95 research outputs found

    Untying Gordian knots: The evolution and biogeography of the large European apomictic polyploid Ranunculus auricomus plant complex

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    Polyploidie, das Vorhandensein von zwei oder mehr vollstĂ€ndigen ChromosomensĂ€tzen, tritt wiederholt ĂŒber den gesamten Baum des Lebens auf. Bei Pflanzen ist die wirtschaftliche, aber vor allem auch die evolutionĂ€re Bedeutung ĂŒberwĂ€ltigend. Polyploidisierungen, wahrscheinlich verbunden mit SchlĂŒsselinnovationen (z.B. die Entwicklung der GefĂ€ĂŸelemente oder des Fruchtblattes), traten in der Evolution der BlĂŒtenpflanzen hĂ€ufig auf. BlĂŒtenpflanzen sind die artenreichste Gruppe im Pflanzenreich mit ca. 370,000 Arten und umfassen 30–70% Neopolyploide. Es wird angenommen, dass Polyploidie und Hybridisierung (Allopolyploidie) besonders zur Entstehung von Biotypen mit neuartiger genomischer Zusammensetzung beitragen und damit SchlĂŒsselfaktoren fĂŒr nachfolgende Artbildungen und Makroevolution sind. Bei Pflanzen sind beide Prozesse hĂ€ufig mit Apomixis, der Reproduktion ĂŒber asexuell gebildete Samen, verbunden. Das rĂ€tselhafte PhĂ€nomen der von Polyploidie und Apomixis begleiteten Artbildung ist jedoch trotz enormer Fortschritte auf dem Gebiet der Genomik noch immer kaum verstanden. Die Frage „Was ist eine Art?“ hat fĂŒr Evolutionsbiologen höchste PrioritĂ€t: Arten sind die Grundlage der BiodiversitĂ€tsforschung, und die evolutionĂ€re und ökologische Forschung stĂŒtzt sich auf gut definierte Einheiten. EvolutionĂ€r junge Artkomplexe bieten eine einzigartige Möglichkeit die Artbildung bei Pflanzen und deren begleitende Prozesse zu erforschen und zu verstehen. Sie umfassen meist wenige sexuelle Stammarten und zahlreiche polyploide, teilweise apomiktische, hybridogene Derivate. Das Fehlen von Rekombination und KreuzbestĂ€ubung in apomiktischen Linien kann zu einer Vielzahl klonaler Hybridlinien mit fixierten morphologischen und ökologischen Merkmalen fĂŒhren (Agamospezies). Selbst das Erkennen und Abgrenzen der sexuellen Stammarten ist aufgrund geringer genetischer Divergenz, eventuellen hybridogenen UrsprĂŒngen, stetigem Genfluss und/oder unvollstĂ€ndiger genetischer Auftrennung der Abstammungslinien (ILS) methodisch herausfordernd. Integrative AnsĂ€tze, die sowohl genomische als auch morphometrische Daten verwenden, um die jungen Stammarten aufzutrennen, fehlen bisher. Die Biogeographie und Evolution der Artkomplexe ist weitaus komplexer. Apomikten besetzen im Vergleich zu ihren sexuellen Verwandten hĂ€ufig grĂ¶ĂŸere Areale oder sind in nördlicheren Regionen verbreitet, ein PhĂ€nomen, das als Geographische Parthenogenese (GP) bezeichnet wird. GP-Muster haben meist einen pleistozĂ€nen Kontext. Klimatische Schwankungen in den gemĂ€ĂŸigten und borealen Zonen boten hĂ€ufig Möglichkeiten zur interspezifischen Hybridisierung, was wahrscheinlich auch zur Entstehung von Apomixis auf der Nordhalbkugel gefĂŒhrt hat. Faktoren, die diese Muster erzeugen, werden immer noch kontrovers diskutiert. GP-Muster wurden bisher oft den Vorteilen apomiktischer Populationen aufgrund von (Allo)polyploidie und uniparentaler Fortpflanzung zugeschrieben: Fixierte, hohe Heterozygotie fĂŒhrt zu einer erhöhten Stresstoleranz, und SelbstfertilitĂ€t bedingt eine bessere KolonisierungsfĂ€higkeit. Einerseits sind die komplexen Wechselwirkungen von genomweiter Heterozygotie, Ploidie, Reproduktionssmodi (sexuell versus asexuell) und klimatischer Umweltfaktoren auf GP-Muster nicht ausreichend untersucht worden, andererseits wurden potentielle Nachteile sexueller Stammarten aufgrund ihres Fortpflanzungssystems auf Fitness und genetische Vielfalt bisher kaum betrachtet. Schließlich sind neben der Biogeographie die retikulate Evolution und die genomische Zusammensetzung und Evolution junger, großer polyploider Pflanzenartenkomplexe noch nicht detailliert entschlĂŒsselt worden. Neben Herausforderungen, die auf eine hohe Anzahl an Polyploidisierungs- und Hybridisierungsereignissen zurĂŒckzufĂŒhren sind, werden bioinformatische Analysen oft durch fehlende Informationen zu sexuellen Stammarten, Ploidiegraden und Reproduktionsmodi erschwert. Der europĂ€ische, polyploid-apomiktische Ranunculus auricomus (Gold-Hahnenfuß) Pflanzenkomplex ist gut geeignet, um alle aufgeworfenen Fragestellungen zu untersuchen. Der Komplex entstand wahrscheinlich durch unzĂ€hlige Hybridisierungen weniger sexueller Stammarten. Bisher wurden mehr als 800 morphologisch sehr diverse Agamospezies (Derivate) beschriebenen. Die sexuellen Stammarten werden weniger als 1.0 Millionen Jahren alt geschĂ€tzt, und die Agamospezies sind wahrscheinlich noch viel jĂŒnger. In meiner Dissertation habe ich unter Verwendung des R. auricomus Komplexes als Modellsystem die bisher wenig verstandenen phylogenetischen, genomischen und biogeographischen Beziehungen junger, polyploider Pflanzengruppen untersucht. Ich habe einen umfassenden theoretischen und bioinformatischen Workflow entwicklelt, beginnend mit der Untersuchung der Evolution der sexuellen Stammarten, ĂŒber die EntschlĂŒsselung der Reproduktionsmodi und Biogeographie polyploid-apomiktischer Derivate bis hin zur Aufdeckung der retikulaten UrsprĂŒnge und Genomzusammensetzung und -evolution des Polyploidkomplexes. Diese Arbeit umfasst 251 Populationen und 87 R. auricomus Taxa europaweit. Die Analysen basieren auf 97,312 genomischen Loci (RADseq), 663 Kerngenen (target enrichment) und 71 Plastidenregionen, und 1,474 Blattploidie-, 4,669 Reproduktions- Samen-, 284 Kreuzungs- (Samenansatz), und 1,593 Morphometrie-Messungen. Phylogenomische Daten basierend auf RADseq, Kerngenen und geometrischer Morphometrie unterstĂŒtzten die Zusammenlegung der zwölf sexuellen Morphospezies in fĂŒnf neu klassifizierte Stammarten. Diese Arten stellen klar unterscheidbare genetische Hauptlinien oder Cluster dar, die sowohl geographisch gut isoliert als auch morphologisch klar differenziert sind: R. cassubicifolius s.l., R. envalirensis s.l., R. flabellifolius, R. marsicus und R. notabilis s.l. Enorme retikulate Beziehungen innerhalb der Kladen, die nicht-vorhandene geographische Isolation und das Fehlen markanter morphologischer Merkmale haben zu diesem taxonomischen Konzept gefĂŒhrt. Allopatrische Artbildungsereignisse fanden interessanterweise vor ca. 0.83–0.58 Millionen Jahren wĂ€hrend enormer klimatischer Schwankungen statt und wurden wahrscheinlich durch Vikarianzprozesse aus einer weit verbreiteten europĂ€ischen Stammart ausgelöst. DarĂŒber hinaus wurde die neue Umschreibung der sexuellen Stammarten durch Populationskreuzungsexperimente unterstĂŒtzt. Kreuzungen zeigten neben Inzuchtdepression, Auszuchtvorteilen und plötzlicher SelbstkompatibilitĂ€t auch völlig fehlende Reproduktionsbarrieren zwischen einigen Morphospezies. DarĂŒber hinaus wurden durchflusszytometrische Ploidy- und Reproduktions-, genomweite RADseq- und klimatische Umweltdaten in einer genetisch-informierten Pfadanalyse basierend auf Generalisierten Linearen Gemischten Modellen (GLMMs) kombiniert. Die Analyse hat ein komplexes europĂ€isches GP-Szenario aufgedeckt, in der Diploide im Vergleich zu Polyploiden eine signifikant höhere SexualitĂ€t (Prozent sexueller Samen), mehr BlĂŒtenblĂ€tter (petaloide NektarblĂ€tter) und bis zu dreimal weniger genomweite Heterozygotie zeigten. Die SexualitĂ€t war ĂŒberaschenderweise positiv mit Sonneneinstrahlung und IsothermalitĂ€t verbunden, und die Heterozygotie zeigte einen positiven Zusammenhang mit der TemperatursaisonalitĂ€t. Die Ergebnisse stimmen mit der sĂŒdlichen Verbreitung diploid-sexueller Populationen ĂŒberein und deuten auf eine höhere Resistenz polyploid-apomiktischer Populationen gegenĂŒber extremeren klimatischen Bedingungen hin. Ein neu entwickelter, multidisziplinĂ€rer Workflow, der alle bisherigen Daten einbezieht, deckte zum ersten Mal den weitestgehend allopolyploiden Ursprung und die Genomzusammensetzung und -evolution des R. auricomus Komplexes auf. Die Taxa waren in nur drei bis fĂŒnf unterstĂŒtzten, nord-sĂŒd verbreiteten Kladen oder Clustern organisiert, die jeweils meistens diploid-sexuelle Stammarten enthielten. Allopolyploidisierungsereignisse bezogen jeweils zwei bis drei verschiedene, diploid-sexuelle Subgenome ein. Es wurde nur ein autotetraploides Ereignis nachgewiesen. Allotetraploide Genome sind gekennzeichnet durch Subgenomdominanz und einer enormen Evolution nach ihrer Entstehung (z.B. Mendelsche Segregation der Hybridgenerationen, RĂŒckkreuzungen zu Elternarten und Genfluss aufgrund fakultativer SexualitĂ€t der Apomikten). Die ĂŒber 800 Taxa des europĂ€ischen R. auricomus-Komplexes sind vermutlich aus vier diploiden Stammarten und eine bisher unbekannte, aktuell wahrscheinlich ausgestorbene Stammart, entstanden. Analysen zeigten auch, dass die Mehrzahl der beschriebenen polyploiden Agamospezies nicht monophyletisch ist und Ă€hnliche Morphotypen wahrscheinlich mehrfach entstanden sind. Eine umfassende taxonomische Überarbeitung des gesamten Komplexes ist daher angebracht. In der Allgemeinen Diskussion kombiniere ich die Ergebnisse meiner Dissertation mit bereits existierenden Pflanzenstudien zur diploid-sexuellen und polyploid-apomiktischen Phylogenetik, Biogeographie und Genomzusammensetzung und -evolution junger Artkomplexe. Ich gebe zudem taxonomische Schlussfolgerungen und erklĂ€re wie Artkomplexe mikro- und makroevolutionĂ€re Prozesse miteinander verbinden. Abschließend gebe ich ein Fazit ĂŒber die Ergebnisse meiner Dissertation und einen Ausblick fĂŒr das laufende Forschungsprojekt und der Forschungsdisziplin der polyploiden Phylogenetik.Polyploidy, the presence of two or more full genomic complements, repeatedly occurs across the tree of life. In plants, not only the economic but particularly the evolutionary importance is overwhelming. Polyploidization events, probably connected to key innovations (e.g., vessel elements or the carpel), occurred frequently in the evolutionary history of flowering plants, which are the most species-rich group in the plant kingdom (ca. 370,000 species) and contain 30–70% neopolyploids. Polyploidy and hybridization (i.e., allopolyploidy) are particularly considered to create biotypes with novel genomic compositions and to be key factors for subsequent speciation and macroevolution. In plants, both processes are frequently connected to apomixis, i.e., the reproduction via asexually-formed seeds. However, the enigmatic phenomenon of plant speciation accompanied by polyploidy and apomixis is still poorly understood despite tremendous progress in the field of genomics. The question of “What is a species?” is of highest priority for evolutionary biologists: Species are the fundamental units for biodiversity, and further evolutionary and ecological research relies on well-defined entities. Evolutionarily young plant species complexes offer a unique opportunity to study plant speciation and accompanying processes. They usually comprise a few sexual progenitor species, and numerous polyploid, partly apomictic, hybrid derivatives. In apomictic lineages, the lack of recombination and cross-fertilization can result in numerous clonal lineages with fixed morphological and ecological traits (agamospecies). Nevertheless, even recognizing and delimiting the sexual progenitors of species complexes is methodically challenging due to low genetic divergence, possible hybrid origins, ongoing gene flow, and/or incomplete lineage sorting (ILS). Integrative approaches using both genomic and morphometric data for disentangling the young progenitors are still lacking so far. The biogeography and evolution of those plant complexes is even more challenging. Apomicts frequently occupy larger areas or more northern regions compared to their sexual relatives, a phenomenon called geographical parthenogenesis (GP). GP patterns usually have a Pleistocene context because climatic range shifts in temperate to boreal zones offered frequent opportunities for interspecific hybridization, probably giving rise to apomixis in the Northern Hemisphere. Factors shaping GP patterns are still controversially discussed. GP has been widely attributed to advantages of apomicts caused by polyploidy and uniparental reproduction, i.e., fixed levels of high heterozygosity leading to increased stress tolerance, and self-fertility leading to better colonizing capabilities. On the one hand, complex interactions of genome-wide heterozygosity, ploidy, reproduction mode (sexual versus asexual), and climatic environmental factors shaping GP have not been studied enough. On the other hand, potential disadvantages of sexual progenitors due to their breeding system on fitness and genetic diversity have received even less attention. Finally, alongside biogeography, the reticulate relationships and genome composition and evolution of young, large polyploid plant species complexes have not yet been deciphered comprehensively. Besides challenges attributed to numerous numbers of polyploidization and hybridization events, bioinformatic analyses are also often hampered by missing information on progenitors, ploidy levels, and reproduction modes. The European apomictic polyploid Ranunculus auricomus (goldilock buttercup) plant complex is well-suited to study all the aforementioned issues. The majority of goldilock buttercups probably arose from hybridization of a few sexual progenitors, leading to more than 800 described, morphologically highly diverse agamospecies. Sexuals are estimated to have speciated less than 1.0 million years ago, and agamospecies are probably much younger. In this thesis, using R. auricomus as a model system, I examined the recalcitrant and hitherto poorly understood phylogenetic, genomic, and biogeographical relationships of young polyploid apomictic plant complexes. I developed a comprehensive theoretical and bioinformatic workflow, starting with analyzing the evolution of the sexual progenitor species, continuing with unraveling reproduction modes and biogeography of apomictic polyploids, and ending up with revealing the reticulate origins and genome composition and evolution of the polyploid complex. Spanning up to 251 populations and 87 R. auricomus taxa Europe-wide, this work gathered data of 97,312 genomic loci (RADseq), 663 nuclear genes (target enrichment), and 71 plastid regions, and 1,474 leaf ploidy, 4,669 reproductive seed, 284 reproductive crossing (seed sets), as well as 1,593 geometric morphometric measurements. First of all, phylogenomics based on RADseq, nuclear gene, and geometric morphometric data supported the lumping of the twelve described sexual morphospecies into five newly circumscribed progenitor species. These species represent clearly distinguishable genetic main lineages or clusters, which are both well geographically isolated and morphologically differentiated: R. cassubicifolius s.l., R. envalirensis s.l., R. flabellifolius, R. marsicus, and R. notabilis s.l. Mainly within-clade reticulate relationships, missing geographical isolation, and a lack of distinctive morphological characters led to this taxonomic treatment. Interestingly, allopatric speciation events took place ca. 0.83–0.58 million years ago during a period of severe climatic oscillations, and were probably triggered by vicariance processes of a widespread European forest-understory ancestor. Sexual species re-circumscriptions were additionally supported by population crossing experiments. Besides inbreeding depression, outbreeding benefits, and sudden self-compatibility, crossings also revealed a lack of reproductive barriers among some of the formerly described morphospecies. Moreover, flow cytometric ploidy and reproductive, RADseq, and environmental data were combined into a genetically informed path analysis based on Generalized Linear Mixed Models (GLMMs). The analysis unveiled a complex European GP scenario, whereby diploids compared to polyploids showed significantly higher sexuality (percent of sexual seeds), more petals (petaloid nectary leaves), and up to three times less genome-wide heterozygosity. Surprisingly, sexuality was positively associated with solar radiation and isothermality, and heterozygosity was positively related to temperature seasonality. Results fit the southern distribution of diploid sexuals and suggest a higher resistance of polyploid apomicts to more extreme climatic conditions. Finally, a self-developed, multidisciplinary workflow incorporating all previously gathered data demonstrated, for the first time, the predominantly allopolyploid origin, genome composition, and post-origin genome evolution of the R. auricomus complex. Taxa were organized in only three to five supported, north-south distributed clades or cluster, each usually containing diploid sexual progenitor species. Allopolyploidizations involved two to three different diploid sexual subgenomes per event. Only one autotetraploid event was detected. Allotetraploids were characterized by subgenome dominance and enormous post-origin evolution, i.e., Mendelian segregation of hybrid generations, back-crossing to parents, and/or gene flow due to facultative sexuality of apomicts. Four diploid sexual progenitors and a previously unknown, nowadays extinct progenitor, probably gave rise to the more than 800 taxa of the European R. auricomus complex. Analyses also showed that the majority of analyzed polyploid agamospecies are non-monophyletic and similar morphotypes probably originated multiple times. The lack of monophyly suggests a comprehensive taxonomic revision of the entire complex. In the General Discussion, I combine my thesis results with existing plant studies on diploid sexual and polyploid apomictic phylogenetics, biogeography, and composition and genome evolution of young species complexes. I explain the taxonomic conclusions and how species complexes link micro- and macroevolutionary processes. Finally, I give conclusions of my thesis and an outlook of the project and the field of polyploid phylogenetics.2021-10-2

    Inferring Haplotypes of Copy Number Variations From High-Throughput Data With Uncertainty

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    Accurate information on haplotypes and diplotypes (haplotype pairs) is required for population-genetic analyses; however, microarrays do not provide data on a haplotype or diplotype at a copy number variation (CNV) locus; they only provide data on the total number of copies over a diplotype or an unphased sequence genotype (e.g., AAB, unlike AB of single nucleotide polymorphism). Moreover, such copy numbers or genotypes are often incorrectly determined when microarray signal intensities derived from different copy numbers or genotypes are not clearly separated due to noise. Here we report an algorithm to infer CNV haplotypes and individuals’ diplotypes at multiple loci from noisy microarray data, utilizing the probability that a signal intensity may be derived from different underlying copy numbers or genotypes. Performing simulation studies based on known diplotypes and an error model obtained from real microarray data, we demonstrate that this probabilistic approach succeeds in accurate inference (error rate: 1–2%) from noisy data, whereas previous deterministic approaches failed (error rate: 12–18%). Applying this algorithm to real microarray data, we estimated haplotype frequencies and diplotypes in 1486 CNV regions for 100 individuals. Our algorithm will facilitate accurate population-genetic analyses and powerful disease association studies of CNVs

    Holocentric plants of the genus Rhynchospora as a new model to study meiotic adaptations to chromosomal structural rearrangements

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    Climate change, world hunger and overpopulation are some of the biggest challenges the world is currently facing. Moreover, they are part of a multidimensional single scenario: as climate change continues to modify our planet, we might see a decrease of arable land and increase in extreme weather patterns, posing a threat to food security. This has a direct impact on regions with high population growth, where food security is already scarce. Considering additionally the unsustainability of intensive global food production and its contribution to greenhouse emissions and biodiversity loss, it®s clear that all these factors are interconnected (Cardinale et al., 2012; Prosekov & Ivanova, 2018; Wiebe et al., 2019). Plants are the main source of staple food in the world and are also the main actors in carbon fixation, they are therefore key protagonists in controlling climate change. Plants are also an essential habitat-defining element balancing our ecosystem. Thus, how we grow plants and crops will, aside from the obvious implications for food security, also have a profound impact on the climate and biodiversity. The natural variability of species is considered an immense pool of genes and traits, and their understanding is key to generate new useful knowledge. For instance, natural populations can be more tolerant to abiotic and biotic stresses, or carry traits that combined together in hybrids, might achieve a higher seed number, or a faster growth. Classical breeding has exploited unrelated varieties to achieve traits of interest like dwarfism and higher grain production. However, only a limited number of crop species have been the focus of recent scientific and technological approaches, and they do not represent the extremely vast natural diversity of species that could generate useful knowledge for future applications (Castle et al., 2006; Pingali, 2012). The key to this natural variability is a process called meiotic recombination, the exchange of genomic material between homologous parental chromosomes. Meiotic recombination takes place during meiosis, a specialized cell division in which sexually reproducing organisms reduce the genomic complement of their gametes by half in preparation for fertilization. Meiotic recombination takes place at the beginning of meiosis, in a stage called prophase I. To exchange DNA sequences, the strands of two homologous chromosomes must be fragmented. This specific process of physiologically induced DNA fragmentation is conserved in the vast majority of eukaryotes (Keeney et al., 1997). After the formation of double-strand breaks, the 3’ ends that are left are targeted by recombinases that help the strands search and invade templates for repair. After invasion, the 3’ end is extended by DNA synthesis, exposing sequences on the opposite strand that can anneal to the other 3’ end of the original double strand break. DNA synthesis at both ends generates a new structure called a double Holliday Junction (dHJ), forming a physical link between homologous chromosomes, named chiasma (Wyatt & West, 2014). The resolutions of these structures are called crossovers (COs), which is the molecular event representing the outcome of meiotic recombination. Other outcomes are possible, like noncrossovers (NCOs). In this case, the invading strand is ejected and anneals to the single-strand 3®end of the original double-strand break (Allers & Lichten, 2001). Crossovers can be divided into two main groups, called class I and class II. COs of the first group are considered to be sensitive to interference, which means that there are mechanisms that prevent two class I COs from happening in proximity of each other. Class II is insensitive to interference. Class I COs are the result of a pathway called ZMM, which involves a group of specialised proteins that are highly conserved among eukaryotes (Lambing et al., 2017; Mercier et al., 2015). Class I COs are the most common, studied and important type of COs. Centromeres are structures, located on regions of the chromosomes, that allow proper chromosome segregation during mitosis and meiosis. Centromeres have a profound effect on plant breeding and crop improvement, as it is known that meiotic recombination is suppressed at centromeres in most eukaryotes. This represents a great limitation for crop improvement, as many possibly useful traits might be in regions not subject to recombination and thus might not be available for breeding purposes. Additionally, the mechanisms behind how recombination is regulated and prevented from happening at centromeres are still unclear. In most model organisms centromeres are single entities localized on specific regions on the chromosomes. This configuration is called monocentric. However, another type of configuration can be found in nature, but is less studied. In fact, some organisms harbour multiple centromeric determinants distributed over their whole chromosomal length. This configuration is called holocentric. The Cyperaceae comprise a vast, diverse family of plants, with a cosmopolitan distribution in all habitats (Spalink et al., 2016). Despite the presence of this family worldwide, knowledge about it is limited. Few genomes are available and molecular insights are scarce. This family is also known to be mainly formed by holocentric species (Melters et al., 2012). Understanding if and how meiotic recombination is achieved in holocentric plants will generate new knowledge that in the future might unlock new traits in elite crops, previously unavailable to breeding, that could help humanity face global climatic, economic and social challenges. Recent studies have reported new knowledge about important meiotic, chromosome and genome adaptions found in species of the Cyperaceae family and in particular the genus Rhynchospora (Marques et al., 2015, 2016a). With the recent publication of the first reference genomes for several Rhynchospora species, we could already perform a comprehensive analysis of their unique genome features and trace the evolutionary history of their karyotypes and how these have been determined by chromosome fusions (Hofstatter et al., 2021, 2022). This new resource paves the way for future research utilising Rhynchospora as a model genus to study adaptations to holocentricity in plants. With this work, my intention is to shed light on the underexplored topic of holocentricity in plants. Using cutting edge techniques, I examine the conservation of meiotic recombination together with other species-specific adaptations like achiasmy and polyploidy in holocentrics. My results reveal new insights into how plant meiotic recombination is regulated when small centromere units are found distributed chromosome-wide, challenging the classic dogma of suppression of recombination at centromeres

    Analysis of association studies and inference of haplotypic phase using hidden Markov models

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    In this thesis I focus on the development and application of hidden Markov model (HMM) to solve problems in statistical genetics. Our method, based on a HMM, models the joint haplotype structure in the samples, where the parameters in the model are estimated by the Baum-Welch EM algorithm. Also, the model does not require pre-defined blocks or a sliding window scheme to define haplotype boundaries. Thus our method is computationally efficient and applicable for either the whole genome sequence or the candidate gene sequence. The first application of this model is for disease association testing using inferred ancestral haplotypes. We employed a HMM to cluster haplotypes into groups of predicted common ancestral haplotypes from diploid genotypes. The results from simulation studies show that our method greatly outperforms single-SNP analyses and has greater power than a haplotype-based method, CLADHC, in most simulation scenarios. The second application is for inferring haplotypic phase and to predict missing genotypes in polyploid organisms. Using a simulation study we demonstrate that the method provides accurate estimates of haplotypic phase and missing genotypes for diploids, triploids and tetraploids. The third application is for joint CNV/SNP haplotype and missing data inference. The results are very encouraging for this application. With the increasing availability of genotype data in both diploid and polyploid organisms, we believe that our programs can facilitate the investigation of genetic variations in genome-wide scale studies

    Disentangling hexaploid genetics : towards DNA-informed breeding for postharvest performance in chrysanthemum

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    DNA-informed selection can strongly improve the process of plant breeding. It requires the detection of DNA polymorphisms, calculation of genetic linkage, access to reliable phenotypes and methods to detect genetic loci associated with phenotypic traits of interest. Cultivated chrysanthemum is an outcrossing hexaploid with an unknown mode of inheritance. This complicates the development of resources and methods that enable the detection of trait loci. Postharvest performance is an essential trait in chrysanthemum, but is difficult to measure. This makes it an interesting but challenging trait to phenotype and detect associated genetic loci. In this thesis I describe the development of resources and methods to enable phenotyping for postharvest performance, genetic linkage map construction and detection of quantitative trait loci in hexaploid chrysanthemum. Postharvest performance is a complicated trait because it is related to many different disorders that reduce quality. One of these disorders in chrysanthemum is disk floret degreening, which occurs after long storage. In chapter 2, we show that degreening can be prevented by feeding the flower heads with sucrose, suggesting carbohydrate starvation plays a role in the degreening process. To investigate the response to carbohydrate starvation of genotypes with different sensitivity to disk floret degreening, we investigated the metabolome of sugar-fed and carbohydrate-starved disk florets by 1H-NMR and HPAEC. We show that the metabolome is severely altered at carbohydrate starvation. In general, starvation results in an upregulation of amino acid and secondary metabolism. Underlying causes of genotypic differences explaining variation in disk floret degreening in the three investigated genotypes remained to be elucidated, but roles of regulation of respiration rate and camphor metabolism were posed as possible candidates. In chapter 3, disk floret degreening was found to be the most important postharvest disorder after 3 weeks of storage among 44 white chrysanthemum cultivars. To investigate the inheritance of disk floret degreening, we crossed two genotypes with opposite phenotypic values of both disk floret degreening and carbohydrate content to obtain a population segregating for disk floret degreening. To phenotype the cultivar panel and the bi-parental population precisely and in a high throughput manner, we developed a method that quantified colour of detached capitula over time. This method was validated with visual observations of disk floret degreening during vase life tests. In a subset of the bi-parental population we measured carbohydrate content of the disk florets at harvest. The amount of total carbohydrates co-segregated with sensitivity to degreening, which shows that the difference in disk floret degreening sensitivity between the parents could be explained by their difference in carbohydrate content. However, the correlation was rather weak, indicating carbohydrate content is not the only factor playing a role. In order to develop resources for DNA-informed breeding, one needs to be able to characterize DNA polymorphisms. In chapter 4, we describe the development of a genotyping array containing 183,000 single nucleotide polymorphisms (SNPs). These SNPs were acquired by sequencing the transcriptome of 13 chrysanthemum cultivars. By comparing the genomic dosage based on the SNP assay and the dosage as estimated by the read depth from the transcriptome sequencing data, we show that alleles are expressed conform the genomic dosage, which contradicts to what is often found in disomic polyploids. In line with this finding, we conclusively show that cultivated chrysanthemum exhibits genome-wide hexasomic inheritance, based on the segregation ratios of large numbers of different types of markers in two different populations. Tools for genetic analysis in diploids are widely available, but these have limited use for polyploids. In chapter 5, we present a modular software package that enables genetic linkage map construction in tetraploids and hexaploids. Because of the modularity, functionality for other ploidy levels can be easily added. The software is written in the programming language R and we named it polymapR. It can generate genetic linkage maps from marker dosage scores in an F1 population, while taking the following steps: data inspection and filtering, linkage analysis, linkage group assignment and marker ordering. It is the first software package that can handle polysomic hexaploid and partial polysomic tetraploid data, and has advantages over other polyploid mapping software because of its scalability and cross-platform applicability. With the marker dosage scores of the bi-parental F1 population from the genotyping array and the developed methods to perform linkage analysis we constructed an integrated genetic linkage map for the hexaploid bi-parental population described in chapter 3 and 4. We describe this process in chapter 6. With this integrated linkage map, we reconstructed the inheritance of parental haplotypes for each individual, and expressed this as identity-by-descent (IBD) probabilities. The phenotypic data on disk floret degreening sensitivity that was acquired as described in chapter 3, was used in addition to three other traits to detect quantitative trait loci (QTL). These QTL were detected based on the IBD probabilities of 1 centiMorgan intervals of each parental homologue. This enabled us to study genetic architecture by estimating the effects of each separate allele within a QTL on the trait. We showed that for many QTL the trait was affected by more than two alleles. In chapter 7, the findings in this thesis are discussed in the context of breeding for heterogeneous traits, the implications of the mode of inheritance for breeding and the advantages and disadvantages of polyploidy in crop breeding. In conclusion, this thesis provides in general a significant step for DNA-informed breeding in polysomic hexaploids, and for postharvest performance in chrysanthemum in particular.</p

    Snow White and Rose Red: Studies on the contrasting evolutionary trajectories of the genera Leucanthemum Mill. and Rhodanthemum B.H. Wilcox & al. (Compositae, Anthemideae)

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    Plant systematics, the study of taxonomy, phylogeny and evolutionary processes in plants has undergone considerable progress in the last decades. The application of modern molecular approaches and DNA-sequencing techniques in the field has led to an improved inventory of biodiversity and a better understanding of evolutionary processes shaping the biological diversity on our planet. The increased availability of molecular and genomic data has particularly facilitated the investigation of shallowly diverged and taxonomically complex taxon-groups, which is challenging due to minor morphological differences, low genetic differentiation and/or hybridization among taxa. The present thesis investigates species delimitation, hybridization and polyploidization in the recently diverged genera Leucanthemum Mill. and Rhodanthemum B.H. Wilcox & al. of the subtribe Leucantheminae K.Bremer & Humphries (Compositae, Anthemideae) by applying Sanger-, 454-pyro-, and restriction site associated DNA (RAD) sequencing, as well as AFLP-fingerprinting and morphometric analyses. The first two parts are focusing on species delimitation and hybridization in the closely-knit taxon groups around L. ageratifolium Pau and R. arundanum B.H. Wilcox & al., respectively. Various analyses based on AFLP fingerprinting, RADseq and multi-locus sequence data demonstrate that the robustness of species delimitation results is considerably influenced by the intensity of hybridization among species and the number of hybrid individuals included. Therefore, a step-by-step approach is performed in both studies, with an initially step of identification and subsequent removal of hybrid individuals, followed by application of different species-delimitation methods. This strategy results in the reliable identification of independent species, subspecies and nothospecies in both taxonomically complex plant groups. The third part of the present thesis compares the contrasting evolutionary trajectories of diploid representatives of both genera in a more comprehensive phylogenetic study. Specific hypotheses for the formation of polyploids in plants are proposed and evaluated to find factors that promote polyploidization in certain plant groups (e.g., Leucanthemum) and not in others (e.g., Rhodanthemum). Multi-locus sequence data from 127 accessions of the subtribe Leucantheminae unveil a significantly higher genetic divergence and hybridization signal among diploid lineages of Leucanthemum compared to Rhodanthemum, in spite of a similar crown age and diversification pattern during the Quaternary. The study demonstrates the importance of genetic differentiation among diploid progenitors and their concurrent affinity for natural hybridization for the formation of a polyploid complex. Furthermore, the role of climate-induced range overlaps on hybridization and polyploid speciation during the Quaternary is discussed

    Genome fractionation and loss of heterozygosity in hybrids and polyploids: Mechanisms, consequences for selection, and link to gene function

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    Hybridization and genome duplication have played crucial roles in the evolution of many animal and plant taxa. The subgenomes of parental species undergo considerable changes in hybrids and polyploids, which often selectively eliminate segments of one subgenome. However, the mechanisms underlying these changes are not well understood, particularly when the hybridization is linked with asexual reproduction that opens up unexpected evolutionary pathways. To elucidate this problem, we compared published cytogenetic and RNAseq data with exome sequences of asexual diploid and polyploid hybrids between three fish species; Cobitis elongatoides, C taenia, and C tanaitica. Clonal genomes remained generally static at chromosome-scale levels but their heterozygosity gradually deteriorated at the level of individual genes owing to allelic deletions and conversions. Interestingly, the impact of both processes varies among animals and genomic regions depending on ploidy level and the properties of affected genes. Namely, polyploids were more tolerant to deletions than diploid asexuals where conversions prevailed, and genomic restructuring events accumulated preferentially in genes characterized by high transcription levels and GC-content, strong purifying selection and specific functions like interacting with intracellular membranes. Although hybrids were phenotypically more similar to C taenia, we found that they preferentially retained C elongatoides alleles. This demonstrates that favored subgenome is not necessarily the transcriptionally dominant one. This study demonstrated that subgenomes in asexual hybrids and polyploids evolve under a complex interplay of selection and several molecular mechanisms whose efficiency depends on the organism's ploidy level, as well as functional properties and parental ancestry of the genomic region.Web of Science38125274525
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