. serrata, and L. wilcoxii to make one of the first comparisons of bacteria and fungi in frog populations and test the hypothesis that differences in microbiomes could explain the differences in patterns of declines and recoveries in the Wet Tropics frog assemblage. I also used qPCR to examine Bd infection prevalence and intensity. Bacterial microbiomes generally had higher operational taxonomic unit (OTU) richness but lower evenness than the fungal microbiomes. Bacterial microbiomes also tended to be less variable within groups of samples (e.g., frog species), resulting in stronger clustering in ordination plots. Nevertheless, fungal and bacterial Bray-Curtis dissimilarities were positively correlated within frog species (i.e., two individuals with similar fungal microbiomes tended to also have similar bacterial microbiomes). Fungal and bacterial richness were also correlated. This is a somewhat novel result that suggests that either one microbiome is driving the other, or both are being affected similarly by environmental variables. Results for associations with Bd were mixed. I did not find associations between Bd and beta-diversity for fungi or bacteria. Also, the relative abundance of bacteria that are inhibitory to Bd (based on previous culturing studies) did not follow the expected patterns of association with Bd. Litoria dayi had the highest relative abundance of inhibitory bacteria despite having never recovered from the outbreak, while L. wilcoxii (which never declined) had a low relative abundance of inhibitory bacteria. Additionally, for L. dayi and L. wilcoxii there were significant positive associations between the relative abundance of inhibitory bacteria and Bd infection intensity. In contrast, OTU richness showed negative associations with Bd infection intensity for both fungi and bacteria. Additionally, for both fungi and bacteria, L. dayi had the lowest OTU richness of any frog species. These results are consistent with a protective effect of OTU richness and suggest that a lack of richness in L. dayi has played a role in its inability to recover from the outbreak. In summary, I found that having large areas of high-quality lowland habitat is likely important for allowing populations to retain genetic diversity during an outbreak, and they should be a focus of conservation efforts. Additionally, neither differences in genetic diversity nor differences in dispersal abilities could explain why L. dayi has been unable to recover from population declines. There was some evidence that L. dayi is in the process of adapting, but this was not conclusive. The microbiome data did not show significant associations between Bd and either total community composition or the relative abundance of inhibitory bacteria, but there were associations with the OTU richness of both fungal and bacterial microbiomes, suggesting that richness may be an important factor in infection dynamics

Emerging model spedies driven by transciptomics

This work is focused on 'emerging model species', i.e. question-driven model species which have sufficient molecular resources to investigate a specific phenomenon in molecular biology, developmental biology, molecular ecology and evolution or related molecular fields. This thesis shows how transcriptomic data can be generated, analyzed, and used to investigate such phenomena of interest even in species lacking a reference genome. The initial ButterflyBase resource has proven to be useful to researchers of species without a reference genome but is limited to the Lepidoptera and supports only the older Sanger sequencing technologies. Thanks to Next Generation Sequencing, transcriptome sequencing is more cost effective but the bottleneck of transcriptomic projects is now the bioinformatic analysis and data mining/dissemination. Therefore, this work continues with presenting novel and innovative approaches which effectively overcome this bottleneck. The est2assembly software produces deeply annotated reference transcriptomes stored in the Chado database. The Drupal Bioinformatic Server Framework and genes4all provide species-neutral and an innovative approach in building standardized online databases and associated web services. All public insect mRNA data were analyzed with est2assembly and genes4all to produce the InsectaCentral. With InsectaCentral, a powerful resource is now available to assist molecular biology in any question-driven model insect species. The software presented here was developed according to specifications of the General Model Organism Database (GMOD) community. All software specifications are species-neutral and can be seamlessly deployed to assist any research community. Further through a case studies chapter, it becomes apparent that the transcriptomic approach is more cost-effective than a genomic approach and therefore sequence-driven evolutionary biology will benefit faster with this field

Widespread Genomic Signatures of Natural Selection in Hominid Evolution

Selection acting on genomic functional elements can be detected by its indirect effects on population diversity at linked neutral sites. To illuminate the selective forces that shaped hominid evolution, we analyzed the genomic distributions of human polymorphisms and sequence differences among five primate species relative to the locations of conserved sequence features. Neutral sequence diversity in human and ancestral hominid populations is substantially reduced near such features, resulting in a surprisingly large genome average diversity reduction due to selection of 19–26% on the autosomes and 12–40% on the X chromosome. The overall trends are broadly consistent with “background selection” or hitchhiking in ancestral populations acting to remove deleterious variants. Average selection is much stronger on exonic (both protein-coding and untranslated) conserved features than non-exonic features. Long term selection, rather than complex speciation scenarios, explains the large intragenomic variation in human/chimpanzee divergence. Our analyses reveal a dominant role for selection in shaping genomic diversity and divergence patterns, clarify hominid evolution, and provide a baseline for investigating specific selective events

O papel dos erros de tradução na aquisição de resistência a antifúngicos

Translation of mRNA by the ribosome is a high-fidelity biological process whose error rates range from 10-3 to 10-4 in eukaryotic cells. Such low error rate generalized the idea that mistranslation is a nuisance to the cell without biological relevance. However, recent studies show that translational errors are regulated and play important roles in adaptation to stress situations. Candida albicans, the most prevalent human fungal pathogen, mistranslates naturally at high level in a dynamic way. Exposure of C. albicans to different growth conditions increases mistranslation levels which results in genomic diversification and increased tolerance to antifungals. In this study, we hypothesized that mistranslation accelerates the acquisition of resistance to fluconazole through genomic alterations. To test this hypothesis, we evolved hypermistranslating and wild-type strains in the absence and presence of fluconazole and compared their resistance trajectories during evolution. Results show that mistranslation increases the frequency of acquisition of fluconazole resistance. Ploidy assessment and genome sequencing revealed that during the course of evolution in fluconazole, the range of genomic diversification was broader in the hypermistranslating strains. Specifically, mutations in efflux, drug target and ergosterol biosynthesis genes seem to speed up the acquisition of antifungal drug resistance in hypermistranslating strains. The present work reveals the pivotal role of mistranslation as a mechanism of adaptation of pathogens to antimicrobials.A tradução do mRNA pelo ribossoma é um processo biológico de elevada fidelidade cujo erro basal é da ordem de 10-3 a 10-4 em células eucarióticas. Este baixo nível de erro generalizou a ideia de que os erros de tradução não têm relevância biológica. Contudo, estudos recentes mostram que tais erros são regulados e são relevantes para a adaptação, em particular em situações de stress. Candida albicans, o fungo patogénico mais prevalente nos humanos, traduz naturalmente o mRNA com elevado nível de erro de modo dinâmico. A exposição de C. albicans a diferentes condições de crescimento aumenta o nível de erro de tradução o que, por sua vez, induz a diversificação do seu genoma e o aparecimento de fenótipos de tolerância a antifúngicos. Neste estudo, colocámos a hipótese de que os erros de tradução aceleram a aquisição de resistência ao fluconazol através de alterações genómicas. Para testar esta hipótese, evoluímos estirpes com elevado erro de tradução e estirpes controlo na ausência e presença de fluconazol e comparámos os seus perfis de resistência durante a evolução. Os resultados mostram que os erros de tradução proporcionam um aumento da frequência de aquisição de resistência ao fluconazol. A avaliação da ploidia e sequenciação dos genomas revelaram que durante a evolução com droga, a diversificação do genoma foi maior nas estirpes com elevado erro de tradução. Especificamente, mutações nos genes de efluxo, alvos da droga e biossíntese de ergosterol parecem acelerar a aquisição de resistência ao fluconazol nestas estirpes. O presente trabalho revela o papel central dos erros de tradução como mecanismo de adaptação dos fungos patogénicos aos antimicóticos.Mestrado em Biomedicina Molecula

The genome, transcriptome, and proteome of the nematode Steinernema carpocapsae: Evolutionary signatures of a pathogenic lifestyle

The entomopathogenic nematode Steinernema carpocapsae has been widely used for the biological control of insect pests. It shares a symbiotic relationship with the bacterium Xenorhabdus nematophila, and is emerging as a genetic model to study symbiosis and pathogenesis. We obtained a high-quality draft of the nematode’s genome comprising 84,613,633 bp in 347 scaffolds, with an N50 of 1.24 Mb. To improve annotation, we sequenced both short and long RNA and conducted shotgun proteomic analyses. S. carpocapsae shares orthologous genes with other parasitic nematodes that are absent in the free-living nematode C. elegans, it has ncRNA families that are enriched in parasites, and expresses proteins putatively associated with parasitism and pathogenesis, suggesting an active role for the nematode during the pathogenic process. Host and parasites might engage in a co-evolutionary arms-race dynamic with genes participating in their interaction showing signatures of positive selection. Our analyses indicate that the consequence of this arms race is better characterized by positive selection altering specific functions instead of just increasing the number of positively selected genes, adding a new perspective to these co-evolutionary theories. We identified a protein, ATAD-3, that suggests a relevant role for mitochondrial function in the evolution and mechanisms of nematode parasitism

Identification and interpretation of pathogenic variants following Next Generation Sequencing (NGS) analysis in human Mendelian disorders

Durante il programma di dottorato, l'attenzione è stata rivolta al supporto del laboratorio di diagnostica nell'implementazione della convalida o nella scoperta di varianti insolite. Questo è di massima importanza per comprendere i meccanismi eziopatogenetici molecolari, ma anche per offrire la migliore consulenza alle famiglie. Di conseguenza, sono stati portati a termine diversi progetti come segue: I) un caso enigmatico di una femmina con un disturbo granulomatoso cronico legato all'X (CGD) con una presunta variante di splicing: (NM_000397:ex9:c.1151+2T>C) nel gene CYBB. II) una nuova presunta variante di splicing emizigote nel gene MAGT1 (NM_032121:c.627+2T>C) situato sul cromosoma X. III) Analisi delle variazioni del numero di copie (CNV) per aumentare il tasso di diagnosi di un pannello NGS per gli errori congeniti dell'immunità, poiché è ben noto che le CNV (inserzioni o eliminazioni di dimensioni comprese tra 2 e 50 megabasi) rappresentano circa il 12% delle anomalie genetiche. Identificare questa ampia variazione è ancora problematico, specialmente con le piattaforme Ion Torrent che utilizziamo per la diagnostica, pertanto abbiamo eseguito un'approfondita analisi in silico utilizzando diversi nuovi software. IV) Otto famiglie con una storia personale o familiare di cancro sono state testate per un pannello di geni multipli osono state sottoposte al sequenziamento completo dell’esoma. Sono state trovate otto varianti patogeniche e verificate tramite sequenziamento di Sanger o MLPA e PCR in Real-time. L'uso di NGS e la rilevazione di CNV hanno migliorato la diagnosi nei pazienti affetti da cancro. Alcune delle famiglie iraniane che soddisfacevano i criteri di Amsterdam sono state incluse in programmi di sorveglianza indipendentemente dal loro stato di portatori di mutazioni prima dei test genetici, mentre dopo la rivelazione del portatore solo i portatori sono stati inclusi, migliorando la conformità e riducendo i costi di gestione.During the PhD program the focus was to support diagnostic lab implementing validation or discover of unusual variants. This is of utmost importance to understand molecular etiopathogenic mechanisms, but also in order to offer the best counselling to families. Thus, different projects were accomplished as follows: I) a puzzling patient of a female with X-linked chronic granulomatous disorder (CGD) with a putative splicing variant: (NM_000397:ex9:c.1151+2T>C) in the CYBB gene. II) a novel hemizygous putative splicing mutation in the MAGT1 gene (NM_032121:c.627+2T>C) located on the X-chromosome. III) Analysis of Copy Number Variations (CNVs) to increase the diagnostic rate of a NGS panel for Inborn errors of immunity, as is well known that CNVs (indels between 2 and 50 megabases), account for roughly 12% of genetic abnormalities. Identifying this large variation is still problematic, especially with the Ion Torrent platforms we use for diagnostic, thus we performed an extensive in silico analysis using multiple new softwares. IV) Eight families possessing a familial or personal history of cancer underwent multigene panel testing or whole exome sequencing. Eight pathogenic variants were found and verified through Sanger sequencing or MLPA and real-time PCR. The use of NGS and CNV detection improved the diagnostic yields in cancer patients. Some of Iranian families who met Amsterdam criteria were enrolled in surveillance programs irrespective of their mutation carrier status before genetic testing, while after carrier detection disclosures only carriers were enrolled improving compliance and decreasing the managing cost

Assess the effect of angiogenesis inhibition in intra-tumor heterogeneity

The genetic diversity of the populations that arise within a tumor following cancer pro- gression is known as intra-tumor heterogeneity. Angiogenesis is the formation of new blood vessels from the existing vascular network. It is involved in different physiolog- ical processes, including wound healing. Tumors induce the excessive production of pro-angiogenic factors that promote the proliferation and variety of cancer cells. The inhibitors of tumor angiogenesis were initially designed to destroy the tumor blood ves- sels, causing the death of cancer cells. However, they have been associated with selecting therapy-resistant cells, thus leading to treatment failure. This thesis aims to study the effect of angiogenesis inhibition in intra-tumor heterogeneity based on an experiment in which a breast cancer cell line, designated as MDA-MB-231, was injected into four mice. Two of them were administered with sunitinib, an anti-angiogenic drug. In partic- ular, this thesis investigates whether the two groups of mice, control and treatment, are distinct. Two variables were analyzed to distinguish between the mice: the intra-tumor heterogeneity and the mutational profiles of the genes. Three different heterogeneity esti- mation methods were chosen: the tumor heterogeneity index, PyClone-VI, and Canopy. These worked with the sequencing data of the mice tumor biopsies, specifically with the somatic mutations and copy number alterations. Dimensionality reduction techniques were applied to extract information from several genes. These relied not only on the mice samples but also on the tumor data of patients stored in The Cancer Genome Atlas, which allowed access to more examples. None of the methods could identify a clear difference between the two groups of mice. Their intra-tumor heterogeneity values were similar, and the mutational profiles of their genes appeared to follow the same pattern. Consid- ering these results, we can assume that destroying the tumor blood vessels of the mice from the treatment group did not drive the diversification of cancer cells. Nonetheless, further research should be conducted to confirm this conclusion. For example, test the latest heterogeneity estimation methods and explore the capabilities of neural networks.A diversidade genética das populações que surgem dentro de um tumor após a progressão do cancro é conhecida como heterogeneidade intra-tumoral. A angiogénese é a formação de novos vasos sanguíneos a partir da rede vascular existente. Está envolvida em dife- rentes processos fisiológicos, incluindo a cicatrização de feridas. Os tumores induzem a produção excessiva de factores pró-angiogénicos que promovem a proliferação e varie- dade de células cancerígenas. Os inibidores da angiogénese tumoral foram inicialmente concebidos para destruir os vasos sanguíneos tumorais, causando a morte de células can- cerígenas. No entanto, têm sido associados à selecção de células resistentes à terapia, levando assim ao fracasso do tratamento. Esta tese visa estudar o efeito da inibição da angiogénese na heterogeneidade intra-tumoral, com base numa experiência em que uma linha celular de cancro da mama, designada por MDA-MB-231, foi injectada em quatro ratos. Dois deles foram administrados com sunitinib, um medicamento anti-angiogénico. Em particular, esta tese investiga se os dois grupos de ratos, controlo e tratamento, são dis- tintos. Duas variáveis foram analisadas para distinguir entre os ratos: a heterogeneidade intra-tumoral e os perfis mutacionais dos genes. Foram escolhidos três métodos diferentes de estimativa da heterogeneidade: o índice de heterogeneidade tumoral, o PyClone-VI, e o Canopy. Estes trabalharam com os dados de sequenciação das biópsias tumorais dos ratos, especificamente com as mutações somáticas e as alterações do número de cópias. Técnicas de redução da dimensionalidade foram aplicadas para extrair informação de vários genes. Estas basearam-se não só nas amostras dos ratos mas também nos dados tumorais de pacientes armazenados no Atlas do Genoma do Cancro, o que permitiu o acesso a mais exemplos. Nenhum dos métodos conseguiu identificar uma diferença clara entre os dois grupos de ratos. Os seus valores de heterogeneidade intra-tumoral eram semelhantes, e os perfis mutacionais dos seus genes pareciam seguir o mesmo padrão. Considerando estes resultados, podemos assumir que a destruição dos vasos sanguíneos tumorais dos ratos do grupo de tratamento não impulsionou a diversificação das células cancerígenas. No entanto, devem ser realizadas mais pesquisas para confirmar esta con- clusão. Por exemplo, testar os métodos de estimativa da heterogeneidade mais recentes e explorar as capacidades de redes neuronais