Genomics of sexual and asexual reproduction in "Daphnia magna"

During my Ph.D., I used the next generation sequencing technology to investigate patterns of recombination and the genetic consequences of different reproductive modes of Daphnia magna. More precisely, I have used Restriction site Associated (RAD) sequencing to construct a high-density genetic map that can be coupled with the draft genome assembly of D. magna, thus, providing an essential tool for genome investigations in this widely used model organism (Chapter I). Such a map has enabled characterization of variation in the meiotic recombination rates across the genome of D. magna for the first time. Since recombination rates are an important parameter in almost any type of genetic research, this newly gained insight into the recombination landscape of D. magna offers a fundamental information for future studies of genome evolution, identification of genes underlying phenotypic traits and population genetic analyses. In addition to sexual reproduction, D. magna can also reproduce asexually to generate clutches of clonal offspring (ameiotic parthenogenesis). This feature of Daphnia biology is extremely useful for scientific experimentation where the genetic variation among tested individuals has to be minimized. However, over the last decade, reports of genome homogenization (loss of heterozygosity - LOH) in asexual lineages of D. pulex have indicated that asexual genomes are not static as it was previously assumed and that some levels of ameiotic recombination, in addition to mutation, may induce genetic variation among putative clones. However, comparing parthenogenetic offspring with their mothers at several thousand genetic markers generated by RAD-sequencing, I was not able to detect any LOH events in D. magna (Chapter II). I cannot exclude the possibility that ameiotic recombination indeed occurs in D. magna, however, my results indicate that such phenomenon is extremely rare or restricted to the very short genomic regions that I was unable to investigate, despite a high-density of markers used in this study. Nevertheless, the implementation of RAD-sequencing protocol for the genome studies of D. magna still enables interrogation of the transmission of genetic information from parents to offspring at unprecedented resolution. For an example, a RAD-sequencing based analysis of reduction in parental heterozygosity among rare ephippial hatchlings (typically produced by sexual reproduction) found in non-male producing populations of D. magna, has enabled differentiation between self-fertilization and automixis (meiotic parthenogenesis), by uncovering the subtle differences in genetic consequences of these reproductive strategies (Chapter III). Harnessing the ability of high-resolution genetic analysis it was demonstrated that, in the absence of males, D. magna can produce diapause eggs by automixis, and an additional type of asexual reproduction that was not previously reported for this species. Finally, RAD-sequencing European populations of D. magna revealed an association of genetic variation with the geographic location of individual samples (Chapter IV), a task which was not previously amenable using mitochondrial or microsatellite markers. This study provided a better insight into population genetic structure of D. magna and suggested that genetic differentiation is mainly driven by geographic distance. These results set a foundation for forthcoming studies aiming to disentangle past and future evolutionary processes shaping populations of this intriguing model organism. Taken together, research presented in my thesis illustrates the practicality of reduced representation genome sequencing for tackling diverse topics in evolutionary biology. By increasing awareness of non-randomness of meiotic recombination across the genome of D. magna, the diversity of reproductive mechanisms it can employ, and its large-scale population structure, I hope this work will contribute to further understanding of the remarkable adaptive capacity this crustacean is famous for

Uncovering cryptic asexuality in Daphnia magna by RAD sequencing

The breeding systems of many organisms are cryptic and difficult to investigate with observational data, yet they have profound effects on a species’ ecology, evolution, and genome organization. Genomic approaches offer a novel, indirect way to investigate breeding systems, specifically by studying the transmission of genetic information from parents to offspring. Here we exemplify this method through an assessment of self-fertilization vs. automictic parthenogenesis in Daphnia magna. Self-fertilization reduces heterozygosity by 50% compared to the parents, but under automixis, whereby two haploid products from a single meiosis fuse, the expected heterozygosity reduction depends on whether the two meiotic products are separated during meiosis I or II (i.e., central vs. terminal fusion). Reviewing the existing literature and incorporating recombination interference, we derive an interchromosomal and an intrachromosomal prediction of how to distinguish various forms of automixis from self-fertilization using offspring heterozygosity data. We then test these predictions using RAD-sequencing data on presumed automictic diapause offspring of so-called nonmale producing strains and compare them with “self-fertilized” offspring produced by within-clone mating. The results unequivocally show that these offspring were produced by automixis, mostly, but not exclusively, through terminal fusion. However, the results also show that this conclusion was only possible owing to genome-wide heterozygosity data, with phenotypic data as well as data from microsatellite markers yielding inconclusive or even misleading results. Our study thus demonstrates how to use the power of genomic approaches for elucidating breeding systems, and it provides the first demonstration of automictic parthenogenesis in Daphnia

Influence of High-Power Ultrasound on Yield of Proteins and Specialized Plant Metabolites from Sugar Beet Leaves (Beta vulgaris subsp. vulgaris var. altissima)

Ultrasound with water as a green solvent is an effective strategy for reducing losses and increasing the utilization of by-products. The extraction of proteins and specialized plant metabolites from sugar beet leaves (Beta vulgaris subsp. vulgaris var. altissima) promotes sustainability in the agro-food chain. Guided by sustainability, samples treated with ultrasound showed lower energy consumption and lower CO2 emissions. Furthermore, the spectrophotometric determination revealed higher protein and phenol yields in ultrasonically treated samples compared to thermally treated ones. The highest yield of total proteins, 147.91 ± 4.58 mg (gd.m.)−1, was observed during ultrasound treatment (amplitude 100%, treatment time 9 min). Under the same extraction conditions, the same trend was observed in the yield of total phenols 17.89 ± 0.38 mg (gd.m.)−1. High-power ultrasound, compared to the thermal extraction method, has increased the yield of proteins and specialized plant metabolites with significantly lower energy consumption and CO2 emissions. The obtained results are in accordance with the foundations of sustainable development. From an economic and environmental point of view, ultrasound with the use of green solvents would be an excellent replacement for conventional extraction methods

Influence of High-Power Ultrasound on Yield of Proteins and Specialized Plant Metabolites from Sugar Beet Leaves (<i>Beta vulgaris</i> subsp. <i>vulgaris</i> var. <i>altissima</i>)

Ultrasound with water as a green solvent is an effective strategy for reducing losses and increasing the utilization of by-products. The extraction of proteins and specialized plant metabolites from sugar beet leaves (Beta vulgaris subsp. vulgaris var. altissima) promotes sustainability in the agro-food chain. Guided by sustainability, samples treated with ultrasound showed lower energy consumption and lower CO2 emissions. Furthermore, the spectrophotometric determination revealed higher protein and phenol yields in ultrasonically treated samples compared to thermally treated ones. The highest yield of total proteins, 147.91 ± 4.58 mg (gd.m.)−1, was observed during ultrasound treatment (amplitude 100%, treatment time 9 min). Under the same extraction conditions, the same trend was observed in the yield of total phenols 17.89 ± 0.38 mg (gd.m.)−1. High-power ultrasound, compared to the thermal extraction method, has increased the yield of proteins and specialized plant metabolites with significantly lower energy consumption and CO2 emissions. The obtained results are in accordance with the foundations of sustainable development. From an economic and environmental point of view, ultrasound with the use of green solvents would be an excellent replacement for conventional extraction methods

A high-density genetic map reveals variation in recombination rate across the genome of Daphnia magna

Background: Recombination rate is an essential parameter for many genetic analyses. Recombination rates are highly variable across species, populations, individuals and different genomic regions. Due to the profound influence that recombination can have on intraspecific diversity and interspecific divergence, characterization of recombination rate variation emerges as a key resource for population genomic studies and emphasises the importance of high-density genetic maps as tools for studying genome biology. Here we present such a high-density genetic map for Daphnia magna, and analyse patterns of recombination rate across the genome. Results: A F2 intercross panel was genotyped by Restriction-site Associated DNA sequencing to construct the third-generation linkage map of D. magna. The resulting high-density map included 4037 markers covering 813 scaffolds and contigs that sum up to 77 % of the currently available genome draft sequence (v2.4) and 55 % of the estimated genome size (238 Mb). Total genetic length of the map presented here is 1614.5 cM and the genome-wide recombination rate is estimated to 6.78 cM/Mb. Merging genetic and physical information we consistently found that recombination rate estimates are high towards the peripheral parts of the chromosomes, while chromosome centres, harbouring centromeres in D. magna, show very low recombination rate estimates. Conclusions: Due to its high-density, the third-generation linkage map for D. magna can be coupled with the draft genome assembly, providing an essential tool for genome investigation in this model organism. Thus, our linkage map can be used for the on-going improvements of the genome assembly, but more importantly, it has enabled us to characterize variation in recombination rate across the genome of D. magna for the first time. These new insights can provide a valuable assistance in future studies of the genome evolution, mapping of quantitative traits and population genetic studies.Science, Faculty ofOther UBCNon UBCZoology, Department ofReviewedFacult

smartpca_run.txt

Descriptions of the creation and commands to use SmartPCA to generate PCA eigenvectors and eigenvalues

Genes mirror geography in Daphnia magna

Identifying the presence and magnitude of population genetic structure remains a major consideration in evolutionary biology as doing so allows one to understand the demographic history of a species as well as make predictions of how the evolutionary process will proceed. Next-generation sequencing methods allow us to reconsider previous ideas and conclusions concerning the distribution of genetic variation, and what this distribution implies about a given species evolutionary history. A previous phylogeographic study of the crustacean Daphnia magna suggested that, despite strong genetic differentiation among populations at a local scale, the species shows only moderate genetic structure across its European range, with a spatially patchy occurrence of individual lineages. We apply RAD sequencing to a sample of D. magna collected across a wide swath of the species' Eurasian range and analyse the data using principle component analysis (PCA) of genetic variation and Procrustes analytical approaches, to quantify spatial genetic structure. We find remarkable consistency between the first two PCA axes and the geographic coordinates of individual sampling points, suggesting that, on a continent-wide scale, genetic differentiation is driven to a large extent by geographic distance. The observed pattern is consistent with unimpeded (i.e. no barriers, landscape or otherwise) migration at large spatial scales, despite the fragmented and patchy nature of favourable habitats at local scales. With high-resolution genetic data similar patterns may be uncovered for other species with wide geographic distributions, allowing an increased understanding of how genetic drift and selection have shaped their evolutionary history

Data from: Genes mirror geography in Daphnia magna

Identifying the presence and magnitude of population genetic structure remains a major consideration in evolutionary biology as doing so allows one to understand the demographic history of a species as well as make predictions of how the evolutionary process will proceed. Next-generation sequencing methods allow us to reconsider previous ideas and conclusions concerning the distribution of genetic variation, and what this distribution implies about a given species evolutionary history. A previous phylogeographic study of the crustacean Daphnia magna suggested that, despite strong genetic differentiation among populations at a local scale, the species shows only moderate genetic structure across its European range, with a spatially patchy occurrence of individual lineages. We apply RAD sequencing to a sample of D. magna collected across a wide swath of the species' Eurasian range and analyse the data using principle component analysis (PCA) of genetic variation and Procrustes analytical approaches, to quantify spatial genetic structure. We find remarkable consistency between the first two PCA axes and the geographic coordinates of individual sampling points, suggesting that, on a continent-wide scale, genetic differentiation is driven to a large extent by geographic distance. The observed pattern is consistent with unimpeded (i.e. no barriers, landscape or otherwise) migration at large spatial scales, despite the fragmented and patchy nature of favourable habitats at local scales. With high-resolution genetic data similar patterns may be uncovered for other species with wide geographic distributions, allowing an increased understanding of how genetic drift and selection have shaped their evolutionary history

Additional file 1: Figure S1. of A high-density genetic map reveals variation in recombination rate across the genome of Daphnia magna

A flow chart of the genetic map construction process. For a detailed description, see Methods section “Linkage analysis”. (PDF 84 kb

Additional file 2: of A high-density genetic map reveals variation in recombination rate across the genome of Daphnia magna

The framework and composite map of Daphnia magna. Listed information include the names of RAD markers; marker alignment position to D. magna genome assembly v2.4; marker assignment as a representative of a segregation pattern (“FRAME” marker), significance level of the segregation ratio distortion (SRD) for each marker based on the p-value of Chi-square test for a difference between the observed and the expected Mendelian ratio (p < 0.1 *, p < 0.05 **, p < 0.01 ***, p < 0.005 ****, p < 0.001 *****, p < 0.0005 ******, p < 0.0001 *******); the number of the linkage group; the position of the marker within the linkage group (in cM, Kosambi corrected); genotype data used for the construction of genetic map. (XLSX 1401 kb