Over the last decades the crustacean aquaculture sector has been steadily growing, in order to meet global demands for its products. A major hurdle for further growth of the industry is the prevalence of viral disease epidemics that are facilitated by the intense culture conditions. A devastating virus impacting on the sector is the White Spot Syndrome Virus (WSSV), responsible for over US $10 billion in losses in shrimp production and trade. The Pathogenicity of WSSV is high, reaching 100 % mortality within 3-10 days in penaeid shrimps. In contrast, the European shore crab Carcinus maenas has been shown to be relatively resistant to WSSV. Uncovering the basis of this resistance could help inform on the development of strategies to mitigate the WSSV threat. C. maenas has been used widely in studies on ecotoxicology and host-pathogen interactions. However, like most aquatic crustaceans, the genomic resources available for this species are limited, impairing experimentation. Therefore, to facilitate interpretations of the exposure studies, we first produced a C. maenas transcriptome and genome scaffold assembly. We also produced a transcriptome for the European lobster (Homarus gammarus), an ecologically and commercially important crustacean species in United Kingdom waters, for use in comparing WSSV responses in this, a susceptible species, and C. maenas. For the C. maenas transcriptome assembly we isolated and pooled RNA from twelve different tissues and sequenced RNA on an Illumina HiSeq 2500 platform. After de novo assembly a transcriptome encompassing 212,427 transcripts was produced. Similar, the H. gammarus transcriptome was based on RNA from nine tissues and contained 106,498 transcripts. The transcripts were filtered and annotated using a variety of tools (including BLAST, MEGAN and RSEM) and databases (including GenBank, Gene Ontology and KEGG). The annotation rate for transcripts in both transcriptomes was around 20-25 % which appears to be common for aquatic crustacean species, as a result of the lack of well annotated gene sequences for this clade. Since it is likely that the host immune system would play an important role in WSSV infection we characterized the IMD, JAK/STAT, Toll-like receptor and other innate immune system pathways. We found a strong overlap between the immune system pathways in C. maenas and H. gammarus. In addition we investigated the sequence diversity of known WSSV interacting proteins amongst susceptible penaeid shrimp/lobster and the more resistant C. maenas. There were differences in viral receptor sequences, like Rab7, that correlate with a less efficient infection by WSSV. To produce the genome scaffold assembly for C. maenas we isolated DNA from muscle tissue and produced both paired-end and mate pair libraries for processing on the Illumina HiSeq 2500 platform. A de novo draft genome assembly consisting of 338,980 scaffolds and covering 362 Mb (36 % of estimated genome size) was produced, using SOAP-denovo2 coupled with the BESST scaffolding system. The generated assembly was highly fragmented due to the presence of repetitive areas in the C. maenas genome. Using a combination of ab initio predictors, RNA-sequencing data from the transcriptome datasets and curated C. maenas sequences we produced a model encompassing 10,355 genes. The gene model for C. maenas Dscam, a gene potentially involved in (pan)crustacean immune memory, was investigated in greater detail as manual curation can improve on the results of ab initio predictors. The scaffold containing C. maenas Dscam was fragmented, thus only contained the latter exons of the gene. The assembled draft genome and transcriptomes for C. maenas and H. gammarus are valuable molecular resources for studies involving these and other aquatic crustacean species. To uncover the basis of their resistance to WSSV, we infected C. maenas with WSSV and measured mRNA and miRNA expression for 7 time points spread over a period of 28 days, using RNA-Seq and miRNA-Seq. The resistance of C. maenas to WSSV infection was confirmed by the fact that no mortalities occurred. In these animals replicating WSSV was latent and detected only after 7 days, and this occurred in five of out 28 infected crabs only. Differential expression of transcripts and miRNAs were identified for each time point. In the first 12 hours post exposure we observed decreased expression of important regulators in endocytosis. Since it is established that WSSV enters the host cells through endocytosis and that interactions between the viral protein VP28 and Rab7 are important in successful infection, it is likely that changes in this process could impact WSSV infection success. Additionally we observed an increased expression of transcripts involved in RNA interference pathways across many time points, indicating a longer term response to initial viral exposure. miRNA sequencing showed several miRNAs that were differentially expressed. The most striking finding was a novel C. maenas miRNA that we found to be significantly downregulated in every WSSV infected individual, suggesting that it may play an important role in mediating the response of the host to the virus. In silico target prediction pointed to the involvement of this miRNA in endocytosis regulation. Taken together we hypothesize that C. maenas resistance to WSSV involves obstruction of viral entry by endocytosis, a process probably regulated through miRNAs, resulting in inefficient uptake of virions.

RNA-seq transcriptome analysis of Chenopodium quinoa at different water treatment levels was conducted in a greenhouse study using four water treatments (field capacity to drought) on a valley ecotype quinoa (variety Ingapirca) and an Altiplano Salares ecotype quinoa (variety Ollague). Physiological results support the earlier findings that the Salares ecotypes display greater tolerance to drought-like stress conditions than the valley ecotypes (as determined by growth rate, photosynthetic rate, stomatal conductance, and stem water potential). cDNA libraries from root tissue sample for each treatment x variety combination were sequenced using Illumina Hi-Seq technology in an RNA-seq experiment. De novo assembly of the transcriptome generated 20,337 unique transcripts. Gene expression analysis of the RNA-seq data identified 462 putative gene products that showed differential expression based on treatment and 27 putative gene products differential expressed based on variety x treatment, including significant increasing expression in the root tissue in response to increasing water stress. BLAST searches and gene ontology analysis show an overlap with drought tolerance stress and other abiotic stress mechanisms.

The rapid development of biotechnology has enabled researchers to collect high-throughput data for studying various biological processes at the genomic level, transcriptomic level, and proteomic level. Due to the large noise in the data and the high complexity of diseases (such as cancer), it is a challenging task for researchers to extract biologically meaningful information that can help reveal the underlying molecular mechanisms. The challenges call for more efforts in developing efficient and effective computational methods to analyze the data at different levels so as to understand the biological systems in different aspects. In this dissertation research, we have developed novel Bayesian approaches to infer alternative splicing mechanisms in biological systems using RNA sequencing data. Specifically, we focus on two research topics in this dissertation: isoform identification and phenotype-specific transcript assembly. For isoform identification, we develop a computational approach, SparseIso, to jointly model the existence and abundance of isoforms in a Bayesian framework. A spike-and-slab prior is incorporated into the model to enforce the sparsity of expressed isoforms. A Gibbs sampler is developed to sample the existence and abundance of isoforms iteratively. For transcript assembly, we develop a Bayesian approach, IntAPT, to assemble phenotype-specific transcripts from multiple RNA sequencing profiles. A two-layer Bayesian framework is used to model the existence of phenotype-specific transcripts and the transcript abundance in individual samples. Based on the hierarchical Bayesian model, a Gibbs sampling algorithm is developed to estimate the joint posterior distribution for phenotype-specific transcript assembly. The performances of our proposed methods are evaluated with simulation data, compared with existing methods and benchmarked with real cell line data. We then apply our methods on breast cancer data to identify biologically meaningful splicing mechanisms associated with breast cancer. For the further work, we will extend our methods for de novo transcript assembly to identify novel isoforms in biological systems; we will incorporate isoform-specific networks into our methods to better understand splicing mechanisms in biological systems. / Ph. D. / The next-generation sequencing technology has significantly improved the resolution of the biomedical research at the genomic level and transcriptomic level. Due to the large noise in the data and the high complexity of diseases (such as cancer), it is a challenging task for researchers to extract biologically meaningful information that can help reveal the underlying molecular mechanisms. In this dissertation, we have developed two novel Bayesian approaches to infer alternative splicing mechanisms in biological systems using RNA sequencing data. We have demonstrated the advantages of our proposed approaches over existing methods on both simulation data and real cell line data. Furthermore, the application of our methods on real breast cancer data and glioblastoma tissue data has further shown the efficacy of our methods in real biological applications.

Transcriptome reconstruction is an important component in the bioinformatical part of transcriptome studies. It is particulary interesting when a reference genome is missing, highly fragmented or incomplete, since in such situations, a simple alignment (or mapping) would not necessarily tell the full story. One species with such a highly fragmented reference genome is the Norway spruce (Picea abies (L.) H. Karst.) -- a conifer, which is very important for Swedish economy. Given its long juvenile phase and irregular cone setting, the demand of cultivated seeds are larger than the supply. This yields a desire to understand the transcriptomal biology behind the cone setting in P. abies. This thesis presents an introduction to this situation, and the biological and bioinformatical background in general, followed by two papers in which this is applied: Paper I introduces a novel de novo transcriptome assembler, with a focus on recovering isoforms, and paper II makes use of this assembler to be able to detect connections between scaffolds in the P. abies genome. Paper I also studies P. abies var acrocona, a mutant with shorter juvenile phase than the wild type, in order to detect how cone setting is initiated.  From differential expression studies of both mRNA and miRNA, a number of genes potentially involved in cone-setting in P. abies were found, and also a set of miRNAs that could be involved in their regulation. / Transkriptomrekonstruktion är en viktig komponent i den bioinformatiska delen av transkriptomstudier. Särskilt intressant är detta när ett referensgenom saknas, är kraftigt fragmenterat eller ofullständigt, ty i dessa situationer skulle inte en vanlig inpassning (eller mappning) kunna berätta allt. En art med ett kraftigt fragmenterat referensgenom är gran (Picea abies (L.) H. Karst.) -- ett barrträd, som är mycket viktigt för svensk ekonomi. På grund av dess långa uppväxtsfas och oregelbundna kottsättning, så är efterfrågan av förädlade fröer större än utbudet. Detta lämnar en önskan att förstå den transkriptomala biologin bakom granens kottsättning. Denna avhandling presenterar en introduktion till denna situation, den generella biologiska och bioinformatiska bakgrunden, följd av två artiklar i vilket detta är tillämpat: Artikel I introducerar en ny de novo transkriptomassembler med fokus på att återskapa isoformer, och artikel II tillämpar denna assembler för att kunna hitta länkar mellan scaffolder (genom-delar som hittills inte kunnat länkas med varandra) i grangenomet. Artikel II studerar även granmutanten acrocona (kottegran), vilken har kortare uppväxtsfas än vildtypen, för att kunna se vad som initierar kottsättning.  Från differentiella expressionsstudier av såväl mRNA som miRNA, hittades ett antal gener potentiellt involverade i granens kottsättning, samt några miRNA som kan vara involverade i dess reglering. / <p>QC 2021-02-12</p>

Chronic Obstructive Pulmonary Disease (COPD) is the 3rd leading cause of death in the US, and idiopathic pulmonary fibrosis (IPF), a type of Interstitial Lung Disease (ILD), is a fast acting, irreversible disease that leads to mortality within 3-5 years. RNA-sequencing provides the opportunity to quantitatively examine the sequences of millions mRNAs, and offers the potential to gain unprecedented insights into the structure of chronic non-malignant lung disease transcriptome. By identifying changes in splicing and novel loci expression associated with disease, we may be able to gain a better understanding of their pathogenesis, identify novel disease-specific biomarkers, and find better targets for therapy. Using RNA-seq data that our group generated on 281 human lung tissue samples (47=Control, 131=COPD, 103=ILD), I initially defined the transcriptomic landscape of lung tissue by identifying which genes were expressed in each tissue sample. I used a mixture model to separate genes into reliable and not reliable expression. Next, I employed reads that overlapped splice junctions in a linear model interaction term to identify disease-specific differential splicing. I identified alternatively spliced genes between control and disease tissues and validated three (PDGFA, NUMB, SCEL) of these genes with qPCR and nanostring (a hybridization-based barcoding technique used to quantify transcripts). Finally, I implemented and improved a pipeline to perform transcriptome assembly using Cufflinks that led to the identification of 1,855 novel loci that did not overlap with UCSC, Vega, and Ensembl annotations. The loci were classified into potential coding and non-coding loci (191 and 1,664, respectively). Expression analysis revealed that there were 120 IPF-associated and 10 emphysema-associated differentially expressed (q < 0.01) novel loci. RNA-seq provides a high-resolution transcript-level view of the pulmonary transcriptome and its modification in lung disease. It has enabled a new understanding of the lung transcriptome structure because it measures not only the transcripts we know but also the ones we do not know. The approaches and improvements I have employed have identified these novel targets and make possible further downstream functional analysis that could identify better targets for therapy and lead to an even better understanding of chronic lung disease pathogenesis. / 2031-01-01T00:00:00Z

Text in English / Corridor disease is an economically important tick-borne disease of cattle in southern Africa. The disease is caused by Theileria parva and transmitted by the vectors, Rhipicephalus appendiculatus and R. zambeziensis. There is currently no vaccine to protect cattle against T. parva that is permitted in South Africa. To develop recombinant anti-tick vaccines against Corridor disease, comprehensive databases of genes expressed in the tick’s salivary glands are required. Therefore, in Chapters 2 and 3, mRNA from the salivary glands of R. appendiculatus and R. zambeziensis was sequenced and assembled using next generation sequencing technologies. Respectively, 12 761 and 13 584 non-redundant protein sequences were predicted from the sialotranscriptomes of R. appendiculatus and R. zambeziensis and uploaded to public sequence domains. This greatly expanded the number of sequences available for the two vectors, which will be invaluable resources for the selection of vaccine candidates in future. Further, in Chapter 3, differential gene expression analysis in R. zambeziensis revealed dynamic expression of secretory protein transcripts during feeding, suggestive of stringent transcriptional regulation of these proteins. Knowledge of these intricate expression profiles will further assist vaccine development in future. In Chapter 4, comparative sialotranscriptomic analyses were performed between R. appendiculatus and R. zambeziensis. The ticks have previously shown varying vector competence for T. parva and this chapter presents the search for correlates of this variance. Phylogenetic analyses were performed using these and other publically available tick transcriptomes, which indicated that R. appendiculatus and R. zambeziensis are closely related but distinct species. However, significant expression differences were observed between the two ticks, specifically of genes involved in tick immunity or pathogen transmission, signifying potential bioinformatic signatures of vector competence. Furthermore, nearly four thousand putative long non-coding RNAs (lncRNAs) were predicted in each of the two ticks. A large number of these showed differential expression and suggested a potential transcriptional regulatory function of lncRNA in tick blood feeding. LncRNAs are completely unexplored in ticks. Finally, in Chapter 5, concluding remarks are given on the potential impact the R. appendiculatus and R. zambeziensis sialotranscriptomes may have on future vaccine developments and some future research endeavours are discussed. / Life and Consumer Sciences / Ph. D. (Life Sciences)

Tesis por compendio / [ES] El pepino dulce (Solanum muricatum) y el tomate de árbol (S. betaceum) pertenecen al grupo de cultivos de la familia Solanaceae. Estos dos cultivos son originarios de América del Sur y actualmente se cultivan en varios países con climas tropicales, subtropicales y mediterráneos. Han sido infrautilizados durante mucho tiempo y han cobrado relevancia solo en los últimos años debido a su alta calidad nutricional. El pepino dulce exhibe niveles significativos de potasio, vitamina C y carotenoides y se informa que presenta propiedades antioxidantes, antidiabéticas, antiinflamatorias y antitumorales. Sus frutos se pueden consumir tanto como postre o en ensaladas. El tomate de árbol también destaca por su alto contenido en compuestos bioactivos como carotenoides, antocianinas, flavonoides y vitaminas. Varios productos como jugos, mermeladas, salsas y productos farmacéuticos son elaborados a partir de sus frutos. Debido a que estos cultivos se han introducido en nuevas regiones, donde pueden estar expuestos a estreses bióticos y abióticos que pueden amenazar su producción, y dado que el pepino dulce se ve especialmente afectado por la escasez de agua, fue necesario realizar un estudio para determinar la respuesta de siete cultivares de pepino dulce a parámetros fisiológicos y bioquímicos al estrés por sequía. Este trabajo puede ayudar a desarrollar programas de selección y mejoramiento que permitan generar nuevas variedades más tolerantes a la sequía. Por otro lado, en los países de clima mediterráneo, el pepino dulce se cultiva como cultivo protegido, aplicando las mismas técnicas agrícolas que otras solanáceas como el tomate y el pimiento. Estos sistemas agrícolas también brindan condiciones óptimas para el desarrollo de enfermedades como Fusarium oxysporum f. sp. lycopersici (FOL), Verticillium dahliae (VE), virus del mosaico del pepino (PepMV) y virus del mosaico del tomate (ToMV), que potencialmente podrían causar grandes daños a los cultivos de pepino dulce. Por tal motivo, se realizó un estudio para evaluar la respuesta de una colección de pepino dulce y sus parientes silvestres contra estas cuatro enfermedades, y encontrar fuentes de resistencia/tolerancia a estos patógenos. Aunque el tomate de árbol es un cultivo frutal importante debido a su valor nutricional y efectos beneficiosos para la salud, actualmente no hay información genómica y transcriptómica disponible públicamente. Por lo tanto, fue fundamental secuenciar el transcriptoma de dos cultivares de tomate de árbol con frutos morados (A21) y frutos anaranjados (A23). Estos dos cultivares han sido ampliamente utilizados y cultivados comercialmente en países de la región andina como Ecuador y Colombia. La obtención del primer transcriptoma de tomate de árbol ha permitido realizar un estudio comparativo entre el tomate de árbol y sus especies cercanas, tomate y patata, identificar genes implicados en la ruta de biosíntesis de carotenoides y desarrollar marcadores de polimorfismo de nucleótido único (SNP). En general, esta Tesis Doctoral aporta información relevante sobre la respuesta del pepino a diversos estreses ambientales, que puede ser utilizada para el desarrollo de nuevas variedades de pepino resistentes a múltiples estreses. Mientras que en tomate de árbol, el desarrollo de herramientas genómicas acelerará los programas de mejoramiento. / [CA] El cogombre dolç (Solanum muricatum) i tomata d'arbre (S. betaceum) pertanyen al grup de cultius de la família Solanaceae. Aquests dos cultius són originaris d'Amèrica del Sud i actualment es cultiven en diversos països amb climes tropicals, subtropicals i mediterranis. Han sigut infrautilitzats durant molt de temps i han cobrat rellevància només en els últims anys a causa de la seua alta qualitat nutricional. El cogombre dolç exhibeix nivells significatius de potassi, vitamina C i carotenoides i s'informa que presenta propietats antioxidants, antidiabètiques, antiinflamatòries i antitumorals. Els seus fruits es poden consumir tant com postres o en ensalades. La tomaca d'arbre també destaca pel seu alt contingut en compostos bioactivos com carotenoides, antocianinas, flavonoides i vitamines. Dels seus fruits s'elaboren diversos productes com a sucs, melmelades, salses i productes farmacèutics. Pel fet que aquests cultius s'han introduït en noves regions on poden estar exposats a estressos biòtics i abiòtics que poden amenaçar la seua producció, atés que el cogombre es veu especialment afectat per l'escassetat d'aigua, va ser necessari realitzar un estudi per a determinar la resposta de set cultivars de cogombre dolç a paràmetres fisiològics i bioquímicos a l'estrés per sequera. Aquest treball pot ajudar a desenvolupar programes de selecció i millorament que permeten generar noves varietats més tolerants a la sequera. D'altra banda, als països de clima mediterrani, el cogombre dolç es cultiva com a cultiu protegit, aplicant les mateixes tècniques agrícoles que unes altres solanáceas com la tomaca i el pimentó. Aquests sistemes agrícoles també brinden condicions òptimes per al desenvolupament de malalties com Fusarium oxysporum f. sp. lycopersici (FOL), Verticillium dahliae (VE), virus del mosaic del cogombre (PepMV) i virus del mosaic de la tomaca (ToMV), que potencialment podrien causar grans danys als cultius de cogombre dolç. Per tal motiu, es va realitzar un estudi per a avaluar la resposta d'una col·lecció de cogombre dolç i els seus parents silvestres contra aquestes quatre malalties, i trobar fonts de resistència/tolerància a aquests patògens. Encara que la tomaca d'arbre és un cultiu fruiter important a causa del seu valor nutricional i efectes beneficiosos per a la salut, actualment no hi ha informació genòmica i transcriptómica disponible públicament. Per tant, va ser fonamental seqüenciar el transcriptoma de dues cultivars de tomaca d'arbre amb fruits morats (A21) i fruits ataronjats (A23). Aquestes dues cultivars han sigut àmpliament utilitzats i cultivats comercialment en països de la regió andina com l'Equador i Colòmbia. L'obtenció del primer transcriptoma de tomaca d'arbre ha permés realitzar un estudi comparatiu entre la tomaca d'arbre i les seues espècies pròximes, tomaca i creïlla, identificar gens implicats en la ruta de biosíntesi de carotenoides i desenvolupar marcadors de polimorfisme de nucleòtid únic (SNP). En general, aquesta Tesi Doctoral aporta informació rellevant sobre la resposta del cogombre a diversos estressos ambientals, que pot ser utilitzada per al desenvolupament de noves varietats de cogombre resistents a múltiples estressos. Mentre que en tomaca d'arbre, el desenvolupament d'eines genòmiques accelerarà els programes de millorament. / [EN] Pepino (Solanum muricatum) and tree tomato (S. betaceum) belong to the group of crops of the Solanaceae family. These two crops are native to South America and currently are grown in various countries with tropical, subtropical and Mediterranean climates. They have been underutilized for a long time and have become relevant only in recent years due to their high nutritional quality. Pepino exhibit significant levels of potassium, vitamin C and carotenoids and it is reported to present antioxidant, antidiabetic, anti-inflammatory and antitumor properties. Its fruits can be consumed both as a dessert or in salads. Tree tomato also highlights high content of bioactive compounds such as carotenoids, anthocyanins, flavonoids and vitamins. Severals products such as juices, jams, sauces and pharmaceutical products are made from its fruits. Due to these crops have been introduced into new regions, where they may be exposed to biotic and abiotic stresses that can threaten their production, and since pepino is specially affected by water scarcity, a study was needed to determine the response of seven pepino cultivars to physiological and biochemical parameters to drought stress. This work can help develop selection and improvement programs that allow the generation of new varieties that are more tolerant to drought. On the other hand, in countries with a Mediterranean climate, pepino is grown as a protected crop, applying the same agricultural techniques as other solanaceous plants such as tomato and pepper. These agricultural systems also provide optimal conditions for the development of diseases such as Fusarium oxysporum f. sp. lycopersici (FOL), Verticillium dahliae (VE), pepino mosaic virus (PepMV) and tomato mosaic virus (ToMV), which could potentially cause great damage to pepino crops. For this reason, a study was performed to evaluate the response of a collection of pepino and their wild relatives against these four diseases, and find sources of resistance/tolerance to those pathogens. Although tree tomato is an important fruit crop due to its nutritional value and beneficial health effects, there is currently no publicly available genomic and transcriptomic information. Therefore, it was essential to sequence the transcriptome of two tree tomato cultivars with purple fruits (A21) and orange fruits (A23). These two cultivars have been widely used and cultivated commercially in countries of the Andean region such as Ecuador and Colombia. Obtaining the first tree tomato transcriptome has made it possible to perform a comparative study between tree tomato and its close species, tomato and potato, identify genes involved in the carotenoid biosynthesis pathway, and develop single nucleotide polymorphism (SNP) markers. In general, this Doctoral Thesis provides relevant information on the response of pepino to various environmental stresses, which can be used for the development of new varieties of pepino resistant to multiple stresses. While in tree tomato, the development of genomic tools will accelerating up breeding programs. / Pacheco Toabanda, JE. (2022). Development of Biotechnological Tools for the Genetic Improvement of Pepino (Solanum Muricatum) and Tree Tomato (S.betaceum) [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/189205 / Compendio