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The use of reference genes is required for relative quantification in gene expression analysis and the stability of these genes can be variable depending on the experimental design. Therefore, it is indispensable to test the reliability of endogenous genes previously to their use. This study evaluated nine candidate reference genes to select the most stable genes to be used as reference in gene expression studies with the femoral cartilage of normal and epiphysiolysis-affected broilers. The femur articular cartilage of 29 male broilers with 35 days of age was collected, frozen and further submitted to RNA extraction and quantitative PCR (qPCR) analysis. The candidate reference genes evaluated were GAPDH, HMBS, HPRT1, MRPS27, MRPS30, RPL30, RPL4, RPL5, and RPLP1. For the gene stability evaluation, three software were used: GeNorm, BestKeeper and NormFinder, and a global ranking was generated using the function RankAggreg. In this study, the RPLP1 and RPL5 were the most reliable endogenous genes being recommended for expression studies with femur cartilage in broilers with epiphysiolysis and possible other femur anomalies., Competing Interests: The authors have declared that no competing interests exist.

Umbilical hernia (UH) is one of the most frequent defects affecting pig production, however, it also affects humans and other mammals. UH is characterized as an abnormal protrusion of the abdominal contents to the umbilical region, causing pain, discomfort and reduced performance in pigs. Some genomic regions associated to UH have already been identified, however, no study involving RNA sequencing was performed when umbilical tissue is considered. Therefore, here, we have sequenced the umbilical ring transcriptome of five normal and five UH-affected pigs to uncover genes and pathways involved with UH development. A total of 13,216 transcripts were expressed in the umbilical ring tissue. From those, 230 genes were differentially expressed (DE) between normal and UH-affected pigs (FDR <0.05), being 145 downregulated and 85 upregulated in the affected compared to the normal pigs. A total of 68 significant biological processes were identified and the most relevant were extracellular matrix, immune system, anatomical development, cell adhesion, membrane components, receptor activation, calcium binding and immune synapse. The results pointed out ACAN, MMPs, COLs, EPYC, VIT, CCBE1 and LGALS3 as strong candidates to trigger umbilical hernias in pigs since they act in the extracellular matrix remodeling and in the production, integrity and resistance of the collagen. We have generated the first transcriptome of the pig umbilical ring tissue, which allowed the identification of genes that had not yet been related to umbilical hernias in pigs. Nevertheless, further studies are needed to identify the causal mutations, SNPs and CNVs in these genes to improve our understanding of the mechanisms of gene regulation., Competing Interests: The authors have declared that no competing interests exist.

Single nucleotide polymorphism (SNP) markers are used to study population structure and conservation genetics, which permits assessing similarities regarding the linkage disequilibrium and information about the relationship among individuals. To investigate the population genomic structure of 300 females and 25 males from a commercial maternal pig line we analyzed linkage disequilibrium extent, inbreeding coefficients using genomic and conventional pedigree data, and population stratification. The average linkage disequilibrium (r2) was 0.291 ± 0.312 for all adjacent SNPs, distancing less than 100 Kb (kilobase) between markers. The average inbreeding coefficients obtained from runs of homozygosity (ROH) and pedigree analyses were 0.119 and 0.0001, respectively. Low correlation was observed between the inbreeding coefficients possibly as a result of genetic recombination effect accounted for the ROH estimates or caused by pedigree identification errors. A large number of long ROHs might indicate recent inbreeding events in the studied population. A total of 36 homozygous segments were found in more than 30% of the population and these ROH harbor genes associated with reproductive traits. The population stratification analysis indicated that this population was possibly originated from two distinct populations, which is a result from crossings between the eastern and western breeds used in the formation of the line. Our findings provide support to understand the genetic structure of swine populations and may assist breeding companies to avoid a high level of inbreeding coefficients to maintain genetic diversity, showing the effectiveness of using genome-wide SNP information for quantifying inbreeding when the pedigree was incomplete or incorrect., Competing Interests: The authors have declared that no competing interests exist.

The use of reference genes is required for relative quantification in gene expression analysis and since the stability of these genes could be variable depending on the experimental design, it has become indispensable to test the reliability of endogenous genes. Therefore, this study evaluated 10 reference candidate genes in two different experimental conditions in order to obtain stable genes to be used as reference in expression studies related to scrotal hernias in pigs. Two independent experiments were performed: one with 30 days-old MS115 pigs and the other with 60 days-old Landrace pigs. The inguinal ring/canal was collected, frozen and further submitted to real-time PCR analysis (qPCR). For the reference genes stability evaluation, four tools were used: GeNorm in the SLqPCR, BestKeeper, NormFinder and Comparative CT. A general ranking was generated using the BruteAggreg function of R environment. In this study, the RPL19 was one of the most reliable endogenous genes for both experiments. The breed/age effects influenced the expression stability of candidate reference genes evaluated in the inguinal ring of pigs. Therefore, this study reinforces the importance of evaluating the stability of several endogenous genes previous their use, since a consensual set of reference genes is not easily obtained. Here, two sets of genes are recommended: RPL19, RPL32 and H3F3A for 30-days MS115 and PPIA and RPL19 for the 60 days-old Landrace pigs. This is the first study using the inguinal ring tissue and the results can be useful as an indicative for other studies working with gene expression in this tissue., Competing Interests: The authors have declared that no competing interests exist.

Apolipoprotein B (APOB) and Adiponectin Receptor 1 (ADIPOR1) are related to the regulation of feed intake, fat metabolism and protein deposition and are candidate genes for genomic studies in birds. In this study, associations of two single nucleotide polymorphisms (SNPs) g.102A>T (APOB) and g.729C>T (ADIPOR1) with carcass, bone integrity and performance traits in broilers were investigated. Genotyping was performed on a paternal line of 1,454 broilers. The SNP detection was carried out by PCR-RFLP technique using the restriction enzymes HhaI for the SNP g.729C>T and MslI for the SNP g.102A>T. The association analyses of the two SNPs with 85 traits were performed using the restricted maximum likelihood (REML) and Generalized Quasi-Likelihood Score (GQLS) methods. For REML the model included the random additive genetic effect of animal and fixed effects of sex, hatch and SNP genotypes. In the GQLS method, a logistic regression was used to associate the genotypes with phenotypes adjusted for fixed effects of sex and hatch. The SNP g.729C>T in the ADIPOR1 gene was associated with thickness of the femur and breast skin yield. Thus, the ADIPOR1 gene seems implicated in the metabolism and/or fat deposition and bone integrity in broilers.