Detection of genetic variation affecting milk coagulation properties in Danish Holstein dairy cattle by analyses of pooled whole-genome sequences from phenotypically extreme samples (pool-seq).

Rennet-induced milk coagulation is an important trait for cheese production. Recent studies have reported an alarming frequency of cows producing poorly coagulating milk unsuitable for cheese production. Several genetic factors are known to affect milk coagulation, including variation in the major milk proteins; however, recent association studies indicate genetic effects from other genomic regions as well. The aim of this study was to detect genetic variation affecting milk coagulation properties, measured as curd-firming rate (CFR) and milk pH. This was achieved by examining allele frequency differences between pooled whole-genome sequences of phenotypically extreme samples (pool-seq).. Curd-firming rate and raw milk pH were measured for 415 Danish Holstein cows, and each animal was sequenced at low coverage. Pools were created containing whole genome sequence reads from samples with "extreme" values (high or low) for both phenotypic traits. A total of 6,992,186 and 5,295,501 SNP were assessed in relation to CFR and milk pH, respectively. Allele frequency differences were calculated between pools and 32 significantly different SNP were detected, 1 for milk pH and 31 for CFR, of which 19 are located on chromosome 6. A total of 9 significant SNP, which were selected based on the possible function of proximal candidate genes, were genotyped in the entire sample set ( = 415) to test for an association. The most significant SNP was located proximal to , explaining 33% of the phenotypic variance. , coding for κ-casein, is the most studied in relation to milk coagulation due to its position on the surface of the casein micelles and the direct involvement in milk coagulation. Three additional SNP located on chromosome 6 showed significant associations explaining 7, 3.6, and 1.3% of the phenotypic variance of CFR. The significant SNP on chromosome 6 were shown to be in linkage disequilibrium with the SNP peaking proximal to ; however, after accounting for the genotype of the peak SNP within this QTL, significant effects (-value < 0.1) could still be detected for 2 of the SNP accounting for 2 and 1% of the phenotypic variance. These 2 interesting SNP were located within introns or proximal to the candidate genes-solute carrier family 4 (sodium bicarbonate cotransporter), member 4 () and LIM and calponin homology domains 1 (), respectively-making them interesting targets for further analysis.

Genome-wide association study using high-density single nucleotide polymorphism arrays and whole-genome sequences for clinical mastitis traits in dairy cattle.

Mastitis is a mammary disease that frequently affects dairy cattle. Despite considerable research on the development of effective prevention and treatment strategies, mastitis continues to be a significant issue in bovine veterinary medicine. To identify major genes that affect mastitis in dairy cattle, 6 chromosomal regions on Bos taurus autosome (BTA) 6, 13, 16, 19, and 20 were selected from a genome scan for 9 mastitis phenotypes using imputed high-density single nucleotide polymorphism arrays. Association analyses using sequence-level variants for the 6 targeted regions were carried out to map causal variants using whole-genome sequence data from 3 breeds. The quantitative trait loci (QTL) discovery population comprised 4,992 progeny-tested Holstein bulls, and QTL were confirmed in 4,442 Nordic Red and 1,126 Jersey cattle. The targeted regions were imputed to the sequence level. The highest association signal for clinical mastitis was observed on BTA 6 at 88.97 Mb in Holstein cattle and was confirmed in Nordic Red cattle. The peak association region on BTA 6 contained 2 genes: vitamin D-binding protein precursor (GC) and neuropeptide FF receptor 2 (NPFFR2), which, based on known biological functions, are good candidates for affecting mastitis. However, strong linkage disequilibrium in this region prevented conclusive determination of the causal gene. A different QTL on BTA 6 located at 88.32 Mb in Holstein cattle affected mastitis. In addition, QTL on BTA 13 and 19 were confirmed to segregate in Nordic Red cattle and QTL on BTA 16 and 20 were confirmed in Jersey cattle. Although several candidate genes were identified in these targeted regions, it was not possible to identify a gene or polymorphism as the causal factor for any of these regions.

Permanent genetic resources added to Molecular Ecology Resources Database 1 August 2012 - 30 September 2012.

This article documents the addition of 83 microsatellite marker loci and 96 pairs of single-nucleotide polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Bembidion lampros, Inimicus japonicus, Lymnaea stagnalis, Panopea abbreviata, Pentadesma butyracea, Sycoscapter hirticola and Thanatephorus cucumeris (anamorph: Rhizoctonia solani). These loci were cross-tested on the following species: Pentadesma grandifolia and Pentadesma reyndersii. This article also documents the addition of 96 sequencing primer pairs and 88 allele-specific primers or probes for Plutella xylostella.

MicroRNA identity and abundance in porcine skeletal muscles determined by deep sequencing.

MicroRNAs (miRNA) are short single-stranded RNA molecules that regulate gene expression post-transcriptionally by binding to complementary sequences in the 3' untranslated region (3' UTR) of target mRNAs. MiRNAs participate in the regulation of myogenesis, and identification of the complete set of miRNAs expressed in muscles is likely to significantly increase our understanding of muscle growth and development. To determine the identity and abundance of miRNA in porcine skeletal muscle, we applied a deep sequencing approach. This allowed us to identify the sequences and relative expression levels of 212 annotated miRNA genes, thereby providing a thorough account of the miRNA transcriptome in porcine muscle tissue. The expression levels displayed a very large range, as reflected by the number of sequence reads, which varied from single counts for rare miRNAs to several million reads for the most abundant miRNAs. Moreover, we identified numerous examples of mature miRNAs that were derived from opposite sides of the same predicted precursor stem-loop structures, and also observed length and sequence heterogeneity at the 5' and 3' ends. Furthermore, KEGG pathway analysis suggested that highly expressed miRNAs are involved in skeletal muscle development and regeneration, signal transduction, cell-cell and cell-extracellular matrix communication and neural development and function.

Evaluation of the inheritance of the complex vertebral malformation syndrome by breeding studies.

To investigate the congenital complex vertebral malformation syndrome (CVM) in Holstein calves, two breeding studies were performed including 262 and 363 cows, respectively. Cows were selected from the Danish Cattle Database based on pedigree and insemination records. Selected cows were progeny of sires with an established heterozygous CVM genotype and pregnant after insemination with semen from another sire with heterozygous CVM genotype. Following calving the breeders should state, if the calf was normal and was requested to submit dead calves for necropsy. In both studies, significantly fewer CVM affected calves than expected were obtained; a finding probably reflecting extensive intrauterine mortality in CVM affected foetuses. The findings illustrate increased intrauterine mortality as a major potential bias in observational studies of inherited disorders.

Expanded retina territory by midbrain transformation upon overexpression of Six6 (Optx2) in Xenopus embryos.

During vertebrate eye development, the expression of the homeobox gene Six6 is restricted to the neural retina and is initiated later than Rx and Pax6 in the presumptive retina field. We show here that overexpression of mouse Six6 in Xenopus embryos can induce transformation of competent tissue of the anterior neural plate into retinal tissue. In Six6 injected embryos, the molecular identity of the presumptive midbrain and rostral hindbrain regions was lost, as shown by the absence of XEn-2 and Xpax2 expression, being replaced by the ectopic expression of the retinal markers Xpax6 and Xrx. When allowed to grow further, Six6 injected embryos developed ectopic eye-like structures in the rostral brain and showed a transformation of the midbrain into retina. Similar results were obtained upon overexpression of Six3 or Xsix3, revealing a possible redundance of Six3 and Six6 activities. Taken together, results obtained suggest that during normal retina development, the relatively late expressed Six6 gene becomes part of a network of retinal homeobox genes that are linked together by positive feedback loops. Furthermore, our results demonstrate that the primitive neural ectoderm of the future midbrain and rostral hindbrain is competent to form retinal tissue.

Increased levels of human papillomavirus type 16 DNA in a subset of prostate cancers.

Whether oncogenic human papilloma viruses (HPVs) are involved in the pathogenesis of prostate cancers has been a subject of great controversy. To clarify the contradictory results of investigations, with the aim of detecting viral nucleic acids in prostate cancers, we have carried out a comparative quantitation of the HPV16-E6 sequence in 84 prostate specimens. Using single-tube quantitative competitive PCR, we characterized 47 prostate cancers and 37 control tissues of benign prostatic hyperplasia. A subgroup of the prostate tumors (10 of 47; 21%) was detected as having significantly higher copy numbers of HPV16-E6 sequences when compared to the control tissue (1 of 37; 3%), using a cutoff value of 300 copies per 12,500 diploid cells (two-sided Fisher's exact test, P = 0.02). Our results indicate that the oncogenic HPV16 might contribute to the development of a subset of prostate tumors.

Single-tube nested competitive PCR with homologous competitor for quantitation of DNA target sequences: theoretical description of heteroduplex formation, evaluation of sensitivity, precision and linear range of the method.

Competitive PCR is a frequently used technique for quantitation of DNA and mRNA. However, the application of the most favourable homologous mutated competitors is impeded by the formation of heteroduplex molecules which complicates the data evaluation and may lead to quantitation errors. Moreover, in most cases a single quantitation of an unknown sample requires multiple competitive reactions for identification of the equivalence point. In the present study, a highly efficient and reliable method as well as the underlying theoretical model is described. The mathematical solutions of this model provide the basis for single-tube quantitation using a homologous mutated competitor. For quantitation of Human Papilloma Virus 16-DNA, it is shown that single tube quantitations using simple PAGE separation and video evaluation for signal analysis permit linear detection within more than two orders of magnitude. In addition, repeated single-tube competitive PCRs exhibited good precision (average standard deviation 5%), even if carried out as nested high cycle PCR for quantitation of low abundant sequences (intraassay sensitivity <2 x 10(2) copies). This evaluation method can be applied to any DNA separation and detection method which is capable of resolving the heteroduplex fraction from both homoduplex molecules.

The Spemann organizer-expressed zinc finger gene Xegr-1 responds to the MAP kinase/Ets-SRF signal transduction pathway.

The transcriptional activity of a set of genes, which are all expressed in overlapping spatial and temporal patterns within the Spemann organizer of Xenopus embryos, can be modulated by peptide growth factors. We identify Xegr-1, a zinc finger protein-encoding gene, as a novel member of this group of genes. The spatial expression characteristics of Xegr-1 during gastrulation are most similar to those of Xbra. Making use of animal cap explants, analysis of the regulatory events that govern induction of Xegr-1 gene activity reveals that, in sharp contrast to transcriptional regulation of Xbra, activation of Ets-serum response factor (SRF) transcription factor complexes is required and sufficient for Xegr-1 gene expression. This finding provides the first indication for Ets-SRF complexes bound to serum response elements to be activated during gastrulation. MAP kinase signalling cascades can induce and sustain expression of both Xegr-1 and Xbra. Ectopic Xbra can induce Xegr-1 transcription by an indirect mechanism that appears to operate via primary activation of fibroblast growth factor secretion. These findings define a cascade of events that links Xbra activity to the signal-regulated control of Xegr-1 transcription in the context of early mesoderm induction in Xenopus laevis.