A frameshift deletion in the GDF9 gene in Icelandic Loa prolific sheep.

Several genetic variants have been shown to affect the mean number of offspring in different sheep breeds. Here, we analyzed samples from Icelandic sheep with the aim of identifying the genetic cause of the Icelandic Loa phenotype using three previously identified prolificacy genes as candidates. We demonstrate that a 4-bp frameshift deletion positioned in the mature region of the GDF9 protein in the Loa animals is a likely causal mutation for the observed increase in prolificacy; however, sequencing showed that not all ewes with a high number of offspring carried the deletion, suggesting the presence of a second mutation segregating within this group of animals.

Deep sequencing of Danish Holstein dairy cattle for variant detection and insight into potential loss-of-function variants in protein coding genes.

BACKGROUND: Over the last few years, continuous development of high-throughput sequencing platforms and sequence analysis tools has facilitated reliable identification and characterization of genetic variants in many cattle breeds. Deep sequencing of entire genomes within a cattle breed that has not been thoroughly investigated would be imagined to discover functional variants that are underlying phenotypic differences. Here, we sequenced to a high coverage the Danish Holstein cattle breed to detect and characterize single nucleotide polymorphisms (SNPs), insertion/deletions (Indels), and loss-of-function (LoF) variants in protein-coding genes in order to provide a comprehensive resource for subsequent detection of causal variants for recessive traits. RESULTS: We sequenced four genetically unrelated Danish Holstein cows with a mean coverage of 27X using an Illumina Hiseq 2000. Multi-sample SNP calling identified 10,796,794 SNPs and 1,295,036 indels whereof 482,835 (4.5 %) SNPs and 231,359 (17.9 %) indels were novel. A comparison between sequencing-derived SNPs and genotyping from the BovineHD BeadChip revealed a concordance rate of 99.6-99.8 % for homozygous SNPs and 93.3-96.5 % for heterozygous SNPs. Annotation of the SNPs discovered 74,886 SNPs and 1937 indels affecting coding sequences with 2145 being LoF mutations. The frequency of LoF variants differed greatly across the genome, a hot spot with a strikingly high density was observed in a 6 Mb region on BTA18. LoF affected genes were enriched for functional categories related to olfactory reception and underrepresented for genes related to key cellular constituents and cellular and biological process regulation. Filtering using sequence derived genotype data for 288 Holstein animals from the 1000 bull genomes project removing variants containing homozygous individuals retained 345 of the LoF variants as putatively deleterious. A substantial number of the putative deleterious LoF variants had a minor allele frequency >0.05 in the 1000 bull genomes data set. CONCLUSIONS: Deep sequencing of Danish Holstein genomes enabled us to identify 12.1 million variants. An investigation into LoF variants discovered a set of variants predicted to disrupt protein-coding genes. This catalog of variants will be a resource for future studies to understand variation underlying important phenotypes, particularly recessively inherited lethal phenotypes.

MicroRNA sequence variation can impact interactions with target mRNA in cattle.

BACKGROUND: Small non-coding microRNAs (miRNAs) are important modulators at post-transcriptional levels. Single-nucleotide polymorphisms (SNPs) located in miRNA genes can alter the secondary structure of pre-miRNA to either impair or promote the miRNA maturation processes. Furthermore, SNPs located in the miRNA seed regions can stabilize or disturb miRNA-target interactions, thereby, quantitatively influence the expression of target genes. Therefore, the main objective of this study was to detect SNPs in bovine miRNAs using the whole-genome re-sequencing datasets of 1632 cattle of five breeds from the 1000 bull genomes project. RESULTS: In total, our study identified 1109, 334, and 130 SNPs in the miRNA precursor, mature, and seed regions, respectively. The heterozygosity values were generally less than 0.3, and the minor allele frequencies (MAFs) were mainly less than 0.1. Most SNPs were in Hardy-Weinberg equilibrium (HWE) (HWE-P > 0.05). Furthermore, we found that the majority of SNPs (MAF > 0.1 and HWE-P > 0.05) in the miRNA seed regions altered the repertoire of target genes, which in turn were enriched in different KEGG pathways or GO terms. Thus target prediction for bta-miR-2888 revealed loss of 309 target genes and gain of 691 target genes. The 691 gained target genes were significantly enriched in 60 KEGG pathways and 21 GO terms. CONCLUSION: In summary, our study identified candidate SNPs in miRNA precursor, mature, and seed regions that are likely to affect target RNA interactions, thereby potentially influencing cattle phenotypic traits.

Integrated Analysis of mRNA and MicroRNA Co-expressed Network for the Differentiation of Bovine Skeletal Muscle Cells After Polyphenol Resveratrol Treatment.

Resveratrol (RSV) has been confirmed to benefit human health. Resveratrol supplemented in the feeds of animals improved pork, chicken, and duck meat qualities. In this study, we identified differentially expressed (DE) messenger RNAs (mRNAs) (n = 3,856) and microRNAs (miRNAs) (n = 93) for the weighted gene co-expression network analysis (WGCNA) to investigate the co-expressed DE mRNAs and DE miRNAs in the primary bovine myoblasts after RSV treatment. The mRNA results indicated that RSV treatments had high correlations with turquoise module (0.91, P-value = 0.01) and blue module (0.93, P-value < 0.01), while only the turquoise module (0.96, P-value < 0.01) was highly correlated with the treatment status using miRNA data. After biological enrichment analysis, the 2,579 DE genes in the turquoise module were significantly enriched in the Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The top two GO terms were actin filament-based process (GO:0030029) and actin cytoskeleton organization (GO:0030036). The top two KEGG pathways were regulation of actin cytoskeleton (bta04810) and tight junction (bta04530). Then, we constructed the DE mRNA co-expression and DE miRNA co-expression networks in the turquoise module and the mRNA-miRNA targeting networks based on their co-expressions in the key module. In summary, the RSV-induced miRNAs participated in the co-expression networks that could affect mRNA expressions to regulate the primary myoblast differentiation. Our study provided a better understanding of the roles of RSV in inducing miRNA and of the characteristics of DE miRNAs in the key co-expressed module in regulation of mRNAs and revealed new candidate regulatory miRNAs and genes for the beef quality traits.

Copy number variation in the bovine genome.

BACKGROUND: Copy 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. RESULTS: We 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. CONCLUSIONS: Together, this analysis provides a useful resource for assessment of the impact of CNVs regarding variation in bovine health and production traits.

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.

Meta-analysis of genome-wide association studies for cattle stature identifies common genes that regulate body size in mammals.

Stature is affected by many polymorphisms of small effect in humans 1 . In contrast, variation in dogs, even within breeds, has been suggested to be largely due to variants in a small number of genes2,3. Here we use data from cattle to compare the genetic architecture of stature to those in humans and dogs. We conducted a meta-analysis for stature using 58,265 cattle from 17 populations with 25.4 million imputed whole-genome sequence variants. Results showed that the genetic architecture of stature in cattle is similar to that in humans, as the lead variants in 163 significantly associated genomic regions (P < 5 × 10-8) explained at most 13.8% of the phenotypic variance. Most of these variants were noncoding, including variants that were also expression quantitative trait loci (eQTLs) and in ChIP-seq peaks. There was significant overlap in loci for stature with humans and dogs, suggesting that a set of common genes regulates body size in mammals.

A linkage map for brown trout (Salmo trutta): chromosome homeologies and comparative genome organization with other salmonid fish.

We report on the construction of a linkage map for brown trout (Salmo trutta) and its comparison with those of other tetraploid-derivative fish in the family Salmonidae, including Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), and Arctic char (Salvelinus alpinus). Overall, we identified 37 linkage groups (2n = 80) from the analysis of 288 microsatellite polymorphisms, 13 allozyme markers, and phenotypic sex in four backcross families. Additionally, we used gene-centromere analysis to approximate the position of the centromere for 20 linkage groups and thus relate linkage arrangements to the physical morphology of chromosomes. Sex-specific maps derived from multiple parents were estimated to cover 346.4 and 912.5 cM of the male and female genomes, respectively. As previously observed in other salmonids, recombination rates showed large sex differences (average female-to-male ratio was 6.4), with male crossovers generally localized toward the distal end of linkage groups. Putative homeologous regions inherited from the salmonid tetraploid ancestor were identified for 10 pairs of linkage groups, including five chromosomes showing evidence of residual tetrasomy (pseudolinkage). Map alignments with orthologous regions in Atlantic salmon, rainbow trout, and Arctic char also revealed extensive conservation of syntenic blocks across species, which was generally consistent with chromosome divergence through Robertsonian translocations.

Dose-response relationship for parathyroid adenoma after exposure to ionizing radiation in infancy.

Several authors have suggested that there is an excess risk of hyperparathyroidism, adenomas or hyperplasia after exposure to ionizing radiation. There is still, however, some uncertainty about this association, because these diseases are often asymptomatic and escape clinical detection if not specially searched for. This study is based on a pooled Swedish cohort of 27,925 persons with skin hemangiomas. The majority received radiation treatment in infancy between 1920 and 1965 in Stockholm and Gothenburg. The mean age at treatment was 6 months and the median thyroid dose was 0.20 Gy (range 0-28.5 Gy). Record linkage with the Swedish Cancer Register for the period 1958-1997 gave 43 cases of parathyroid adenoma in the cohort. Analyses of excess relative risk (ERR) models were performed using Poisson regression methods. Clinical records were scrutinized to determine if the childhood radiation exposure was known (biased cases) at the time of diagnosis. Seven of the cases of parathyroid adenoma were classified as biased cases. The standardized incidence ratio (SIR) was 2.10 (95% confidence interval 1.52-2.82) when all cases were included and 1.76 (95% CI 1.23-2.43) with the biased cases excluded. A linear dose-response model with stratification for sex fitted the data best. The ERR per gray was 3.84 (95% CI 1.56-8.99) with all cases and 1.56 (95% CI 0.36-4.45) with the biased cases excluded. There was a significant difference in the ERR per gray between the two subcohorts, probably because of different diagnostic activity in the regions. Our findings confirm that there is a dose-response relationship for radiation-induced parathyroid adenomas.

Cancer incidence among Swedish patients exposed to radioactive thorotrast: a forty-year follow-up survey.

Thorotrast is an alpha-particle-emitting radiological contrast medium that caused chronic exposure to internal alpha-particle radiation when it was administered systemically. Cancer incidence in 432 Swedish patients exposed to Thorotrast was evaluated by computerized linkage of the cohort with the Swedish Cancer Register. Standardized incidence ratios (SIRs) were calculated as the ratio of observed cases in the cohort to expected cases in the general population. A total of 170 cancers occurring in 152 individuals were reported, whereas only 57 cases were expected. The SIR was significantly increased for cancer at all sites (3.0), with the largest excesses noted for primary liver and gallbladder cancer (SIR = 39.2). Other significantly elevated risks were observed for liver cancer not specified as primary, small intestine cancer, stomach cancer, leukemia, kidney cancer, CNS tumors, and pancreatic cancer. Among women, there was a significantly increased risk for lung cancer, based on a small number. Our results show that cumulative radiation exposure is directly related to carcinogenesis in the liver and gallbladder, which is consistent with earlier findings. In addition, there may be a relationship between radiation exposure and the development of other solid tumors.