Clonal genome evolution of the marbled crayfish, Procambarus virginalis

Marbled crayfish (Procambarus virginalis) are the only freshwater crayfish known to reproduce by cloning (apomictic parthenogenesis). Notably, among genetically identical offspring raised in the same environment, distinct phenotypic differences can be observed. These unique characteristics establish the marbled crayfish as a particularly interesting laboratory model. Additionally, parthenogenetic reproduction enables the marbled crayfish to rapidly spread and form stable populations, which poses a serious threat in many freshwater habitats. A further understanding of this organism requires the accessibility of its 3.5 Gbp large genome sequence. This doctoral thesis provides the first de novo genome assembly of the marbled crayfish. Multiple shotgun and long jumping distance libraries were generated from one individual female, with a single base coverage of over 100x. Sequencing data was used for a first genome assembly with a length weighted median scaffold size (N50) of over 40 kbp. The estimated genome wide heterozygosity rate of 0.53% is substantially higher compared to other arthropod genomes. Transcriptome data enabled the refinement of genetic structures. Eventually, a total of 87.8% complete and 7.4% fragmented single-copy arthropod orthologs were identified using the benchmarking software BUSCO. Single nucleotide variations were analyzed to verify clonality in geographically isolated populations. Results indicate an evolution from a single origin. Moreover, detailed insights into genotype distributions support the theory of asexual speciation by autopolyploidization. Comparison of three Procambarus species indicates detectable genetic separation between marbled crayfish and the closest relative Procambarus fallax. Automatic annotation of 21,000 genes using the annotation pipeline MAKER provides a detailed overview of genetic features. For example, a cellulase gene was identified which potentially plays a key role in omnivorousness. Genomic data and several online services are provided by a central web resource. This thesis provides detailed genetic insights into the unknown but very versatile order of decapod crustaceans. Considered economically and ecologically relevant keystone species, a representative genome sequence provides an important resource for future research

Phylogeographic reconstruction of the marbled crayfish origin

The marbled crayfish (Procambarus virginalis) is a triploid and parthenogenetic freshwater crayfish species that has colonized diverse habitats around the world. Previous studies suggested that the clonal marbled crayfish population descended as recently as 25 years ago from a single specimen of P. fallax, the sexually reproducing parent species. However, the genetic, phylogeographic, and mechanistic origins of the species have remained enigmatic. We have now constructed a new genome assembly for P. virginalis to support a detailed phylogeographic analysis of the diploid parent species, Procambarus fallax. Our results strongly suggest that both parental haplotypes of P. virginalis were inherited from the Everglades subpopulation of P. fallax. Comprehensive whole-genome sequencing also detected triploid specimens in the same subpopulation, which either represent evolutionarily important intermediate genotypes or independent parthenogenetic lineages arising among the sexual parent population. Our findings thus clarify the geographic origin of the marbled crayfish and identify potential mechanisms of parthenogenetic speciation

The methylome of the marbled crayfish links gene body methylation to stable expression of poorly accessible genes

Background: The parthenogenetic marbled crayfish (Procambarus virginalis) is a novel species that has rapidly invaded and colonized various different habitats. Adaptation to different environments appears to be independent of the selection of genetic variants, but epigenetic programming of the marbled crayfish genome remains to be understood. Results: Here, we provide a comprehensive analysis of DNA methylation in marbled crayfish. Whole-genome bisulfite sequencing of multiple replicates and different tissues revealed a methylation pattern that is characterized by gene body methylation of housekeeping genes. Interestingly, this pattern was largely tissue invariant, suggesting a function that is unrelated to cell fate specification. Indeed, integrative analysis of DNA methylation, chromatin accessibility and mRNA expression patterns revealed that gene body methylation correlated with limited chromatin accessibility and stable gene expression, while low-methylated genes often resided in chromatin with higher accessibility and showed increased expression variation. Interestingly, marbled crayfish also showed reduced gene body methylation and higher gene expression variability when compared with their noninvasive mother species, Procambarus fallax. Conclusions: Our results provide novel insights into invertebrate gene body methylation and its potential role in adaptive gene regulation

Impact of DLK1-DIO3 imprinted cluster hypomethylation in smoker patients with lung cancer.

DNA methylation is important for gene expression and genome stability, and its disruption is thought to play a key role in the initiation and progression of cancer and other diseases. The DLK1-DIO3 cluster has been shown to be imprinted in humans, and some of its components are relevant to diverse pathological processes. The purpose of this study was to assess the methylation patterns of the DLK1-DIO3 cluster in patients with lung cancer to study its relevance in the pathogenesis of this disease. We found a characteristic methylation pattern of this cluster in smoking associated lung cancer, as compared to normal lung tissue. This methylation profile is not patent however in lung cancer of never smokers nor in lung tissue of COPD patients. We found 3 deregulated protein-coding genes at this locus: one was hypermethylated (DIO3) and two were hypomethylated (DLK1 and RTL1). Statistically significant differences were also detected in two different families of SNORDs, two miRNA clusters and four lncRNAs (MEG3, MEG8, MEG9 and LINC00524). These findings were validated using data from the cancer genome atlas (TCGA) database. We have then showed an inverse correlation between DNA methylation and expression levels in 5 randomly selected genes. Several targets of miRNAs included in the DLK1-DIO3 cluster have been experimentally verified as tumor suppressors. All of these results suggest that the dysmethylation of the imprinted DLK1-DIO3 cluster could have a relevant role in the pathogenesis of lung cancer in current and former smokers and may be used for diagnostic and/or therapeutic purposes.SMP is funded by Fondo de Investigacion Sanitaria (CD1100153), Consejeria de Salud y Bienestar Social (PI2009-0224 and PI-0046-2012), and Fundacion Mutua Madrilena (2014). LPA is funded by Fondo de Investigacion Sanitaria (PI1102688 and 1401964) and RTICC (R12/0036/0028). AC lab was supported by grants to from the Spanish Ministry of Economy and Competitivity, Plan Nacional de I+D+I 2008-2011, Plan Estatal de I+D+I 2013-2016, ISCIII (Fis: PI12/00137, PI15/00045, RTICC: RD12/0036/0028) co-funded by FEDER from Regional Development European Funds (European Union), Consejeria de Ciencia e Innovacion (CTS-6844 and CTS-1848) and Consejeria de Salud of the Junta de Andalucia (PI-0135-2010 and PI-0306-2012). CC and EJL are supported by grants from PN I+D+I 2008-2011, Spain, Instituto de Salud Carlos III, (PI12/02838), Subdireccion General de Redes y Centros de Investigacion Cooperativa, Red Tematica de Investigacion Cooperativa en Cancer (RD12/0036/0025). This work has been also possible thanks to the Plan Estatal de I+D+i 2013-2016, Grant PIE13/0004 co-funded by the ISCIII and FEDER funds. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Genome analysis of the monoclonal marbled crayfish reveals genetic separation over a short evolutionary timescale

The marbled crayfish (Procambarus virginalis) represents a very recently evolved parthenogenetic freshwater crayfish species that has invaded diverse habitats in Europe and in Madagascar. However, population genetic analyses have been hindered by the homogeneous genetic structure of the population and the lack of suitable tools for data analysis. We have used whole-genome sequencing to characterize reference specimens from various known wild populations. In parallel, we established a whole-genome sequencing data analysis pipeline for the population genetic analysis of nearly monoclonal genomes. Our results provide evidence for systematic genetic differences between geographically separated populations and illustrate the emerging differentiation of the marbled crayfish genome. We also used mark-recapture population size estimation in combination with genetic data to model the growth pattern of marbled crayfish populations. Our findings uncover evolutionary dynamics in the marbled crayfish genome over a very short evolutionary timescale and identify the rapid growth of marbled crayfish populations as an important factor for ecological monitoring.peerReviewe

Differentiation‐related epigenomic changes define clinically distinct keratinocyte cancer subclasses

Abstract Keratinocyte cancers (KC) are the most prevalent malignancies in fair‐skinned populations, posing a significant medical and economic burden to health systems. KC originate in the epidermis and mainly comprise basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC). Here, we combined single‐cell multi‐omics, transcriptomics, and methylomics to investigate the epigenomic dynamics during epidermal differentiation. We identified ~3,800 differentially accessible regions between undifferentiated and differentiated keratinocytes, corresponding to regulatory regions associated with key transcription factors. DNA methylation at these regions defined AK/cSCC subtypes with epidermal stem cell‐ or keratinocyte‐like features. Using cell‐type deconvolution tools and integration of bulk and single‐cell methylomes, we demonstrate that these subclasses are consistent with distinct cells‐of‐origin. Further characterization of the phenotypic traits of the subclasses and the study of additional unstratified KC entities uncovered distinct clinical features for the subclasses, linking invasive and metastatic KC cases with undifferentiated cells‐of‐origin. Our study provides a thorough characterization of the epigenomic dynamics underlying human keratinocyte differentiation and uncovers novel links between KC cells‐of‐origin and their prognosis

Methylation profiling identifies two subclasses of squamous cell carcinoma related to distinct cells of origin

Cutaneous squamous cell carcinoma (cSCC) is a skin cancer that normally progresses from UV-induced actinic keratosis (AK). Here, the authors investigate the epigenomics of cSCC and highlight two distinct subclasses of AK and cSCC originating from distinct keratinocyte differentiation stages