Microsatellite markers for the palaeo-temperature indicator Pentapharsodinium dalei (Dinophyceae)

Pentapharsodinium dalei is a widely distributed cold-water dinoflagellate, which is used in palaeoecology as an indicator of relatively warmer conditions in polar and sub-polar regions. This species has been proposed to be one of the first indicators of global warming at high latitudes. We developed the first microsatellite markers for P. dalei to facilitate the study of spatial and temporal population genetic changes. Single cysts were isolated from surface sediments in Koljö Fjord, Sweden. After cyst germination, single vegetative cells were isolated for establishing monoclonal cultures. Six dinucleotide polymorphic microsatellite markers were developed as multiplex polymerase chain reactions and were genotyped in 32 strains. The number of alleles per locus varied between 4 and 12, and the estimated gene diversity varied from 0.588 to 0.891. The haploid state of the vegetative cells was confirmed. The six selected microsatellites will be useful to explore population dynamics in P. dalei from contemporary planktonic and revived benthic samples to enable, for example, detailed studies into the evolutionary consequences of anthropogenic and climate-driven habitat changes.     

Outlier SNP markers reveal fine‐scale genetic structuring across European hake populations (Merluccius merluccius)

Shallow population structure is generally reported for most marine fish and explained as a consequence of high dispersal, connectivity and large population size. Targeted gene analyses and more recently genome‐wide studies have challenged such view, suggesting that adaptive divergence might occur even when neutral markers provide genetic homogeneity across populations. Here, 381 SNPs located in transcribed regions were used to assess large‐ and fine‐scale population structure in the European hake (Merluccius merluccius), a widely distributed demersal species of high priority for the European fishery. Analysis of 850 individuals from 19 locations across the entire distribution range showed evidence for several outlier loci, with significantly higher resolving power. While 299 putatively neutral SNPs confirmed the genetic break between basins (F_CT = 0.016) and weak differentiation within basins, outlier loci revealed a dramatic divergence between Atlantic and Mediterranean populations (F_CT range 0.275–0.705) and fine‐scale significant population structure. Outlier loci separated North Sea and Northern Portugal populations from all other Atlantic samples and revealed a strong differentiation among Western, Central and Eastern Mediterranean geographical samples. Significant correlation of allele frequencies at outlier loci with seawater surface temperature and salinity supported the hypothesis that populations might be adapted to local conditions. Such evidence highlights the importance of integrating information from neutral and adaptive evolutionary patterns towards a better assessment of genetic diversity. Accordingly, the generated outlier SNP data could be used for tackling illegal practices in hake fishing and commercialization as well as to develop explicit spatial models for defining management units and stock boundaries.     

Megalin–deficiency causes high myopia,retinal pigment epithelium-macromelanosomes and abnormal development of the ciliary body in mice

In man, mutations of the megalin-encoding gene causes the rare Donnai-Barrow/Facio-Oculo-Acoustico-Renal Syndrome, which is partially characterized by high-grade myopia. Previous studies of renal megalin function have established that megalin is crucial for conservation of renal filtered nutrients including vitamin A; however, the role of megalin in ocular physiology and development is presently unknown. Therefore, we investigate ocular megalin expression and the ocular phenotype of megalin-deficient mice. Topographical and subcellular localization of megalin as well as the ocular phenotype of megalin-deficient mice were examined with immunological techniques using light, confocal and electron microscopy. We identified megalin in the retinal pigment epithelium (RPE) and non-pigmented ciliary body epithelium (NPCBE) in normal mouse eyes. Immunocytochemical investigations furthermore showed that megalin localizes to vesicular structures in the RPE and NPCBE cells. Histological investigations of ocular mouse tissue also identified a severe myopia phenotype as well as enlarged RPE melanosomes and abnormal ciliary body development in the megalin-deficient mice. In conclusion, the complex ocular phenotype observed in the megalin-deficient mice suggests that megalin-mediated developmental abnormalities may contribute to the high myopia phenotype observed in the Donnai-Barrow Syndrome patients and, thus, that megalin harbors important roles in ocular development and physiology. Finally, our data show that megalin-deficient mice may provide a valuable model for future studies of megalin in ocular physiology and pathology.     

Glutamate Dehydrogenase Isoforms with N-Terminal (His)6- or FLAG-Tag Retain Their Kinetic Properties and Cellular Localization

Glutamate dehydrogenase (GDH) is a crucial enzyme on the crossroads of amino acid and energy metabolism and it is operating in all domains of life. According to current knowledge GDH is present only in one functional isoform in most animals, including mice. In addition to this housekeeping enzyme (hGDH1 in humans), humans and apes have acquired a second isoform (hGDH2) with a distinct tissue expression profile. In the current study we have cloned both mouse and human GDH constructs containing FLAG and (His)₆ small genetically-encoded tags, respectively. The hGDH1 and hGDH2 constructs containing N-terminal (His)₆ tags were successfully expressed in Sf9 cells and the recombinant proteins were isolated to ≥95 % purity in a two-step procedure involving ammonium sulfate precipitation and Ni²⁺-based immobilized metal ion affinity chromatography. To explore whether the presence of the FLAG and (His)₆ tags affects the cellular localization and functionality of the GDH isoforms, we studied the subcellular distribution of the expressed enzymes as well as their regulation by adenosine diphosphate monopotassium salt (ADP) and guanosine-5′-triphosphate sodium salt (GTP). Through immunoblot analysis of the mitochondrial and cytosolic fraction of the HEK cells expressing the recombinant proteins we found that neither FLAG nor (His)₆ tag disturbs the mitochondrial localization of GDH. The addition of the small tags to the N-terminus of the mature mitochondrial mouse GDH1 or human hGDH1 and hGDH2 did not change the ADP activation or GTP inhibition pattern of the proteins as compared to their untagged counterparts. However, the addition of FLAG tag to the C-terminus of the mouse GDH left the recombinant protein fivefold less sensitive to ADP activation. This finding highlights the necessity of the functional characterization of recombinant proteins containing even the smallest available tags.     

Description of Ulvella elegans sp. nov. and U. islandica sp. nov. (Ulvellaceae,Ulvophyceae) from Iceland – a study based on morphology of species in culture and tufA gene sequences

Two new Ulvella species, U. elegans R. Nielsen & K. Gunnarsson and U. islandica R. Nielsen & K. Gunnarsson are described. These microfilamentous marine green algae were found in the sublittoral zone in northern Iceland, epiphytic on Euthora cristata and associated with a calcareous polychaete tube, respectively. Unialgal cultures were established from field-collected material for morphological observations. In culture, Ulvella elegans was characterized by rosettes of monostromatic pseudoparenchyma consisting of radiating filaments with a margin of mutually free filaments. Each cell had one pyrenoid. Hairs were not observed. Ulvella islandica had a heterotrichous morphology, consisting of dense tufts of upright broad branches and much narrower, rhizoid-like branches. Acrochaete-type hairs occurred; these are hyaline non-septate merocytic extensions from a more or less bulbous base, which may be separated from the vegetative cell below. Most cells had one pyrenoid except for a few broad cells which had two or three. In a phylogenetic reconstruction based on the chloroplast-encoded tufA gene, the sequences for the two species were clearly distinct from any other Ulvella sequence available for this gene. Ulvella islandica was placed in a clade together with U. lens, U. wittrockii, U. reticulata and U. pseudorepens. Ulvella elegans occupied a branch deep in the phylogeny but the position was poorly supported.     

Concordance analysis for QTL detection in dairy cattle: a case study of leg morphology

Background

The present availability of sequence data gives new opportunities to narrow down from QTL (quantitative trait locus) regions to causative mutations. Our objective was to decrease the number of candidate causative mutations in a QTL region. For this, a concordance analysis was applied for a leg conformation trait in dairy cattle. Several QTL were detected for which the QTL status (homozygous or heterozygous for the QTL) was inferred for each individual. Subsequently, the inferred QTL status was used in a concordance analysis to reduce the number of candidate mutations.

Methods

Twenty QTL for rear leg set side view were mapped using Bayes C. Marker effects estimated during QTL mapping were used to infer the QTL status for each individual. Subsequently, polymorphisms present in the QTL regions were extracted from the whole-genome sequences of 71 Holstein bulls. Only polymorphisms for which the status was concordant with the QTL status were kept as candidate causative mutations.

Results

QTL status could be inferred for 15 of the 20 QTL. The number of concordant polymorphisms differed between QTL and depended on the number of QTL statuses that could be inferred and the linkage disequilibrium in the QTL region. For some QTL, the concordance analysis was efficient and narrowed down to a limited number of candidate mutations located in one or two genes, while for other QTL a large number of genes contained concordant polymorphisms.

Conclusions

For regions for which the concordance analysis could be performed, we were able to reduce the number of candidate mutations. For part of the QTL, the concordant analyses narrowed QTL regions down to a limited number of genes, of which some are known for their role in limb or skeletal development in humans and mice. Mutations in these genes are good candidates for QTN (quantitative trait nucleotides) influencing rear leg set side view.     

Genomic relationships based on X chromosome markers and accuracy of genomic predictions with and without X chromosome markers

Background

Although the X chromosome is the second largest bovine chromosome, markers on the X chromosome are not used for genomic prediction in some countries and populations. In this study, we presented a method for computing genomic relationships using X chromosome markers, investigated the accuracy of imputation from a low density (7K) to the 54K SNP (single nucleotide polymorphism) panel, and compared the accuracy of genomic prediction with and without using X chromosome markers.

Methods

The impact of considering X chromosome markers on prediction accuracy was assessed using data from Nordic Holstein bulls and different sets of SNPs: (a) the 54K SNPs for reference and test animals, (b) SNPs imputed from the 7K to the 54K SNP panel for test animals, (c) SNPs imputed from the 7K to the 54K panel for half of the reference animals, and (d) the 7K SNP panel for all animals. Beagle and Findhap were used for imputation. GBLUP (genomic best linear unbiased prediction) models with or without X chromosome markers and with or without a residual polygenic effect were used to predict genomic breeding values for 15 traits.

Results

Averaged over the two imputation datasets, correlation coefficients between imputed and true genotypes for autosomal markers, pseudo-autosomal markers, and X-specific markers were 0.971, 0.831 and 0.935 when using Findhap, and 0.983, 0.856 and 0.937 when using Beagle. Estimated reliabilities of genomic predictions based on the imputed datasets using Findhap or Beagle were very close to those using the real 54K data. Genomic prediction using all markers gave slightly higher reliabilities than predictions without X chromosome markers. Based on our data which included only bulls, using a G matrix that accounted for sex-linked relationships did not improve prediction, compared with a G matrix that did not account for sex-linked relationships. A model that included a polygenic effect did not recover the loss of prediction accuracy from exclusion of X chromosome markers.

Conclusions

The results from this study suggest that markers on the X chromosome contribute to accuracy of genomic predictions and should be used for routine genomic evaluation.     

Mortality after Parental Death in Childhood: A Nationwide Cohort Study from Three Nordic Countries

Background

Bereavement by spousal death and child death in adulthood has been shown to lead to an increased risk of mortality. Maternal death in infancy or parental death in early childhood may have an impact on mortality but evidence has been limited to short-term or selected causes of death. Little is known about long-term or cause-specific mortality after parental death in childhood.

Methods and Findings

This cohort study included all persons born in Denmark from 1968 to 2008 (n = 2,789,807) and in Sweden from 1973 to 2006 (n = 3,380,301), and a random sample of 89.3% of all born in Finland from 1987 to 2007 (n = 1,131,905). A total of 189,094 persons were included in the exposed cohort when they lost a parent before 18 years old. Log-linear Poisson regression was used to estimate mortality rate ratio (MRR). Parental death was associated with a 50% increased all-cause mortality (MRR = 1.50, 95% CI 1.43–1.58). The risks were increased for most specific cause groups and the highest MRRs were observed when the cause of child death and the cause of parental death were in the same category. Parental unnatural death was associated with a higher mortality risk (MRR = 1.84, 95% CI 1.71–2.00) than parental natural death (MRR = 1.33, 95% CI 1.24–1.41). The magnitude of the associations varied according to type of death and age at bereavement over different follow-up periods. The main limitation of the study is the lack of data on post-bereavement information on the quality of the parent-child relationship, lifestyles, and common physical environment.

Conclusions

Parental death in childhood or adolescence is associated with increased all-cause mortality into early adulthood. Since an increased mortality reflects both genetic susceptibility and long-term impacts of parental death on health and social well-being, our findings have implications in clinical responses and public health strategies. Please see later in the article for the Editors'' Summary     

A 660-Kb Deletion with Antagonistic Effects on Fertility and Milk Production Segregates at High Frequency in Nordic Red Cattle: Additional Evidence for the Common Occurrence of Balancing Selection in Livestock

In dairy cattle, the widespread use of artificial insemination has resulted in increased selection intensity, which has led to spectacular increase in productivity. However, cow fertility has concomitantly severely declined. It is generally assumed that this reduction is primarily due to the negative energy balance of high-producing cows at the peak of lactation. We herein describe the fine-mapping of a major fertility QTL in Nordic Red cattle, and identify a 660-kb deletion encompassing four genes as the causative variant. We show that the deletion is a recessive embryonically lethal mutation. This probably results from the loss of RNASEH2B, which is known to cause embryonic death in mice. Despite its dramatic effect on fertility, 13%, 23% and 32% of the animals carry the deletion in Danish, Swedish and Finnish Red Cattle, respectively. To explain this, we searched for favorable effects on other traits and found that the deletion has strong positive effects on milk yield. This study demonstrates that embryonic lethal mutations account for a non-negligible fraction of the decline in fertility of domestic cattle, and that associated positive effects on milk yield may account for part of the negative genetic correlation. Our study adds to the evidence that structural variants contribute to animal phenotypic variation, and that balancing selection might be more common in livestock species than previously appreciated.