Christian Bendixen

Design of a high density SNP genotyping assay in the pig using SNPs identified and characterized by next generation sequencing technology

Background The dissection of complex traits of economic importance to the pig industry requires the availability of a significant number of genetic markers, such as single nucleotide polymorphisms (SNPs). This study was conducted to discover several hundreds of thousands of porcine SNPs using next generation sequencing technologies and use these SNPs, as well as others from different public sources, to design a high-density SNP genotyping assay. Methodology/Principal Findings A total of 19 reduced representation libraries derived from four swine breeds (Duroc, Landrace, Large White, Pietrain) and a Wild Boar population and three restriction enzymes (AluI, HaeIII and MspI) were sequenced using Illuminas Genome Analyzer (GA). The SNP discovery effort resulted in the de novo identification of over 372K SNPs. More than 549K SNPs were used to design the Illumina Porcine 60K+SNP iSelect Beadchip, now commercially available as the PorcineSNP60. A total of 64,232 SNPs were included on the Beadchip. Results from genotyping the 158 individuals used for sequencing showed a high overall SNP call rate (97.5%). Of the 62,621 loci that could be reliably scored, 58,994 were polymorphic yielding a SNP conversion success rate of 94%. The average minor allele frequency (MAF) for all scorable SNPs was 0.274. Conclusions/Significance Overall, the results of this study indicate the utility of using next generation sequencing technologies to identify large numbers of reliable SNPs. In addition, the validation of the PorcineSNP60 Beadchip demonstrated that the assay is an excellent tool that will likely be used in a variety of future studies in pigs.

Ancient human genome sequence of an extinct Palaeo-Eskimo

We report here the genome sequence of an ancient human. Obtained from ∼4,000-year-old permafrost-preserved hair, the genome represents a male individual from the first known culture to settle in Greenland. Sequenced to an average depth of 20×, we recover 79% of the diploid genome, an amount close to the practical limit of current sequencing technologies. We identify 353,151 high-confidence single-nucleotide polymorphisms (SNPs), of which 6.8% have not been reported previously. We estimate raw read contamination to be no higher than 0.8%. We use functional SNP assessment to assign possible phenotypic characteristics of the individual that belonged to a culture whose location has yielded only trace human remains. We compare the high-confidence SNPs to those of contemporary populations to find the populations most closely related to the individual. This provides evidence for a migration from Siberia into the New World some 5,500 years ago, independent of that giving rise to the modern Native Americans and Inuit.

Ancient DNA Reveals Lack of Continuity between Neolithic Hunter-Gatherers and Contemporary Scandinavians

The driving force behind the transition from a foraging to a farming lifestyle in prehistoric Europe (Neolithization) has been debated for more than a century [1-3]. Of particular interest is whether population replacement or cultural exchange was responsible [3-5]. Scandinavia holds a unique place in this debate, for it maintained one of the last major hunter-gatherer complexes in Neolithic Europe, the Pitted Ware culture [6]. Intriguingly, these late hunter-gatherers existed in parallel to early farmers for more than a millennium before they vanished some 4,000 years ago [7, 8]. The prolonged coexistence of the two cultures in Scandinavia has been cited as an argument against population replacement between the Mesolithic and the present [7, 8]. Through analysis of DNA extracted from ancient Scandinavian human remains, we show that people of the Pitted Ware culture were not the direct ancestors of modern Scandinavians (including the Saami people of northern Scandinavia) but are more closely related to contemporary populations of the eastern Baltic region. Our findings support hypotheses arising from archaeological analyses that propose a Neolithic or post-Neolithic population replacement in Scandinavia [7]. Furthermore, our data are consistent with the view that the eastern Baltic represents a genetic refugia for some of the European hunter-gatherer populations.

Paleo-Eskimo mtDNA Genome Reveals Matrilineal Discontinuity in Greenland

The Paleo-Eskimo Saqqaq and Independence I cultures, documented from archaeological remains in Northern Canada and Greenland, represent the earliest human expansion into the New Worlds northern extremes. However, their origin and genetic relationship to later cultures are unknown. We sequenced a mitochondrial genome from a Paleo-Eskimo human by using 3400-to 4500-year-old frozen hair excavated from an early Greenlandic Saqqaq settlement. The sample is distinct from modern Native Americans and Neo-Eskimos, falling within haplogroup D2a1, a group previously observed among modern Aleuts and Siberian Sireniki Yuit. This result suggests that the earliest migrants into the New Worlds northern extremes derived from populations in the Bering Sea area and were not directly related to Native Americans or the later Neo-Eskimos that replaced them.

Complex Vertebral Malformation in Holstein Calves

A recently observed lethal congenital defect of purebred Holstein calves is reported. Eighteen genetically related calves were necropsied. One calf had been aborted on gestation day 159, and the others were delivered between day 250 and day 285. Birth weights were reduced. The defect was characterized by shortening of the cervical and thoracic parts of the vertebral column due to multiple hemivertebrae, fused and misshaped vertebrae, and scoliosis. Symmetrical flexures of the carpal joints and the metacarpophalangeal joint in combination with a slight lateral rotation of the phalanges also were present. Similar low-grade arthrogryposis was present in the posterior limbs. Fifty percent of the calves had heart malformation. Other malformations occurred in a few calves. Complex vertebral malformation (CVM) is proposed as the designation for this defect. A genetic etiology is indicated because cases occurred following breeding between genetically related individuals. Two common ancestors were found; both were former elite sires of US Holstein origin. Because of the widespread international use of semen from sires occurring in the pedigrees of affected calves, CVM is expected to occur in several countries.

Developmental kinetics of the first cell cycles of bovine in vitro produced embryos in relation to their in vitro viability and sex

The development of bovine IVP-embryos was observed in a time-lapse culture system to determine cell cycle lengths of 1) embryos that developed into compact morulae (CM) or blastocysts (BL) within 174 h after insemination (viable), 2) embryos that arrested during earlier stages (nonviable) and 3) male and female embryos. In 4 replicates, inseminated oocytes were cultured on a microscope stage in 3 to 4 groups on a granulosa cell monolayer in supplemented TCM 199. Images were sequentially recorded and stored at 30-min intervals. All embryos that could be identified throughout the culture period were included (n = 392), and the times of cleavage events noted. After culture, 100 CM or BL were randomly selected for sexing by PCR. BL developed equally well in the time-lapse and control culture systems (36 vs 38%). The respective lengths of the first 4 cell cycles of viable embryos were 32.0 +/- 3.9, 8.8 +/- 1.6, 10.8 +/- 4.7 and 47.7 +/- 11.8 h. The subsequent intervals between the 9- to 16-cell, early morula, CM and BL stages lasted 16.2 to 18.2 h. Blastomeres of 2-, 4- and 8-cell embryos cleaved asynchronously with < 1, 2.6 +/- 2.5 and 9.2 +/- 4.5 h intervals, respectively, between the first and last blastomere to cleave. The interval from insemination to tight compaction and formation of a blastocoel was 128.4 +/- 10.7 and 145.8 +/- 12.5 h, respectively. The first 3 cell cycles were approximately 3 h shorter (P < 0.1) while the fourth cycle was 5 h shorter (P = 0.06) for the viable vs nonviable embryos. On this basis it was possible to define time windows in which the proportion of viable 2-, 3- to 4-, 5- to 8- and 9- to 16- cell embryos were at their highest. No differences were found between the cleavage intervals of male and female embryos. We conclude 1) that the time-lapse culture system allows for detailed observation of the developmental kinetics of several embryo groups at the same time, and 2) that these embryos can be manipulated at the end of culture, thus allowing a linkage between early cleavage events and other developmental parameters such as embryo sex or viability after transfer.

Copy number variation in the bovine genome

BackgroundCopy number variations (CNVs), which represent a significant source of genetic diversity in mammals, have been shown to be associated with phenotypes of clinical relevance and to be causative of disease. Notwithstanding, little is known about the extent to which CNV contributes to genetic variation in cattle.ResultsWe designed and used a set of NimbleGen CGH arrays that tile across the assayable portion of the cattle genome with approximately 6.3 million probes, at a median probe spacing of 301 bp. This study reports the highest resolution map of copy number variation in the cattle genome, with 304 CNV regions (CNVRs) being identified among the genomes of 20 bovine samples from 4 dairy and beef breeds. The CNVRs identified covered 0.68% (22 Mb) of the genome, and ranged in size from 1.7 to 2,031 kb (median size 16.7 kb). About 20% of the CNVs co-localized with segmental duplications, while 30% encompass genes, of which the majority is involved in environmental response. About 10% of the human orthologous of these genes are associated with human disease susceptibility and, hence, may have important phenotypic consequences.ConclusionsTogether, this analysis provides a useful resource for assessment of the impact of CNVs regarding variation in bovine health and production traits.

Advances in porcine genomics and proteomics—a toolbox for developing the pig as a model organism for molecular biomedical research

Our current knowledge of human biology is often based on studying a wide range of animal species. In particular, for understanding human diseases, the development of adequate animal models is of immediate importance. Although genetic strains and transgenic animal model organisms like fruit fly (Drosophila), zebrafish and rodents are highly informative about the function of single genes and proteins, these organisms do not always closely reflect human biology, and alternative animal models are thus in great demand. The pig is a non-primate mammal that closely resembles man in anatomy, physiology and genetics. Pigs, although not easily kept for laboratory research, are, however, readily available for biomedical research through the large scale industrial production of pigs produced for human consumption. Recent research has facilitated the biological experimentation with pigs, and helped develop the pig into a novel model organism for biomedical research. This toolbox includes the near completion of the pig genome, catalogues of genes and genetic variation in pigs, extensive characterization of pig proteomes and transcriptomes, as well as the development of transgenic disease models. The aim of this review is to highlight the current progress of these ongoing areas of research, which are mandatory for successful development of biomedical pig models that are in demand for understanding human biology in health and disease.

Homozygosity for a single base-pair mutation in the oocyte-specific GDF9 gene results in sterility in Thoka sheep

The control of fecundity is critical in determining mammalian offspring survival. It is regulated principally by the ovulation rate, so that primates and large farm species commonly have a single offspring. Previously, several mutations have been identified in sheep which increase the naturally low ovulation rate; although in some cases homozygous ewes are infertile. In the present study we present a detailed characterization of a novel mutation in growth differentiation factor 9 (GDF9), found in Icelandic Thoka sheep. This mutation is a single base change (A1279C) resulting in a nonconservative amino acid change (S109R) in the C-terminus of the mature GDF9 protein, which is normally expressed in oocytes at all stages of development. Genotyping all animals for which reproductive records were available confirmed this mutation to be associated with increased fecundity in heterozygous ewes and infertility in homozygotes. Analysis of homozygote ovarian morphology and a number of genes normally activated in growing follicles showed that GDF9 was not involved in oocyte activation, but in subsequent development of the follicle. This study highlights the importance of oocyte factors in regulating fertility and provides new information for structural analysis and investigation of the potentially important sites of dimerization or translational modifications required to produce biologically active GDF9. It also provides the basis for the utilization of these animals to enhance sheep production.

A snapshot of CNVs in the pig genome.

Recent studies of mammalian genomes have uncovered the extent of copy number variation (CNV) that contributes to phenotypic diversity, including health and disease status. Here we report a first account of CNVs in the pig genome covering part of the chromosomes 4, 7, 14, and 17 already sequenced and assembled. A custom tiling oligonucleotide array was used with a median probe spacing of 409 bp for screening 12 unrelated Duroc boars that are founders of a large family material. After a strict CNV calling pipeline, 37 copy number variable regions (CNVRs) across all four chromosomes were identified, with five CNVRs overlapping segmental duplications, three overlapping pig unigenes and one overlapping a RefSeq pig mRNA. This CNV snapshot analysis is the first of its kind in the porcine genome and constitutes the basis for a better understanding of porcine phenotypes and genotypes with the prospect of identifying important economic traits.