Demography, Selection and Evolution in Conifers

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 12-09-2017La presente tesis doctoral la componen un total de 3 capítulos dedicados a inferir diferentes aspectos de la adaptación molecular en las coníferas. En el primer capítulo introducimos el trabajo y lo contextualizamos en el actual entendimiento de la genética molecular de las coníferas. En el segundo capítulo nos centramos en el estudio de la demografía y los procesos de selección natural del pino Carrasco (Pinus halepensis Mill) a lo largo de su distribución natural. Ahondamos en el efecto de la historia demográfica y del “gene surfing” (fenómeno estocástico por el que genes de baja frecuencia pueden alcanzar altas frecuencias a causa de colonizaciones a larga distancia) y de cómo esto dificulta la detección de las señales de selección. En el tercer capítulo estudiamos los procesos de selección a escala local, para lo cual utilizamos pares de poblaciones procedentes de zonas cuyas características ambientales contrastan entre sí, dentro del hábitat del pino Carrasco. Para desarrollar este capítulo comparamos diferentes metodologías estadísticas para inferir SNPs con frecuencias atípicas potencialmente bajo selección. Usamos lo aprendido hasta ahora para inferir selección en otras especies de coníferas, haciendo uso de datos genómicos. En el cuarto capítulo investigamos los MicroRNAs (un tipo de Pequeño ARN no codificante) en coníferas como una potencial y clave dirección para entender la adaptación molecular. Exploramos cómo establecer una ontología de genes para estas moléculas y así facilitar la transferencia de conocimientos de especies modelo, como es Arabidopsis thaliana (L.) Heynh, a otras especies a día de hoy menos estudiadas como son el grupo de coníferas. Keywords: Molecular Ecology, Selection, Adaptation, Genomics, Conifers

Genomic epidemiology of salmonid alphavirus in Norwegian aquaculture reveals recent subtype-2 transmission dynamics and novel subtype-3 lineages

Viral disease poses a major barrier to sustainable aquaculture, with outbreaks causing large economic losses and growing concerns for fish welfare. Genomic epidemiology can support disease control by providing rapid inferences on viral evolution and disease transmission. In this study, genomic epidemiology was used to investigate salmonid alphavirus (SAV), the causative agent of pancreas disease (PD) in Atlantic salmon. Our aim was to reconstruct SAV subtype-2 (SAV2) diversity and transmission dynamics in recent Norwegian aquaculture, including the origin of SAV2 in regions where this subtype is not tolerated under current legislation. Using nanopore sequencing, we captured ~90% of the SAV2 genome for n = 68 field isolates from 10 aquaculture production regions sampled between 2018 and 2020. Using time-calibrated phylogenetics, we infer that, following its introduction to Norway around 2010, SAV2 split into two clades (SAV2a and 2b) around 2013. While co-present at the same sites near the boundary of Møre og Romsdal and Trøndelag, SAV2a and 2b were generally detected in non-overlapping locations at more Southern and Northern latitudes, respectively. We provide evidence for recent SAV2 transmission over large distances, revealing a strong connection between Møre og Romsdal and SAV2 detected in 2019/20 in Rogaland. We also demonstrate separate introductions of SAV2a and 2b outside the SAV2 zone in Sognefjorden (Vestland), connected to samples from Møre og Romsdal and Trøndelag, respectively, and a likely 100 km Northward transmission of SAV2b within Trøndelag. Finally, we recovered genomes of SAV2a and SAV3 co-infecting single fish in Rogaland, involving novel SAV3 lineages that diverged from previously characterized strains >25 years ago. Overall, this study demonstrates useful applications of genomic epidemiology for tracking viral disease spread in aquaculture

Single cell transcriptomics of Atlantic salmon (Salmo salar L.) liver reveals cellular heterogeneity and immunological responses to challenge by Aeromonas salmonicida

Copyright © 2022 Taylor, Ruiz Daniels, Dobie, Naseer, Clark, Henderson, Boudinot, Martin and Macqueen.Peer reviewedPublisher PD

Transcriptomic landscape of Atlantic salmon (Salmo salar L.) skin

In this study, we present the first spatial transcriptomic atlas of Atlantic salmon skin using the Visium Spatial Gene Expression protocol. We utilized frozen skin tissue from four distinct sites, namely the operculum, pectoral and caudal fins, and scaly skin at the flank of the fish close to the lateral line, obtained from two Atlantic salmon (150 g). High quality frozen tissue sections were obtained by embedding tissue in O.C.T media prior to freezing and sectioning. Further, we generated libraries and spatial transcriptomic maps, achieving a minimum of 80 million reads per sample with mapping efficiencies ranging from 79.3% to 89.4%. Our analysis revealed the detection of over 80.000 transcripts and nearly 30.000 genes in each sample. Among the tissue types observed in the skin, the epithelial tissues exhibited the highest number of transcripts (UMI-counts), followed by muscle tissue, loose and fibrous connective tissue, and bone. Notably, the widest nodes in the transcriptome network were shared among the epithelial clusters, while dermal tissues showed less consistency, which is likely attributable to the presence of multiple cell types at different body locations. Additionally, we identified collagen type 1 as the most prominent gene family in the skin, while keratins were found to be abundant in the epithelial tissue. Furthermore, we successfully identified gene markers specific to epithelial tissue, bone, and mesenchyme. To validate their expression patterns, we conducted a meta-analysis of the microarray database, which confirmed high expression levels of these markers in mucosal organs, skin, gills, and the olfactory rosette.publishedVersio

Mapping the cellular landscape of Atlantic salmon head kidney by single cell and single nucleus transcriptomics

Single-cell transcriptomics is the current gold standard for global gene expression profiling, not only in mammals and model species, but also in non-model fish species. This is a rapidly expanding field, creating a deeper understanding of tissue heterogeneity and the distinct functions of individual cells, making it possible to explore the complexities of immunology and gene expression on a highly resolved level. In this study, we compared two single cell transcriptomic approaches to investigate cellular heterogeneity within the head kidney of healthy farmed Atlantic salmon (Salmo salar). We compared 14,149 cell transcriptomes assayed by single cell RNA-seq (scRNA-seq) with 18,067 nuclei transcriptomes captured by single nucleus RNA-Seq (snRNA-seq). Both approaches detected eight major cell populations in common: granulocytes, heamatopoietic stem cells, erythrocytes, mononuclear phagocytes, thrombocytes, B cells, NK-like cells, and T cells. Four additional cell types, endothelial, epithelial, interrenal, and mesenchymal cells, were detected in the snRNA-seq dataset, but appeared to be lost during preparation of the single cell suspension submitted for scRNA-seq library generation. We identified additional heterogeneity and subpopulations within the B cells, T cells, and endothelial cells, and revealed developmental trajectories of heamatopoietic stem cells into differentiated granulocyte and mononuclear phagocyte populations. Gene expression profiles of B cell subtypes revealed distinct IgM and IgT-skewed resting B cell lineages and provided insights into the regulation of B cell lymphopoiesis. The analysis revealed eleven T cell sub-populations, displaying a level of T cell heterogeneity in salmon head kidney comparable to that observed in mammals, including distinct subsets of cd4/cd8-negative T cells, such as tcrγ positive, progenitor-like, and cytotoxic cells. Although snRNA-seq and scRNA-seq were both useful to resolve cell type-specific expression in the Atlantic salmon head kidney, the snRNA-seq pipeline was overall more robust in identifying several cell types and subpopulations. While scRNA-seq displayed higher levels of ribosomal and mitochondrial genes, snRNA-seq captured more transcription factor genes. However, only scRNA-seq-generated data was useful for cell trajectory inference within the myeloid lineage. In conclusion, this study systematically outlines the relative merits of scRNA-seq and snRNA-seq in Atlantic salmon, enhances understanding of teleost immune cell lineages, and provides a comprehensive list of markers for identifying major cell populations in the head kidney with significant immune relevance.</p

Cell atlas of the Atlantic salmon spleen reveals immune cell heterogeneity and cellspecific responses to bacterial infection

The spleen is a conserved secondary lymphoid organ that emerged in parallel to adaptive immunity in early jawed vertebrates. Recent studies have applied single cell transcriptomics to reveal the cellular composition of spleen in several species, cataloguing diverse immune cell types and subpopulations.In this study, 51,119 spleen nuclei transcriptomes were comprehensively investigated in the commercially important teleost Atlantic salmon (Salmo salar L.), contrasting control animals with those challenged with the bacterial pathogen Aeromonas salmonicida. We identified clusters of nuclei representing the expected major cell types, namely T cells, B cells, natural killer-like cells, granulocytes, mononuclear phagocytes, endothelial cells, mesenchymal cells, erythrocytes and thrombocytes. We discovered heterogeneity within several immune lineages, providing evidence for resident macrophages and melanomacrophages, infiltrating monocytes, several candidate dendritic cell subpopulations, and B cells at distinct stages of differentiation, including plasma cells and an igt+ subset. We provide evidence for twelve candidate T cell subsets, including cd4+ T helper and regulatory T cells, one cd8+ subset, three γδT subsets, and populations double negative for cd4 and cd8. The number of genes showing differential expression during the early stages of Aeromonas infection was highly variable across immune cell types, with the largest changes observed in macrophages and infiltrating monocytes, followed by resting mature B cells. Our analysis provides evidence for a local inflammatory response to infection alongside B cell maturation in the spleen, and upregulation of ccr9 genes in igt+ B cells, T helper and cd8+ cells, and monocytes, consistent with the recruitment of immune cell populations to the gut to deal with Aeromonas infection. Overall, this study provides a new cell-resolved perspective of the immune actions of Atlantic salmon spleen, highlighting extensive heterogeneity hidden to bulk transcriptomics. We further provide a large catalogue of cell-specific marker genes that can be leveraged to further explore the function and structural organization of the salmonid immune system

Looking for local adaptation:Convergent microevolution in aleppo pine (pinus halepensis)

Finding outlier loci underlying local adaptation is challenging and is best approached by suitable sampling design and rigorous method selection. In this study, we aimed to detect outlier loci (single nucleotide polymorphisms, SNPs) at the local scale by using Aleppo pine (Pinus halepensis), a drought resistant conifer that has colonized many habitats in the Mediterranean Basin, as the model species. We used a nested sampling approach that considered replicated altitudinal gradients for three contrasting sites. We genotyped samples at 294 SNPs located in genomic regions selected to maximize outlier detection. We then applied three different statistical methodologies-Two Bayesian outlier methods and one latent factor principal component method-To identify outlier loci. No SNP was an outlier for all three methods, while eight SNPs were detected by at least two methods and 17 were detected only by one method. From the intersection of outlier SNPs, only one presented an allelic frequency pattern associated with the elevational gradient across the three sites. In a context of multiple populations under similar selective pressures, our results underline the need for careful examination of outliers detected in genomic scans before considering them as candidates for convergent adaptation