Functional Classification of Skeletal Muscle Networks. I. Normal Physiology

Extensive measurements of the parts list of human skeletal muscle through transcriptomics and other phenotypic assays offer the opportunity to reconstruct detailed functional models. Through integration of vast amounts of data present in databases and extant knowledge of muscle function combined with robust analyses that include a clustering approach, we present both a protein parts list and network models for skeletal muscle function. The model comprises the four key functional family networks that coexist within a functional space; namely, excitation-activation family (forward pathways that transmit a motoneuronal command signal into the spatial volume of the cell and then use Ca2+ fluxes to bind Ca2+ to troponin C sites on F-actin filaments, plus transmembrane pumps that maintain transmission capacity); mechanical transmission family (a sophisticated three-dimensional mechanical apparatus that bidirectionally couples the millions of actin-myosin nanomotors with external axial tensile forces at insertion sites); metabolic and bioenergetics family (pathways that supply energy for the skeletal muscle function under widely varying demands and provide for other cellular processes); and signaling-production family (which represents various sensing, signal transduction, and nuclear infrastructure that controls the turn over and structural integrity and regulates the maintenance, regeneration, and remodeling of the muscle). Within each family, we identify subfamilies that function as a unit through analysis of large-scale transcription profiles of muscle and other tissues. This comprehensive network model provides a framework for exploring functional mechanisms of the skeletal muscle in normal and pathophysiology, as well as for quantitative modeling

드노보 전장유전체 조립 및 분석을 통한 해양 절지동물 진화 사례 연구

학위논문 (박사) -- 서울대학교 대학원 : 자연과학대학 생명과학부, 2020. 8. 김원.The de novo genome assembly has become an essential approach for studying non-model organisms since the post-genome era arrived. The reported cases of de novo genome assemblies of non-model arthropods have increased dramatically in recent days. The marine arthropod, however, is one of the least sequenced animal groups despite of their surprisingly high taxonomic and morphological diversity. The de novo genome studies on these marine arthropods remain mostly limited in terms of their cases and quality of assemblies up to now. This study therefore conducted the first case of de novo genome research focusing to the under-sampled marine arthropod groups, the Class Pycnogonida and the Infraorder Brachyura in Korea. In this study, one mitochondrial genome and four whole-genomes were de novo assembled and their genomic characteristics were discussed. While the two cases of de novo genomes assembled by using short read-length sequencing showed limited assembly quality, the long read-length based assemblies of Nymphon striatum and Chionoecetes opilio provided significantly informative, high-qualitied genomes. The preliminary phylogenomic research of this study which firstly included the representative genomes of pycnogonid and brachyuran decapod, also implied that recent hypothesis of xiphosuran nested in the most derived clade, Arachnopulmonate, is indeedly plausible. Furthermore, the limitations of de novo genome researches on the laboratory experiment lacking bioinformatics background were discussed to establish an optimized research workflow for the genomic study on non-model marine arthropod.포스트게놈 시대의 도래에 따라 드노보 유전체 조립은 비모델 생명체의 생명현상을 연구하는데 필수적인 과정이 되었다. 비모델 절지동물의 드노보 조립된 유전체의 사례는 근래에 들어 급격하게 증가했다. 그러나, 해양 절지동물은 놀라울 정도로 다양한 분류군과 형태를 가짐에도 불구하고, 가장 드노보 유전체 조립 연구가 미흡한 분류군 중 하나이다. 현재까지 보고된 해양 절지동물의 드노보 유전체 조립 연구는 대부분이 그 양과 질 모두가 제한적이다. 그러므로, 본 연구는 국내에서 최초로 선행 연구가 미흡한 해양 절지동물 분류군인 바다거미 강과 단미 하목에 초점을 맞춰 드노보 유전체 조립 및 분석을 실시하였다. 본 연구의 결과로, 1건의 미토콘드리아 유전체와 4건의 전장유전체가 드노보 조립되었으며, 조립된 유전체의 특징이 기술되었다. 단서열 염기서열결정법으로 조립된 두 건의 유전체의 품질은 비교적 낮았으나, 장서열 염기서열결정법을 주로하여 조립된 Nymphon striatum과 Chionoecetes opilio 유전체가 매우 풍부한 고품질 유전체 정보를 제공한다는 것이 밝혀졌다. 본 연구에서 수행된 기초적인 계통유전체학 연구는 바다거미 강과 십각 목을 각각 대표하는 드노보 조립된 유전체를 최초로 포함했으며, 이를 통해 최근 논란의 대상인 거미강에 속하는 투구게류 가설을 지지하는 결과를 나타내는 것으로 밝혀졌다. 더 나아가, 비생물정보학 연구실 환경에서 이루어지는 드노보 유전체 연구에서 발생하는 제한요인들을 분석함으로써 비모델 해양 절지동물의 드노보 유전체 연구에 최적화된 안정적인 연구방법론을 제시하였다.BACKGROUNDS 1 CHAPTER 1. THE PILOT RESEARCHES FOR EVOLUTIONARY STUDIES ON MARINE ARTHROPOD GENOMES 17 1.1. The preliminary genomic studies on Liparis tanakae and its genomic characteristics 19 1.1.1. Introduction 19 1.1.2. Materials and Methods 22 1.1.3. Results 29 1.1.4. Discussion 35 1.2. The de novo Mitochondrial genome assembly of Chionoecetes opilio : The manual curation of predicted genes and the phylogenomic analyses with large datasets 38 1.2.1. Introduction 38 1.2.2. Materials and Methods 40 1.2.3. Results 43 1.2.4. Discussion 49 CHAPTER 2. THE DE NOVO GENOME ASSEMBLIES OF THREE MARINE ARTHROPODS 53 2.1. The first de novo assembled genome of Portunus trituberculatus indicating the bottlenecks in researching non-model marine arthropods 55 2.1.1. Introduction 55 2.1.2. Materials and Methods 57 2.1.3. Results 63 2.1.4. Discussion 69 2.2. The high-qualitied marine arthropod assemblies : De novo assembled Chionoecetes opilio and Nymphon striatum genomes and their characteristics 71 2.2.1. Introduction 71 2.2.2. Materials and Methods 76 2.2.3. Results 86 2.3. General discussion 100 2.3.1. The ab initio prediction and annotation of marine arthropod Hox genes 100 2.3.2. The optimizied workflow of de novo whole-genome researches of marine arthropods 104 CHAPTER 3. THE CASE STUDY OF THE ARTHROPOD EVOLUTION THROUGH THE COMPARATIVE WHOLE-GENOME ANALYSES 111 3.1. The preliminary chelicerate phylogenomic analyses incorporating under-sampled taxa 113 3.1.1. Introduction 113 3.1.2. Materials and Methods 120 3.1.3. Results and Discussion 125 CONCLUSION 131 REFERENCES 135 APPENDIX 157 Appendix 1. Detailed list of sequenced animal genomes with their Scientific names visible 159 ABSTRACT (In Korean) 180Docto

Genomic basis for skin phenotype and cold adaptation in the extinct Steller’s sea cow

Steller’s sea cow, an extinct sirenian and one of the largest Quaternary mammals, was described by Georg Steller in 1741 and eradicated by humans within 27 years. Here, we complement Steller’s descriptions with paleogenomic data from 12 individuals. We identified convergent evolution between Steller’s sea cow and cetaceans but not extant sirenians, suggesting a role of several genes in adaptation to cold aquatic (or marine) environments. Among these are inactivations of lipoxygenase genes, which in humans and mouse models cause ichthyosis, a skin disease characterized by a thick, hyperkeratotic epidermis that recapitulates Steller’s sea cows’ reportedly bark-like skin. We also found that Steller’s sea cows’ abundance was continuously declining for tens of thousands of years before their description, implying that environmental changes also contributed to their extinction

포유류 유전체 내 선택압에 의한 적응 흔적 및 특성 발굴

학위논문(박사)--서울대학교 대학원 :자연과학대학 협동과정 생물정보학전공,2019. 8. 김희발.The central goal of evolutionary biology is to understand the genetic basis of evolutionary processes and adaptive traits. In this regard, the recent advances in sequencing technologies and the explosion of sequence data provide a better opportunity to reach this goal. Various genomic variations are now easily and precisely obtained for large-scale of samples. They are expanding the scope of typical genomic studies, allowing us to take into account diverse evolutionary processes. The aim of this thesis is to demonstrate the applications of such genomic variations while taking into account diverse evolutionary scenarios and time scales. As such, this thesis will fill in the gaps in the knowledge of mammalian genetic background underlying adaptive traits through genome-wide scan and comparative genome analysis. This thesis consists of five chapters and includes results of genome analysis for detecting evolutionary signatures in three mammal species; pinnipeds, primates, and cattle. The basic background, terminologies and recent example studies related to this thesis were introduced in chapter 1. Chapter 2 and 3 focused on divergence between species (macroevolution), while chapter 4 and 5 focused on polymorphism within species (microevolution). Pinnipeds are a remarkable group of marine animals with unique adaptations to semi-aquatic life. However, their genomes are poorly characterized. In chapter 2, evolutionary signatures of pinnipeds have been investigated using amino acid substitutions. Novel genome assemblies of 3 pinniped species; Phoca largha, Callorhinus ursinus, and Eumetopias jubatus have been generated. These genome assemblies have been used to detect rapidly evolving genes and substitutions unique to pinnipeds associated with their specificities. As a result, unique substitutions were found within the TECTA gene and are likely related to the adaptation to amphibious sound perception in pinnipeds. In addition, several genes (FASN, KCNA5, and IL17RA) containing substitutions specific to pinnipeds were found to be potential candidates of phenotypic convergence in all marine mammals. It indicates the weak link between molecular and phenotypic convergence, and confirms the results of previous studies. This study provides candidate targets for future studies of gene function, as well as backgrounds for convergent evolution of marine mammals. Humans have the largest brain among extant primates with specialized neuronal connections. However, how the human brain rapidly evolved compared to that of closely related primates is not fully understood. In chapter 3, a genome-wide survey has been performed to find an explanation for the rapid evolution of human brain. Based on the hypothesis that tandem repeats could play a key role in introducing genetic variations due to their unstable nature, a genome-wide survey detected 152 human-specific TRs (HSTR) that have emerged only in the human lineage. The HSTRs are associated with biological functions in brain development and synapse function, and the expression level of HSTR-associated genes in brain tissues was significantly higher in human than in other primates. These results suggest a possibility that de novo emergence of TRs might have contributed to the rapid evolution of human brain. The genetic history of cattle is complex, but contains plentiful information to comprehend mammalian evolutionary process such as domestication, and environmental adaptations. In chapter 4, the genomic influence of recent artificial selection has been examined in the case of Korean native cattle, Hanwoo. Using runs of homozygosity (ROH), an increase of inbreeding for decades has been shown, and at the same time, it has been demonstrated that inbreeding has been of little influence on body weight trait. In chapter 5, admixture between two cattle populations; Bos taurus, and Bos indicus has been examined in Indigenous African cattle populations., Several evidences based on single nucleotide polymorphism (SNP) support that adaptive admixture is at the root of the success of African cattles rapid dispersion across African continent. The findings in this thesis demonstrated applications of various genomic variations under diverse evolutionary scenarios and time scales, and thus may contribute to the understanding of evolutionary processes in mammals.진화 생물학의 핵심 목표는 진화 과정과 적응 형질의 유전적 기초를 이해하는 것이다. 이와 관련하여 최근 시퀀싱 기술의 진보와 서열 데이터의 폭발적 증가는 이러한 목표를 달성 할 수 있는 더 좋은 기회를 제공하고 있다. 실제로 시퀀싱 기술의 발달로 대규모 시료에 대하여 좀 더 쉽고 정확하게 다양한 유전변이를 얻을 수 있게 되었으며, 이 덕분에 일반적인 유전체 연구의 범위가 확장되었고 다양한 진화 과정을 고려할 수도 있게 되었다. 이에 이 논문의 목적은 다양한 진화과정 하에서 여러 유전변이의 유용성을 보여주고, 전장 유전체 및 비교 유전체 분석을 통해 포유류 적응 형질의 유전적 배경을 밝히는 것이다. 이 논문은 세 가지 포유 동물 종(기각류, 영장류, 소)의 유전체 분석 결과를 포함한 5 개의 장으로 구성되어있다. 제 1장에서는 이 논문과 관련된 배경 지식과 최근의 연구 사례를 소개하고 있다. 전반부 (제 2, 3장)는 종간 비교분석에 중점을 두었고, 후반부(제4, 5장)는 종내의 다형성에 초점을 두고 있다. 기각류는 반 수생 환경에 적응한 특징적인 해양 동물이다. 그러나 그 유전체는 특성이 잘 알려져 있지 않다. 제2 장에서는 아미노산 치환 정보를 이용하여 기각류의 진화 및 적응 흔적을 조사하였다. 구체적으로 기각류3 종의 새로운 유전체를 이용하여 기각류의 생활환경과 관련된 양성선택 유전자 및 아미노산 치환 흔적을 발굴하였다. 특히 TECTA 유전자 내의 고유한 아미노산 치환 흔적은 기각류의 청각과 밀접한 관련이 있을 것으로 예상된다. 또한, 이전 연구결과와 같이 해양 포유류에서 표현형의 수렴진화와 직접적으로 연관되어 있는 서열 수렴은 흔하지 않다는 것을 확인하였다. 예를 들어, FASN, KCNA5 및 IL17RA는 기각류에 특이적인 아미노산 치환을 포함하지만 모든 해양 포유 동물에서 공통적으로 표현형 수렴진화 (두꺼운 지방조직, 저산소 적응 및 병원체에 대한 면역 반응)가 일어났을 것으로 예상된다. 이러한 연구 결과들은 해양 포유류의 수렴 진화 특성에 대한 지식을 제공함과 동시에 유전자 기능 연구에 대한 후보 표적을 제공할 것으로 기대된다. 인간은 현존하는 영장류 중에서 가장 큰 두뇌를 가지고 있다. 그러나 인간의 뇌가 어떻게 영장류 중에서 특히 빠르게 진화했는지 는 완전히 밝혀지지 않았다. 제 3 장에서는 인간 두뇌의 급속한 진화에 대한 가설을 찾기 위해 유전체 분석을 수행하였다. 반복서열이 그 불안정한 성질 때문에 급속한 유전적 변이를 일으키는 데 핵심적인 역할을 할 수 있다는 가설에 근거하여, 유전체 비교분석에서 인간 특이적인 152 개의 반복서열을 검출하였다. 특이하게도, 이러한 반복서열들은 뇌 발달 및 시냅스 기능과 관련이 있었으며, 뇌 조직에서 해당 반복서열과 관련된 유전자의 발현 수준은 다른 영장류보다 인간에서 유의하게 높았다. 이러한 결과는 반복서열이 인간 두뇌의 급속한 진화에 기여하였을 수도 있다는 하나의 가능성을 제시한다. 소의 유전적 역사는 복잡하지만 가축화 및 환경 적응과 같은 포유 동물의 진화 과정을 이해할 수 있는 풍부한 정보를 담고있다. 제 4 장에서는 한국 토종 소품종인 한우의 유전체 선발이 한우 집단에 미친 유전적 영향을 조사 하였다. Runs of humozygosity를 이용하여, 최근에 일어난 근친 교배의 증가를 보여 주었고 동시에, 근교약세가 체중에 영향을 미칠 만큼 크지 않았다는 것을 유전정보를 통해 보여주었다. 제 5 장에서는 소의 두 아종 (Bos taurus, Bos indicus)사이의 유전적 혼합을 아프리카 토착 소의 단일 염기 다형성 자료를 통해 분석하였다. 이를 통해 아프리카 소의 환경에 대한 빠른 적응의 원인은 유전적 혼합에 있다는 여러 증거를 제시하였다. 이 논문은 다양한 진화과정 하에서 다양한 유전변이의 적용사례를 보여주고, 또한 이를 통해 포유 동물의 다양한 진화 과정을 이해하는 데에 기여할 수 있을 것으로 기대된다.ABSTRACT I CONTENTS V LIST OF TABLES VII LIST OF FIGURES IX GENERAL INTRODUCTION XIV CHAPTER 1. LITERATURE REVIEW 1 1.1 GENOMIC VARIATIONS 2 1.2 SIGNATURES OF POSITIVE SELECTION 7 1.3 SIGNATURES OF INTROGRESSION 13 CHAPTER 2. DECIPHERING THE EVOLUTIONARY SIGNATURES OF PINNIPEDS USING NOVEL GENOME SEQUENCES: THE FIRST GENOMES OF PHOCA LARGHA, CALLORHINUS URSINUS, AND EUMETOPIAS JUBATUS 17 2.1 ABSTRACT 18 2.2 INTRODUCTION 19 2.3 MATERIALS AND METHODS 23 2.4 RESULTS 37 2.5 DISCUSSION 66 CHAPTER 3. DE NOVO EMERGENCE AND POTENTIAL FUNCTION OF HUMAN-SPECIFIC TANDEM REPEATS IN BRAIN-RELATED LOCI 70 3.1 ABSTRACT 71 3.2 INTRODUCTION 72 3.3 MATERIALS AND METHODS 75 3.4 RESULTS 89 3.5 DISCUSSION 109 CHAPTER 4. ARTIFICIAL SELECTION INCREASED BODY WEIGHT BUT INDUCED INCREASE OF RUNS OF HOMOZYGOSITY IN HANWOO CATTLE 114 4.1 ABSTRACT 115 4.2 INTRODUCTION 116 4.3 MATERIALS AND METHODS 121 4.4 RESULTS 128 4.5 DISCUSSION 150 CHAPTER 5. THE MOSAIC GENOME ARCHITECTURE OF INDIGENOUS AFRICAN CATTLE AS A UNIQUE GENETIC RESOURCE FOR ADAPTATION TO LOCAL ENVIRONMENTS 154 5.1 ABSTRACT 155 5.2 INTRODUCTION 157 5.3 MATERIALS AND METHODS 161 5.4 RESULTS 174 5.5 DISCUSSION 199 GENERAL DISCUSSION 204 REFERENCES 207 요약(국문초록) 234Docto

Genome annotation, comparative genomics and evolution of the model grass genus Brachypodium (Poaceae)

INTRODUCCIÓNEn esta Tesis Doctoral se han llevado a cabo estudios evolutivos, biogeográficos, genómicos, transcriptómicos y de expresión génica en los taxones que integran el género Brachypodium (Poaceae) con el objetivo de descifrar los eventos de especiación que han dado lugar a las especies de dicho género. La tesis está constituida por cuatro capítulos con sus respectivos cuatro apéndices. DESARROLLO TEÓRICOCapítulo 1. Reconstrucción de los orígenes y la biogeografía de los genomas de las especies del género modelo de gramíneas Brachypodium, altamente reticulado y rico en especies alopoliploides, mediante métodos de evolución mínima, coalescencia y máxima verosimilitud (Reconstructing the origins and the biogeography of species’ genomes in the highly reticulate allopolyploid-rich model grass genus Brachypodium using minimum evolution, coalescence and maximum likelihood approaches).Se ha reconstruido la filogenia y la historia biogeográfica de las especies reconocidas de Brachypodium mediante el análisis evolutivo del gen nuclear copia simple GIGANTEA (GI), y de otros genes tanto nucleares (ITS, ETS) como plastídicos (ndhF, trnL-F). Para ello se han desarrollado análisis de redes haplotípicas, de mapeo de los alelos poliploides en el árbol de especies diploides por evolución mínima y de construcción del árbol de especies (de genomas y subgenomas), así como la reconstrucción biogeográfica y el cálculo de los tiempos de divergencia de los subgenomas homeólogos por métodos máximo verosímiles y bayesianos, respectivamente, y la estimación por coalescencia de los posibles tiempos en los que se produjeron los eventos de hibridación que dieron lugar a las especies alopoliploides.Nuestros resultados apoyan la naturaleza alopoliploide de las especies poliploides del género, así como un escenario espacio-temporal de diversas divergencias y fusiones de los genomas en las diferentes áreas ancestrales, mostrando un claro predominio de la dispersión de los genomas diploides frente a los alopoliploides. También han sido inferidos los tiempos de divergencia de todas los linajes estudiados, desde el más ancestral, B. stacei (6,8 Ma) hasta los más recientes del “core perennial” (0,7-0,3 Ma).Capítulo 2. Mapeos sinténicos contra genomas de referencia y estudios filogenómicos basados en análisis multigénicos revelan los ancestros de los subgenomas homeologos de especies alopoliploides del género Brachypodium. (Reference-genome syntenic mapping and multigene-based phylogenomics reveal the ancestry of homeologous subgenomes in grass Brachypodium allopolyploids).Se llevaron a cabo mapeos sinténicos de datos transcriptómicos y de genotipado genómico (GBS) contra los genomas de referencia de Brachypodium desarrollando nuestros propios algoritmos y herramientas bioinformáticas. Con esos datos se ha reconstruido la filogenia y se han datado los orígenes de los genomas y subgenomas presentes en especies diploides y alopoliploides de Brachypodium empleando métodos bayesianos y de máxima verosimilitud. Adicionalmente, se ha obtenido y analizado el pan-transcriptoma de las especies estudiadas, identificando genes potencialmente exclusivos de determinados grupos de especies.Los análisis filogenéticos basados en datos transcriptómicos junto con la obtención de tamaños genómicos nos ha permitido elucidar complejos escenarios de hibridación para los subgenomas homeologos de las seis especies alopoliploides de Brachypodium estudiadas, mostrando distintos eventos de hibridación que implican únicamente a genomas ancestrales, a genomas ancestrales y recientes, y únicamente a genomas recientes. Nuestros resultados apoyan el origen ancestral de B. mexicanum, el intermedio de B. boissieri y B. retusum, y la rápida radiación de las especies del “core perennial”.Los capítulos 1 y 2 suponen un completo análisis del género Brachypodium, en especial de las especies alopoliploides, cuyos resultados apoyan y datan los eventos evolutivos que han dado lugar a la compleja y reticulada historia evolutiva que presenta dicho género así como los posibles linajes progenitores de las especies alopoliploides. Capítulo 3. Genómica comparada del plastoma y filogenómica de Brachypodium: improntas de los tiempos de floración, introgresión y recombinación en los ecotipos recientemente evolucionados. (Comparative plastome genomics and phylogenomics of Brachypodium: flowering time signatures, introgression and recombination in recently diverged ecotypes).Se han ensamblado, anotado y analizado los genomas organulares (plastomas) de un amplio número de ecotipos de las especies anuales de Brachypodium, y se ha reconstruido su filogenia, comparándola con la de sus genomas nucleares. Se ha dilucidado la diversificación de los ecotipos y su relación con factores de tiempos de floración y geográficos. El estudio comparativo de los 57 genomas cloroplásticos ensamblados y anotados, 53 B. distachyon, 3 B. hybridum y 1 B. stacei, ha revelado reordenamientos genómicos, como la inserción de 1161 pb y la deleción de una de las copias del gen rps19, característicos de las especies B. stacei y B. hybridum (plastoma tipo stacei), respecto de las líneas de B. distachyon. Se han estimado sus orígenes mediante datación anidada dentro del marco evolutivo de las gramíneas y, para las líneas de B. distachyon, se ha comparado la filogenia plastómica con la nuclear, detectándose una divergencia clara de dos linajes intra-específicos, relacionada con sus tiempos de floración, y una subestructuración más reciente relacionada con su geografía, así como la evidencia de capturas cloroplásticas e introgresión entre clados distantes. Estos datos apoyan que la tendencia a la microespeciación se ve frenada por procesos de introgresión recurrente.Capítulo 4. Características de las redes de co-expresión y los genes diferencialmente expresados explican los patrones de respuesta a sequía en la gramínea modelo Brachypodium distachyon. (Co-expression network features and differentially expressed genes explain drought-response patterns in the model grass Brachypodium distachyon).Se han identificado y analizado genes funcionales implicados en la respuesta ambiental a estrés hídrico mediante el análisis de redes de co-expresión génica y de genes diferencialmente expresados en diversos ecotipos de la planta modelo Brachypodium distachyon.Se han analizado tanto la topología de dichas redes como los genes e isoformas más interconectadas, detectando 38 módulos en la red de co-expresión bajo condiciones de sequía, con 628 genes altamente interconectados (820 transcritos), y 30 módulos en la red de co-expresión en condiciones de riego con 839 genes altamente interconectados (1072 transcritos). Además se han identificado los procesos biológicos en los que están implicados los genes co-expresados, encontrando cinco módulos exclusivos de la red bajo condiciones de sequía de los cuales tres están directamente relacionados con procesos de respuesta a estrés hídrico. Estos resultados han sido correlacionados con datos pan-genómicos observando que la mayoría de los genes co-expresados son genes “core”, conservados en todos la líneas estudiadas, o “soft-core”, conservados en más del 90% de las líneas estudiadas.CONCLUSIONES1-Los análisis evolutivos y biogeográficos de los 20 taxones reconocidos del género Brachypodium empleando cinco genes (tres nucleares y dos plastídicos) indican que aproximadamente la mitad de las especies son diploides y la otra mitad alopoliploides. El análisis de evolución mínima de “injerto” de alelos alopoliploides en las ramas del árbol diploide recobra los linajes homeólogos (subgenomas) de los alopoliploides. Las sucesivas divergencias de los linajes de las diploides anuales (B. stacei, B. distachyon) tuvieron lugar durante el Mioceno tardío-Plioceno en la cuenca Mediterránea, mientras que las de los linajes de las diploides perennes (B. arbuscula, B. genuense, B. sylvaticum, B. glaucovirens, B. pinnatum-2x y B. rupestre-2x) ocurrieron durante el Cuaternario en las regiones Mediterránea y Euroasiática, con colonizaciones esporádicas de otros continentes. Las respectivas divergencias de los linajes homeólogos de los alopoliploides tuvieron lugar en distintos tiempos evolutivos. Nuestro escenario biogeográfico apoya la existencia de dispersiones a larga distancia únicamente en los linajes diploides, mientras que todos los eventos de hibridación y duplicación genómica ocurrieron dentro de las áreas ancestrales progenitoras más recientes, sin posteriores expansiones de área.2- Los análisis filogenómicos mediante datos de RNA-seq y GBS han identificado a B. mexicanum como la especie alopoliploide más antigua mostrando subgenomas de tipo ancestral (A) y materno de tipo stacei (B) (Mioceno medio-tardío). Los alopoliploides de elevado nivel de ploidía, B. boissieri y B. retusum, muestran tres y cuatro subgenomas respectivamente. Ambas especies presentan subgenomas A y B así como el subgenoma intermedio tipo distachyon (C) (Mioceno-Plioceno) (heredado maternalmente en B. boissieri). B. retusum también presenta un subgenoma materno tipo core perennial recientemente evolucionado (D) (Cuaternario). Los alotetraploides del clado core perennial B. rupestre y B. phoenicoides muestran únicamente subgenomas recientemente evolucionados tipo C y D (Cuaternario), siendo los diploides perennes B. pinnatum y B. sylvaticum sus respectivos progenitores maternos. El reciente alopoliploide B. hybridum se formó repetidamente y mediante cruzamientos bidireccionales durante el Cuaternario y es el único alopoliploide del que se conocen ambos progenitores diploides actuales, B. distachyon y B. stacei.3- Los análisis pan-transcriptómicos de 5202 conjuntos de tránscritos del género Brachypodium muestran genes expresados exclusivamente en los grupos de especies perennes (30), anuales (49), poliploides (14), alopoliploides más antiguos (143), especies ancestrales (14) y especies recientemente evolucionadas (52). Los tránscritos exclusivos de los alopoliploides antiguos podrían estar asociados con su genoma ancestral tipo A. Los tránscritos anotados como subunidad ARN polimerasa, encontrados únicamente en todas las especies anuales de Brachypodium, podrían indicar la existencia de diferencias en los niveles de expresión de las ARN polimerasas entre las especies anuales y perennes, o la pérdida de copias ancestrales en las especies perennes más recientemente evolucionadas.4- Los análisis pan-genómicos de los plastomas de 53 ecotipos de B. distachyon, 3 de B. hybridum y 1 de B. stacei han detectado una inserción (1161 pb) y una deleción en una de las copias del gen rps19 que diferencian a los plastomas de B. stacei y B. hybridum con respecto a los de B. distachyon, sin que se haya observado variación en el contenido génico entre los plastomas de B. distachyon.5- El árbol filogenómico de los plastomas de B. distachyon muestra la divergencia de dos linajes principales, correspondientes a los clados Extremely Delayed Flowering (EDF+) y Spanish (S+) – Turkish (T+), sugiriendo que el tiempo de floración es un factor decisivo en la divergencia intra-específica de B. distachyon. La comparación topológica entre las filogenias nucleares y plastídicas de esta especie revela nueve eventos de captura cloroplástica y dos de introgresión y micro-recombinación entre esos clados, apoyando la existencia de flujo génico entre linajes previamente aislados. Los intercambios de plastomas entre los tres grupos, EDF+, T+, S+, probablemente hayan sido el resultado de retro-cruzamientos aleatorios seguidos de estabilización por presión selectiva.6- Los análisis mediante redes ponderadas de co-expresión génica llevados a cabo en 33 ecotipos de B. distachyon bajo condiciones de sequía y riego identificaron cinco módulos exclusivos de la red de sequía, incluyendo 465 isoformas y 11 genes altamente interconectados (hubs). El análisis seleccionó genes candidatos y factores de transcripción (bHLH, ABF1, MADS box) potencialmente implicados en la regulación de la respuesta a sequía, tales como la síntesis de prolina y las respuestas a carencias de agua o fosfato, así como a estímulos por temperatura. Los análisis de expresión diferencial de genes en los ecotipos han detectado 4941 tránscritos, de los cuales dos terceras partes están sobre-expresados en las plantas en condiciones de sequía con respecto a las sometidas a condiciones de riego. Los análisis pan-transcriptómicos muestran que la mayoría de los genes expresados en ambas condiciones son genes del core, presentes en todos los ecotipos estudiados, mientras que una fracción de los genes hub corresponden a genes soft-core y shell, encontrados únicamente en algunos ecotipos. <br /

Development and application of RNA-seq bioinformatic tools to explore non-model organisms in ageing research

Im Rahmen dieser Arbeit habe ich eine Software entwickelt, die alle notwendigen Schritte vollzieht um von Sequenzierungsdaten zu Transkriptkatalogen zu gelangen. Dazu werden sowohl öffentlich verfügbare als auch selbst angefertigte Programme effizient miteinander verbunden, um die Transkriptomassemblierung und -annotation vorzunehmen, sowie Schwachstellen der Assemblierung auszubessern. Die Wettbewerbsfähigkeit von FRAMA im Vergleich zu genombasierten Verfahren wurde durch Anwendung auf Sequenzierungsdaten des Nacktmulls demonstriert. Der Nacktmull rückt immer stärker in den Fokus der Alternsforschung, durch seine außergewöhnlich Lebensspanne von >30 Jahren in Gefangenschaft, die von einer extremen Widerstandskraft gegen altersbedingten Zerfall und einer lebenslangen Fruchtbarkeit begleitet wird. In Anbetracht seiner eusozialen Lebensweise, ist eine lebenslange Fruchtbarkeit bemerkenswert, denn in einer Kolonie trägt ein einzelnes Weibchen die metabolische Last der Fortpflanzung und wird dabei mindestens genauso alt wie ihre fortpflanzungsinaktiven Geschwister. Als Teil dieser Arbeit habe ich mit Hilfe von Genexpressionsanalysen und basierend auf dem zuvor angefertigten Transkriptkatalog, fortpflanzungaktive und -inaktive Nacktmulle verglichen. Weiterhin habe ich FRAMA auf Sequenzierungsdaten von Saisonfischen der Gattung Nothobranchius angewandt. Diese Gattung zeigt, vermutlich in Anpassung an die kurze Verfügbarkeit von Wasser in ihrem Habitat, eine sehr kurze Lebensspanne und umfasst eines der kurzlebigstes bekannten Wirbeltiere. Die Transkriptkataloge dienten als Grundlage zur Analyse positiver Selektion um genetische Determinanten kurzer Lebensspannen zu identifizieren. Beide Analysen haben interessante Genkandidaten in Bezug zur Alterung aufgedeckt und sowohl Einblicke in die sexuelle Reifung von Nacktmullen als auch der Evolution kurzer Lebenspannen in Saisonfischen gegebe

Symbiodinium genomes reveal adaptive evolution of functions related to coral-dinoflagellate symbiosis

Symbiosis between dinoflagellates of the genus Symbiodinium and reef-building corals forms the trophic foundation of the world’s coral reef ecosystems. Here we present the first draft genome of Symbiodinium goreaui (Clade C, type C1: 1.03 Gbp), one of the most ubiquitous endosymbionts associated with corals, and an improved draft genome of Symbiodinium kawagutii (Clade F, strain CS-156: 1.05 Gbp) to further elucidate genomic signatures of this symbiosis. Comparative analysis of four available Symbiodinium genomes against other dinoflagellate genomes led to the identification of 2460 nuclear gene families (containing 5% of Symbiodinium genes) that show evidence of positive selection, including genes involved in photosynthesis, transmembrane ion transport, synthesis and modification of amino acids and glycoproteins, and stress response. Further, we identify extensive sets of genes for meiosis and response to light stress. These draft genomes provide a foundational resource for advancing our understanding of Symbiodinium biology and the coral-algal symbiosis.H.L. was supported by an Australian Research Council grant (DP150101875) awarded to M.A. R. and C.X.C. T.G.S. is supported by an Australian Government Research Training Program Scholarship. R.A.G.-P. is supported by an International Postgraduate Research Scholarship and a University of Queensland Centenary Scholarship. This project was supported by the computational resources of the Australian National Computational Infrastructure (NCI) National Facility systems through the NCI Merit Allocation Scheme (Project d85) awarded to M.A.R. and C.X.C. The data used in this project were funded by the Great Barrier Reef Foundation’s Resilient Coral Reefs Successfully Adapting to Climate Change research and development program in collaboration with the Australian Government, Bioplatforms Australia through the National Collaborative Research Infrastructure Strategy (NCRIS), Rio Tinto and a family foundation. The authors also acknowledge the work done by the Reef Future Genomics (ReFuGe) 2020 Consortium. Access to data generated by the consortium can be accessed via reefgenomics.org. In memory of S.F., our friend and colleague who is sorely missed

Visualization Tools for Comparative Genomics applied to Convergent Evolution in Ash Trees

Assembly and analysis of whole genomes is now a routine part of genetic research, but effective tools for the visualization of whole genomes and their alignments are few. Here we present two approaches to allow such visualizations to be done in an efficient and user-friendly manner. These allow researchers to spot problems and patterns in their data and present them effectively. First, FluentDNA is developed to tackle single full genome visualization and assembly tasks by representing nucleotides as colored pixels in a zooming interface. This enables users to identify features without relying on algorithmic annotation. FluentDNA also supports visualizing pairwise alignments of wellassembled whole genomes from chromosome to nucleotide resolution. Second, Pantograph is developed to tackle the problem of visualizing variation among large numbers of whole genome sequences. This uses a graph genome approach, which addresses many of the technical challenges of whole genome multiple sequence alignments by representing aligned sequences as nodes which can be shared by many individuals. Pantograph is capable of scaling to thousands of individuals and is applied to SARS and A. thaliana pangenomes. Alongside the development of these new genomics tools, comparative genomic research was undertaken on worldwide species of ash trees. I assembled 13 ash genomes and used FluentDNA to quality check the results and discovered contaminants and a mitochondrial integration. I annotated protein coding genes in 28 ash assemblies and aligned their gene families. Using phylogenetic analysis, I identified gene duplications that likely occurred in an ancient whole genome duplication shared by all ash species. I examined the fate of these duplicated genes, showing that losses are concentrated in a subset of gene families more often than predicted by a null model simulation. I conclude that convergent evolution has occurred in the loss and retention of duplicated genes in different ash species.BBSRC BB/S004661/

Strukturell variasjon som påvirker genetisk miljøtilpasning i laksefisk

Structural variations (SVs), e.g. deletions, insertions, inversions and duplications of sequences, are a major source of genomic variation affecting more base pairs in the genome than single nucleotide polymorphisms (SNPs). Despite their increasingly recognised importance in adaptive evolution and species diversification, SVs are vastly understudied in most species. Long-read sequencing, together with recently developed bioinformatic tools, have provided step-change improvements in the precision and recall of SV detection and allow us to increase the detected SVs manyfold across the species range. In addition, long-reads represent a major shift in our ability to build continuous genome assemblies as fundamental resources for most genome wide studies. The work in this thesis utilises long-read data to generate multiple genome sequences for the two salmonid species Atlantic salmon (Salmo salar) and lake whitefish (Coregonus clupeaformis). We present the first pan-genome for Atlantic salmon, comprising 11 long-read-based assemblies across the species range. Among these, the highest quality genome has 2.55 Gbp assembled into chromosome sequences, 259 Mbp more sequence than in the previous Atlantic salmon reference genome. The genome has a highly improved continuity with contig N50 increasing from 58 kbp to 28.06 Mbp (484-fold). The detection of SVs in these 11 individuals, revealed 1,061,452 SVs, with an average of ~77.4 Mbp of sequence differing per sample. The Atlantic salmon has adapted to different river environment across a large geographical distribution. To investigate genomic variation underlying these adaptations, we associated SVs and environmental data in a dataset of 366 short-read samples genotyped using genome graph analyses. These analyses highlighted multiple SVs contributing to environmental adaptations, including an 18 kbp deletion encompassing a polymorphic segmental duplication of three genes associated with annual precipitation. Next, we use the Atlantic salmon pan-genome to study the emergence of supergenes. Because supergenes can be maintained over millions of years by balancing selection and typically exhibit strong recombination suppression, their underlying functional variants and how they are formed are largely unknown. Inversions are type of rearrangement commonly associated with supergenes, and by directly comparing multiple highly continuous genome assemblies we were able to detect a number of large inversions in Atlantic salmon. A 3 Mb inversion, estimated to be ~15,000-year-old, and segregating in North American populations, displayed supergene signatures with adaptive variation captured within the standard arrangement of the inversion, as well as other adaptive variation accumulating after the inversion occurred. Characterization of other inversions with matched repeat structures at the breakpoints did not show any supergene signatures, suggesting that shared breakpoint repeats may obstruct the supergene formation. Lastly, we created long-read based genome assemblies for sympatric species pairs (Dwarf and Normal) belonging to lake whitefish (Coregonus clupeaformis). The species pairs offer a suitable model system for studying genomic patterns of differentiation and in particular the role of SVs in speciation. By combining long-reads, direct assembly, and short-read methods we detect 89,909 high-confidence SVs in the species pair across two lakes, covering five times more sequence in the genome compared to SNPs. In the study, we highlight shared outliers of differentiation between the lakes, indicating that they contribute to speciation. Interestingly, we find that more than 70% of SVs differentiating between the Normal and Dwarf species pairs of lake whitefish are overlapping transposable elements. This work demonstrates that SVs may play an important role for the differentiation and speciation of sympatric species pairs in lake whitefish.Strukturell variasjon (SVer), for eksempel delesjoner, insersjoner, inversjoner og duplikasjoner av sekvens, er en viktig kilde til genomisk variasjon som samplet sett påvirker flere basepar i genomet enn punktmutasjoner (SNPs). Til tross for en økende annerkjennelse for at SVer spiller en viktig rolle i genetisk tilpassing til ulikt miljø og artsdannelse har denne typen variasjon vært lite studert i mange arter. Ny DNA-sekvenseringsteknologi med lengre leselengder (long-read sequencing), samt utvikling av nye bioinformatiske verktøy, har ført til drastiske forbedringer i deteksjonen av SVer. ‘Long-read’ sekvensering gjør det også mulig å lage mer komplette og sammenhengende genomsekvenser enn tidligere. I denne avhandlingen benytter vi oss av ‘long-read’ data til å lage flere genomsekvenser av høy kvalitet for to ulike laksefiskarter: Atlanterhavslaks (Salmo salar) og en Nordamerikansk type sik ‘lake whitefish’ (Coregonus clupeaformis). Her rapporterer vi det første pan-genomet for Atlanterhavslaks. Det består av 11 assemblier basert på ‘long- read’ sekvensering av individer fra fire ulike fylogeografiske grupper av villaks. Assembliet av høyest kvalitet inkluderer 2,55 Gbp sekvens i kromosomer, 259 Mbp mer enn det forrige referansegenomet til Atlanterhavslaks. I tillegg ble andelen sammenhengende sekvens, målt som contig N50, økt fra 58 kbp til 28,06 Mbp (484 ganger høyere). Vi fant 1.061.452 SVer på tvers av de 11 individene med ~77,4 Mbp gjennomsnittlig sekvensforskjell per prøve. Atlanterhavslaksen har over tid tilpasset miljøet i ulike elver. For å studere underliggende genetisk variasjon for denne tilpasningen assosierte vi SVer med ulike miljøvariabler i et datasett bestående av 366 ‘short-read’ sekvenserte prøver ved bruk av en genom-graf. Ved hjelp av disse analysene fant vi flere SVer som bidrar til miljøtilpasning, blant annet en 18 kbp lang delesjon som inneholder tre gener assosiert med mengden nedbør i området. Vi brukte så pan-genomet for Atlanterhavsaks til å studere dannelsen av ‘supergener’. Supergener er en sammenkobling av genetisk variasjon i koblingsulikevekt som for eksempel kan oppstå ved hjelp av store inversjoner. Her utnyttet vi 11 genomassemblier til å identifisere og karakterisere en rekke store inversjoner i Atlanterhavslaks. En av inversjonene på 3 Mbp, estimert til å være ~15.000 år gammel, viste signaturer for utvikling som supergen. For de andre inversjonene som var flankert av repetert DNA fant vi ikke karakteristiske trekk på supergener, noe som tyder på at det repetitive DNA forhindrer en dannelse av supergener. Til slutt lagde vi genomsekvenser for ulike former (‘Normal’ og ‘Dwarf’) av ‘lake whitefish’ (Coregonus clupeaformis) som lever i de samme innsjøene i Nord-Amerika. Genomsekvensene muliggjør studier av genomiske mekanismene bak artsdannelse i denne laksefisken. Ved å kombinere ‘long-read’ data, direkte sammenlikning av assemblier, og ‘short-read’ data fant vi 89,909 SVer som skilte de to formene av ‘lake whitefish’ i to innsjøer. SVene omfatter mer enn fem ganger flere basepar i genomet sammenlignet med SNPs. I studiet fant vi flere SVer med avvikende forekomst (‘outliers’) i de to formene av ‘lake whitefish’, noe som indikerer at disse SVene bidrar til artsdannelse. Videre fant vi at 70 % av SVene overlappet en form av repetert DNA kalt transposable elementer. Dette arbeidet understreker at SVer kan spille en viktig rolle for artsdannelse i ’lake whitefish’