Anna Castelló

Real-time quantitative PCR-based system for determining transgene copy number in transgenic animals

In this paper, we describe a rapid and accurate real-time quantitative PCR-based system to determine transgene copy number in transgenic animals. We used the 2(-deltadeltaCt) method to analyze different transgenic lines without the requirement of a control sample previously determined by Southern blot analysis. To determine the transgene copy number in several mouse lines carrying a goat beta-Lactoglobulin transgene, we developed a TaqMan assay in which a goat genomic DNA sample was used as a calibrator. Moreover, we used the glucagon gene as a reference control because this gene is highly conserved between species and amplifies with the same efficiency and sensitivity in goat as in mouse. With this assay, we provide an alternative simple method to determine the transgene copy number, avoiding the traditional and tedious blotting techniques. The assays discrimination ability from our results is of at least six copies and, similar to the limitations of the blotting techniques, the accuracy of the quantification diminishes when the transgene copy number is high.

Copy number variation in the porcine genome inferred from a 60 k SNP BeadChip.

BackgroundRecent studies in pigs have detected copy number variants (CNVs) using the Comparative Genomic Hybridization technique in arrays designed to cover specific porcine chromosomes. The goal of this study was to identify CNV regions (CNVRs) in swine species based on whole genome SNP genotyping chips.ResultsWe used predictions from three different programs (cnvPartition, PennCNV and GADA) to analyze data from the Porcine SNP60 BeadChip. A total of 49 CNVRs were identified in 55 animals from an Iberian x Landrace cross (IBMAP) according to three criteria: detected in at least two animals, contained three or more consecutive SNPs and recalled by at least two programs. Mendelian inheritance of CNVRs was confirmed in animals belonging to several generations of the IBMAP cross. Subsequently, a segregation analysis of these CNVRs was performed in 372 additional animals from the IBMAP cross and its distribution was studied in 133 unrelated pig samples from different geographical origins. Five out of seven analyzed CNVRs were validated by real time quantitative PCR, some of which coincide with well known examples of CNVs conserved across mammalian species.ConclusionsOur results illustrate the usefulness of Porcine SNP60 BeadChip to detect CNVRs and show that structural variants can not be neglected when studying the genetic variability in this species.

Liver transcriptome profile in pigs with extreme phenotypes of intramuscular fatty acid composition

BackgroundNew advances in high-throughput technologies have allowed for the massive analysis of genomic data, providing new opportunities for the characterization of the transcriptome architectures. Recent studies in pigs have employed RNA-Seq to explore the transcriptome of different tissues in a reduced number of animals. The main goal of this study was the identification of differentially-expressed genes in the liver of Iberian x Landrace crossbred pigs showing extreme phenotypes for intramuscular fatty acid composition using RNA-Seq.ResultsThe liver transcriptomes of two female groups (H and L) with phenotypically extreme intramuscular fatty acid composition were sequenced using RNA-Seq. A total of 146 and 180 unannotated protein-coding genes were identified in intergenic regions for the L and H groups, respectively. In addition, a range of 5.8 to 7.3% of repetitive elements was found, with SINEs being the most abundant elements. The expression in liver of 186 (L) and 270 (H) lncRNAs was also detected. The higher reproducibility of the RNA-Seq data was validated by RT-qPCR and porcine expression microarrays, therefore showing a strong correlation between RT-qPCR and RNA-Seq data (ranking from 0.79 to 0.96), as well as between microarrays and RNA-Seq (r=0.72). A differential expression analysis between H and L animals identified 55 genes differentially-expressed between groups. Pathways analysis revealed that these genes belong to biological functions, canonical pathways and three gene networks related to lipid and fatty acid metabolism. In concordance with the phenotypic classification, the pathways analysis inferred that linolenic and arachidonic acids metabolism was altered between extreme individuals. In addition, a connection was observed among the top three networks, hence suggesting that these genes are interconnected and play an important role in lipid and fatty acid metabolism.ConclusionsIn the present study RNA-Seq was used as a tool to explore the liver transcriptome of pigs with extreme phenotypes for intramuscular fatty acid composition. The differential gene expression analysis showed potential gene networks which affect lipid and fatty acid metabolism. These results may help in the design of selection strategies to improve the sensorial and nutritional quality of pork meat.

Genome-wide association study for intramuscular fatty acid composition in an Iberian × Landrace cross

The lipid content and fatty acid (FA) profile have an important impact in human health as well as in the technological transformation and nutritional and organoleptic quality of meat. A genome-wide association study (GWAS) on 144 backcross pigs (25% Iberian × 75% Landrace) was performed for 32 traits associated with intramuscular FA composition and indices of FA metabolism. The GWAS was carried out using Qxpak 5.0 and the genotyping information obtained from the Porcine SNP60K BeadChip (Illumina Inc., San Diego, CA). Signals of significant association considering a false- discovery rate (q-value < 0.05) were observed in 15 of the 32 analyzed traits, and a total of 813 trait-associated SNP (TAS), distributed in 43 chromosomal intervals on almost all autosomes, were annotated. According to the clustering analysis based on functional classification, several of the annotated genes are related to FA composition and lipid metabolism. Some interesting positional concordances among TAS and previously reported QTL for FA compositions and/or other lipid traits were also found. These common genomic regions for different traits suggest pleiotropic effects for FA composition and were found primarily on SSC4, SSC8, and SSC16. These results contribute to our understanding of the complex genetic basis of FA composition and FA metabolism.

Transcriptome architecture across tissues in the pig

BackgroundArtificial selection has resulted in animal breeds with extreme phenotypes. As an organism is made up of many different tissues and organs, each with its own genetic programme, it is pertinent to ask: How relevant is tissue in terms of total transcriptome variability? Which are the genes most distinctly expressed between tissues? Does breed or sex equally affect the transcriptome across tissues?ResultsIn order to gain insight on these issues, we conducted microarray expression profiling of 16 different tissues from four animals of two extreme pig breeds, Large White and Iberian, two males and two females. Mixed model analysis and neighbor – joining trees showed that tissues with similar developmental origin clustered closer than those with different embryonic origins. Often a sound biological interpretation was possible for overrepresented gene ontology categories within differentially expressed genes between groups of tissues. For instance, an excess of nervous system or muscle development genes were found among tissues of ectoderm or mesoderm origins, respectively. Tissue accounted for ~11 times more variability than sex or breed. Nevertheless, we were able to confidently identify genes with differential expression across tissues between breeds (33 genes) and between sexes (19 genes). The genes primarily affected by sex were overall different than those affected by breed or tissue. Interaction with tissue can be important for differentially expressed genes between breeds but not so much for genes whose expression differ between sexes.ConclusionEmbryonic development leaves an enduring footprint on the transcriptome. The interaction in gene × tissue for differentially expressed genes between breeds suggests that animal breeding has targeted differentially each tissues transcriptome.

Analysis of porcine adipose tissue transcriptome reveals differences in de novo fatty acid synthesis in pigs with divergent muscle fatty acid composition

BackgroundIn pigs, adipose tissue is one of the principal organs involved in the regulation of lipid metabolism. It is particularly involved in the overall fatty acid synthesis with consequences in other lipid-target organs such as muscles and the liver. With this in mind, we have used massive, parallel high-throughput sequencing technologies to characterize the porcine adipose tissue transcriptome architecture in six Iberian x Landrace crossbred pigs showing extreme phenotypes for intramuscular fatty acid composition (three per group).ResultsHigh-throughput RNA sequencing was used to generate a whole characterization of adipose tissue (backfat) transcriptome. A total of 4,130 putative unannotated protein-coding sequences were identified in the 20% of reads which mapped in intergenic regions. Furthermore, 36% of the unmapped reads were represented by interspersed repeats, SINEs being the most abundant elements. Differential expression analyses identified 396 candidate genes among divergent animals for intramuscular fatty acid composition. Sixty-two percent of these genes (247/396) presented higher expression in the group of pigs with higher content of intramuscular SFA and MUFA, while the remaining 149 showed higher expression in the group with higher content of PUFA. Pathway analysis related these genes to biological functions and canonical pathways controlling lipid and fatty acid metabolisms. In concordance with the phenotypic classification of animals, the major metabolic pathway differentially modulated between groups was de novo lipogenesis, the group with more PUFA being the one that showed lower expression of lipogenic genes.ConclusionsThese results will help in the identification of genetic variants at loci that affect fatty acid composition traits. The implications of these results range from the improvement of porcine meat quality traits to the application of the pig as an animal model of human metabolic diseases.

Determination of Reference microRNAs for Relative Quantification in Porcine Tissues

Relative quantification is the strategy of choice for processing RT-qPCR data in microRNAs (miRNAs) expression studies. Normalisation of relative quantification data is performed by using reference genes. In livestock species, such as pigs, the determination of reference miRNAs and the optimal number of them has not been widely studied. In this study, the stability of ten miRNAs (Ssc-let-7a, Ssc-miR-103, Ssc-miR-17-3p, Hsa-miR-25, Hsa-miR-93, Ssc-miR-106a, Ssc-miR-191, Ssc-miR-16, Ssc-miR-26a and Ssc-miR-17-5p) was investigated by RT-qPCR in different tissues (skeletal muscle, kidney, liver, ovary and uterus) and in different pig breeds (Iberian, Landrace, Large White, Meishan and Vietnamese) as variation factors. Stability values were calculated with geNorm and NormFinder algorithms obtaining high correlation between them (r2 = 0.99). The analyses showed that tissue is an important variability factor in miRNAs expression stability whereas breed is not a determinant factor. All ten miRNAs analysed had good stability values and, therefore, can be used as reference miRNAs. When all tissues were considered, miR-93 was the most stable miRNA. Dividing data set by tissues, let-7a was the most stable in skeletal muscle and ovary, miR-17-5p in kidney, miR-26a in liver and miR-103 in uterus. Moreover, the optimal number of reference miRNAs to be used for proper normalisation data was determined. It is suggested the use of five reference miRNAs (miR-93, miR-25, miR-106a, miR-17-5p and miR-26a) in multi-tissue experimental designs and the use of three reference miRNAs as the optimal number in single tissues studies (let-7a, miR-17-5p and miR-25 in skeletal muscle; miR-17-5p, miR-93 and miR-26a in kidney, miR-26a, miR-103 and let-7a in liver, let-7a, miR-25 and miR-106a in ovary and miR-103, let-7a and miR-93 in uterus). Overall, this study provides valuable information about the porcine reference miRNAs that can be used in order to perform a proper normalisation when relative quantification by RT-qPCR studies is undertaken.

From SNP co-association to RNA co-expression: Novel insights into gene networks for intramuscular fatty acid composition in porcine

BackgroundFatty acids (FA) play a critical role in energy homeostasis and metabolic diseases; in the context of livestock species, their profile also impacts on meat quality for healthy human consumption. Molecular pathways controlling lipid metabolism are highly interconnected and are not fully understood. Elucidating these molecular processes will aid technological development towards improvement of pork meat quality and increased knowledge of FA metabolism, underpinning metabolic diseases in humans.ResultsThe results from genome-wide association studies (GWAS) across 15 phenotypes were subjected to an Association Weight Matrix (AWM) approach to predict a network of 1,096 genes related to intramuscular FA composition in pigs. To identify the key regulators of FA metabolism, we focused on the minimal set of transcription factors (TF) that the explored the majority of the network topology. Pathway and network analyses pointed towards a trio of TF as key regulators of FA metabolism: NCOA2, FHL2 and EP300. Promoter sequence analyses confirmed that these TF have binding sites for some well-know regulators of lipid and carbohydrate metabolism. For the first time in a non-model species, some of the co-associations observed at the genetic level were validated through co-expression at the transcriptomic level based on real-time PCR of 40 genes in adipose tissue, and a further 55 genes in liver. In particular, liver expression of NCOA2 and EP300 differed between pig breeds (Iberian and Landrace) extreme in terms of fat deposition. Highly clustered co-expression networks in both liver and adipose tissues were observed. EP300 and NCOA2 showed centrality parameters above average in the both networks. Over all genes, co-expression analyses confirmed 28.9% of the AWM predicted gene-gene interactions in liver and 33.0% in adipose tissue. The magnitude of this validation varied across genes, with up to 60.8% of the connections of NCOA2 in adipose tissue being validated via co-expression.ConclusionsOur results recapitulate the known transcriptional regulation of FA metabolism, predict gene interactions that can be experimentally validated, and suggest that genetic variants mapped to EP300, FHL2, and NCOA2 modulate lipid metabolism and control energy homeostasis in pigs.

Genome-wide linkage analysis of QTL for growth and body composition employing the PorcineSNP60 BeadChip

BackgroundThe traditional strategy to map QTL is to use linkage analysis employing a limited number of markers. These analyses report wide QTL confidence intervals, making very difficult to identify the gene and polymorphisms underlying the QTL effects. The arrival of genome-wide panels of SNPs makes available thousands of markers increasing the information content and therefore the likelihood of detecting and fine mapping QTL regions. The aims of the current study are to confirm previous QTL regions for growth and body composition traits in different generations of an Iberian x Landrace intercross (IBMAP) and especially identify new ones with narrow confidence intervals by employing the PorcineSNP60 BeadChip in linkage analyses.ResultsThree generations (F3, Backcross 1 and Backcross 2) of the IBMAP and their related animals were genotyped with PorcineSNP60 BeadChip. A total of 8,417 SNPs equidistantly distributed across autosomes were selected after filtering by quality, position and frequency to perform the QTL scan. The joint and separate analyses of the different IBMAP generations allowed confirming QTL regions previously identified in chromosomes 4 and 6 as well as new ones mainly for backfat thickness in chromosomes 4, 5, 11, 14 and 17 and shoulder weight in chromosomes 1, 2, 9 and 13; and many other to the chromosome-wide signification level. In addition, most of the detected QTLs displayed narrow confidence intervals, making easier the selection of positional candidate genes.ConclusionsThe use of higher density of markers has allowed to confirm results obtained in previous QTL scans carried out with microsatellites. Moreover several new QTL regions have been now identified in regions probably not covered by markers in previous scans, most of these QTLs displayed narrow confidence intervals. Finally, prominent putative biological and positional candidate genes underlying those QTL effects are listed based on recent porcine genome annotation.

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.