Probabilidad de detección de pedigrees erróneos mediante polimorfismos bioquímicos en razas caninas españolas

Mediante técnicas convencionales de electroforesis, se realiza un estudio poblacional de once marcadores genéticos sanguíneos -polimorfismos bioquímicos- para un total de 484 individuos, pertenecientes a diez razas caninas españolas. La información aportada a partir de los valores de las frecuencias génicas de los once sistemas polimórficos, nos permite estimar, en cada una de las razas, las probabilidades de detección de errores en el pedigree. Considerando los once polimorfismos conjuntamente, y en el caso de un único individuo analizado por camada, se obtienen unos valores que oscilan entre el 72,02% para la raza Ca de Bestiar y el 83,02% para la raza Mastín Español, con un valor promedio del 78,29% para el conjunto de las razas. La probabilidad de exclusión de falsas filiaciones se incrementa hasta un valor promedio, para las diez razas, del 92,07%, con unos valores extremos del 88,55% para Ca de Bestiar y del 94,68% para Mastín Español, si se analizan cinco descendientes por camada. Se discute asimismo, la relación eficacia/coste de cada uno de los sistemas de detección (electroforesis en gel de almidón, en gel de poliacrilamida y bidimensional) en el protocolo de control de parentesco.A total of 11 genetic loci encoding enzymes and other blood proteins has been assayed by conventional techniques al electrophoresis in 484 indiuiduals, belonging to 10 spanish breeds of dogs. In each breed the information provided by the values of the allelic frequencies in the 11 polymorphic systems can be used to assess the probabilities to detect erroneous pedigrees. Using the whole set of the 11 polymorpbisms, and in the case 011 individual analyzed per litter, the values ranged between 72,02% for the "Ca de Bestiar" breed and 83,02% in the "Mastín Español" breed, with an average al 78,29%. The probabilities to exclude false filiations increase to an average 0192, O7%lar the 10 breeds, from 88,55% in "Ca de Bestiar" to 94,68% in "Mastín Español", if 5 offsprings are used per litter in the analysis. The relationship between the efficiency and the cost of each system ofdetection (electrophoresis in starch gel) in polyacrilamyde gel and bidimensional) in the protocol of control of relationship is also discussed

Microbiota profiling with long amplicons using Nanopore sequencing : full-length 16S rRNA gene and the 16S-ITS-23S of the rrn operon

Background: Profiling the microbiome of low-biomass samples is challenging for metagenomics since these samples are prone to contain DNA from other sources (e.g. host or environment). The usual approach is sequencing short regions of the 16S rRNA gene, which fails to assign taxonomy to genus and species level. To achieve an increased taxonomic resolution, we aim to develop long-amplicon PCR-based approaches using Nanopore sequencing. We assessed two different genetic markers: the full-length 16S rRNA (~1,500 bp) and the 16S-ITS-23S region from the rrn operon (4,300 bp). Methods: We sequenced a clinical isolate of Staphylococcus pseudintermedius, two mock communities and two pools of low-biomass samples (dog skin). Nanopore sequencing was performed on MinION™ using the 1D PCR barcoding kit. Sequences were pre-processed, and data were analyzed using EPI2ME or Minimap2 with rrn database. Consensus sequences of the 16S-ITS-23S genetic marker were obtained using canu. Results: The full-length 16S rRNA and the 16S-ITS-23S region of the rrn operon were used to retrieve the microbiota composition of the samples at the genus and species level. For the Staphylococcus pseudintermedius isolate, the amplicons were assigned to the correct bacterial species in ~98% of the cases with the16S-ITS-23S genetic marker, and in ~68%, with the 16S rRNA gene when using EPI2ME. Using mock communities, we found that the full-length 16S rRNA gene represented better the abundances of a microbial community; whereas, 16S-ITS-23S obtained better resolution at the species level. Finally, we characterized low-biomass skin microbiota samples and detected species with an environmental origin. Conclusions: Both full-length 16S rRNA and the 16S-ITS-23S of the rrn operon retrieved the microbiota composition of simple and complex microbial communities, even from the low-biomass samples such as dog skin. For an increased resolution at the species level, targeting the 16S-ITS-23S of the rrn operon would be the best choice

Characterization of the Impact of Density Gradient Centrifugation on the Profile of the Pig Sperm Transcriptome by RNA-Seq

Altres ajuts: CERCA Programme/Generalitat de CatalunyaRNA-Seq data from human semen suggests that the study of the sperm transcriptome requires the previous elimination from the ejaculates of somatic cells carrying a larger load of RNA. Semen purification is also carried to study the sperm transcriptome in other species including swine and it is often done by density gradient centrifugation to obtain viable spermatozoa from fresh ejaculates or artificial insemination doses, thereby limiting the throughput and remoteness of the samples that can be processed in one study. The aim of this work was to evaluate the impact of purification with density gradient centrifugation by BoviPure TM on porcine sperm. Four boar ejaculates were purified with BoviPure TM and their transcriptome sequenced by RNA-Seq was compared with the RNA-Seq profiles of their paired non-purified sample. Seven thousand five hundred and nineteen protein coding genes were identified. Correlation, cluster, and principal component analysis indicated high-although not complete-similarity between the purified and the paired non-purified ejaculates. 372 genes displayed differentially abundant RNA levels between treatments. Most of these genes had lower abundances after purification and were mostly related to translation, transcription and metabolic processes. We detected a significant change in the proportion of genes of epididymal origin within the differentially abundant genes (1.3%) when compared with the catalog of unaltered genes (0.2%). In contrast, the proportion of testis-specific genes was higher in the group of unaltered genes (4%) when compared to the list of differentially abundant genes (0%). No proportion differences were identified for prostate, white blood, lymph node, tonsil, duodenum, skeletal muscle, liver, and mammary gland. Altogether, these results suggest that the purification impacts on the RNA levels of a small number of genes which are most likely caused by the removal of epididymal epithelial cells but also premature germinal cells, immature or abnormal spermatozoa or seminal exosomes with a distinct load of RNAs

Non-synonymous genetic variation in exonic regions of canine Toll-like receptors

Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) considered to be the primary sensors of pathogens in innate immunity. Genetic variants could be associated to differences in breed innate immune response to pathogens and thus to susceptibility to infections or autoimmune diseases. There is therefore great interest in the characterization of canine TLRs. Polymorphisms in canine TLRs have been characterized by massive sequencing after enrichment of their exonic regions. DNAs from 335 dogs (seven different breeds) and 100 wolves (two different populations) were used in pools. The ratio of SNP discovery was 76.5% (in relation to CanFam 3.1); 155 out of 204 variants identified were new. Functional annotation identified 64 non-synonymous variants (43 new), 73 synonymous variants (56 new) and 67 modifier variants (57 new). 12 out of 64 non-synonymous variants are breed or wolf specific. TLR5 has been found to be the most polymorphic among canine TLRs. Finally, a TaqMan OpenArray® plate containing 64 SNPs with a possible functional effect in the protein (4 frameshifts and 60 non-synonymous codons) has been designed and validated. Non-synonymous genetic variation has been characterized in exonic regions of canine Toll-like Receptors. The TaqMan OpenArray® plate developed to capture the individual variability that affects protein function will allow high-throughput genotyping either to study association to infection susceptibility or even TLR evolution in the canine genome. The online version of this article (doi:10.1186/2052-6687-1-11) contains supplementary material, which is available to authorized users

Evaluation of the capability of the PCV2 genome to encode miRNAs : lack of viral miRNA expression in an experimental infection

Porcine circovirus type 2 (PCV2) is a ssDNA virus causing PCV2-systemic disease (PCV2-SD), one of the most important diseases in swine. MicroRNAs (miRNAs) are a new class of small non-coding RNAs that regulate gene expression post-transcriptionally. Viral miRNAs have recently been described and the number of viral miRNAs has been increasing in the past few years. In this study, small RNA libraries were constructed from two tissues of subclinically PCV2 infected pigs to explore if PCV2 can encode viral miRNAs. The deep sequencing data revealed that PCV2 does not express miRNAs in an in vivo subclinical infection

A Genetic Predictive Model for Canine Hip Dysplasia : Integration of Genome Wide Association Study (GWAS) and Candidate Gene Approaches

Canine hip dysplasia is one of the most prevalent developmental orthopedic diseases in dogs worldwide. Unfortunately, the success of eradication programs against this disease based on radiographic diagnosis is low. Adding the use of diagnostic genetic tools to the current phenotype-based approach might be beneficial. The aim of this study was to develop a genetic prognostic test for early diagnosis of hip dysplasia in Labrador Retrievers. To develop our DNA test, 775 Labrador Retrievers were recruited. For each dog, a blood sample and a ventrodorsal hip radiograph were taken. Dogs were divided into two groups according to their FCI hip score: control (A/B) and case (D/E). C dogs were not included in the sample. Genetic characterization combining a GWAS and a candidate gene strategy using SNPs allowed a case-control population association study. A mathematical model which included 7 SNPs was developed using logistic regression. The model showed a good accuracy (Area under the ROC curve = 0.85) and was validated in an independent population of 114 dogs. This prognostic genetic test represents a useful tool for choosing the most appropriate therapeutic approach once genetic predisposition to hip dysplasia is known. Therefore, it allows a more individualized management of the disease. It is also applicable during genetic selection processes, since breeders can benefit from the information given by this test as soon as a blood sample can be collected, and act accordingly. In the authors' opinion, a shift towards genomic screening might importantly contribute to reducing canine hip dysplasia in the future. In conclusion, based on genetic and radiographic information from Labrador Retrievers with hip dysplasia, we developed an accurate predictive genetic test for early diagnosis of hip dysplasia in Labrador Retrievers. However, further research is warranted in order to evaluate the validity of this genetic test in other dog breeds

miRNA Expression Profile Analysis in Kidney of Different Porcine Breeds

MicroRNAs (miRNAs) are important post-transcriptional regulators in eukaryotes that target mRNAs repressing their expression. The uncertain process of pig domestication, with different origin focuses, and the selection process that commercial breeds suffered, have generated a wide spectrum of breeds with clear genetic and phenotypic variability. The aim of this work was to define the miRNAs expression profile in kidney of several porcine breeds. Small RNA libraries from kidney were elaborated and high-throughput sequenced with the 454 Genome Sequencer FLX (Roche). Pigs used were classified into three groups: the European origin group (Iberian breed and European Wild Boar ancestor), European commercial breeds (Landrace, Large White and Piétrain breeds) and breeds with Asian origin (Meishan and Vietnamese breeds). A total of 229 miRNAs were described in the pig kidney miRNA profile, including 110 miRNAs out of the 257 previously described pig miRNAs and 119 orthologous miRNAs. The most expressed miRNAs in pig kidney microRNAome were Hsa-miR-200b-3p, Ssc-miR-125b and Ssc-miR-23b. Moreover, 5 novel porcine miRNAs and 3 orthologous miRNAs could be validated through RT-qPCR. miRNA sequence variation was determined in 116 miRNAs, evidencing the presence of isomiRs. 125 miRNAs were differentially expressed between breed groups. The identification of breed-specific miRNAs, which could be potentially associated to certain phenotypes, is becoming a new tool for the study of the genetic variability underlying complex traits and furthermore, it adds a new layer of complexity to the interesting process of pig evolution

Rapid genome resequencing of an atoxigenic strain of Aspergillus carbonarius

In microorganisms, Ion Torrent sequencing technology has been proved to be useful in whole-genome sequencing of bacterial genomes (5 Mbp). In our study, for the first time we used this technology to perform a resequencing approach in a whole fungal genome (36 Mbp), a non-ochratoxin A producing strain of Aspergillus carbonarius. Ochratoxin A (OTA) is a potent nephrotoxin which is found mainly in cereals and their products, but it also occurs in a variety of common foods and beverages. Due to the fact that this strain does not produce OTA, we focused some of the bioinformatics analyses in genes involved in OTA biosynthesis, using a reference genome of an OTA producing strain of the same species. This study revealed that in the atoxigenic strain there is a high accumulation of nonsense and missense mutations in several genes. Importantly, a two fold increase in gene mutation ratio was observed in PKS and NRPS encoding genes which are suggested to be involved in OTA biosynthesis

Endometrial gene expression profile of pregnant sows with extreme phenotypes for reproductive efficiency

Prolificacy can directly impact porcine profitability, but large genetic variation and low heritability have been found regarding litter size among porcine breeds. To identify key differences in gene expression associated to swine reproductive efficiency, we performed a transcriptome analysis of sows' endometrium from an Iberian x Meishan F2 population at day 30-32 of gestation, classified according to their estimated breeding value (EBV) as high (H, EBV > 0) and low (L, EBV < 0) prolificacy phenotypes. For each sample, mRNA and small RNA libraries were RNA-sequenced, identifying 141 genes and 10 miRNAs differentially expressed between H and L groups. We selected four miRNAs based on their role in reproduction, and five genes displaying the highest differences and a positive mapping into known reproductive QTLs for RT-qPCR validation on the whole extreme population. Significant differences were validated for genes: PTGS2 (p = 0.03; H/L ratio = 3.50), PTHLH (p = 0.03; H/L ratio = 3.69), MMP8 (p = 0.01; H/L ratio = 4.41) and SCNN1G (p = 0.04; H/L ratio = 3.42). Although selected miRNAs showed similar expression levels between H and L groups, significant correlation was found between the expression level of ssc-miR-133a (p < 0.01) and ssc-miR-92a (p < 0.01) and validated genes. These results provide a better understanding of the genetic architecture of prolificacy-related traits and embryo implantation failure in pigs