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Next-generation sequencing RNA-Seq technology is a powerful tool that creates new possibilities for whole-transcriptome analysis. In our study, the RNA-Seq method was applied to analyze global changes in transcriptome from muscle tissue (m. semimembranosus) in two pig breeds (Pietrain and Polish Landrace, PL). The breeds differ in terms of muscularity, growth rate and reproduction traits. Using three different approaches (deseq, cufflinks and edger) and taking into account the most restrictive criteria, 35 genes differentially expressed between Pietrain and PL pigs were identified. In both breeds, the most abundant were transcripts encoding ribosomal and cytoskeletal proteins (TPM3, TCAP, TMOD4, TPM2, TNNC1) and calcium-binding proteins involved in muscle contraction, calcium-mediated signaling or cation transport (CASQ1, MLC2V, SLC25A4, MYL3). In PL pigs, we identified up-regulation of several genes that play crucial roles in reproduction: female gamete generation (BDP1, PTPN21, USP9X), fertilization (EGFR) and embryonic development (CPEB4). In the Pietrain breed, only seven genes were over-expressed (CISH, SPP1, TUBA8, ATP6V1C2, IGKC, predicted LOC100510960 and LOC100626400), and they play important roles in, for example, negative regulation of apoptosis, immune response, cell-cell signaling, cell growth and migration as well as the metabolic process. The functions of the majority of selected genes were consistent with phenotypic variation in investigated breeds; thus, we proposed a new panel of candidate genes that can be associated with economically important pig traits.

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The objective of the present study was to analyze the level of intramuscular fat (IMF) in loin (musculus longissimus dorsi) and ham (musculus semimembranosus) and the level of subcutaneous fat in these cuts depending on breed, age and the expression level of FABP3 and LEPR genes. The results obtained showed that only the breed influenced on the level of both intramuscular and subcutaneous fat to the same extent (P ≤ 0.001). The age of animals had an effect on fat content of the cuts (P ≤ 0.001) and to a lower extent on the level of IMF in both muscles (P ≤ 0.05). We confirmed highly significant effect of breed and age on the LEPR mRNA abundance—the expression of the this gene increased significantly (P ≤ 0.01) with age and the highest expression was found for the Puławska breed in m. longissimus dorsi and for the Polish Landrace breed in m. semimembranosus. We observed the high correlations between the transcript level of the LEPR gene and the fat content of individual cuts (P ≤ 0.01). The expression level of FABP3 gene influenced the level of IMF (P ≤ 0.01), but not the level of subcutaneous fat in loin and ham.

Gene expression analysis by Real-Time PCR requires careful selection of endogenous reference genes to obtain accurate results. We evaluated usefulness of six reference genes to expression studies of porcine stomach. We selected genes which were recently described as very stable (RPL27, RPS29, RPS13, OAZ1) and two commonly used housekeeping genes (GAPDH, ACTB). Our results indicate that OAZ1, RPS29 and RPL27 are more suitable reference genes than ACTB and GAPDH in expression studies of porcine stomach. In our study the most stable genes were OAZ1 (M=0.856), RPS29 (M=0.862) and RPL27 (M=0.892), while ACTB (M=1.03) and GAPDH (M=1.005) were less stable. RPS13 (M=1.913) appeared to be highly unstable.Set of three the most stable genes has been used to compare ghrelin (GHRL) expression in two different regions (diverticulum ventriculi and fundus ventriculi) of porcine stomach. Ghrelin mRNA was highly expressed in both regions of stomach, however level of GHRL mRNA was approximately 5-fold higher in fundus ventriculi than in diverticulum ventriculi.

The Drosophila-like homolog 1 (DLK1), a transmembrane signal protein similar to other members of the Notch/Delta/Serrate family, regulates the differentiation process in many types of mammalian cells. Callipyge sheep and DLK1 knockout mice are excellent examples of a fundamental role of the gene encoding DLK1 in muscle growth and fat deposition. DLK1 is located within co-regulated imprinted clusters (the DLK1/DIO3 domain), along with other imprinted genes. Some of these, e.g. the RNA coding MEG3 gene, presumedly interfere with DLK1 transcription. The aim of our study was to analyze DLK1 and MEG3 gene expression in porcine tissues (muscle, liver, kidney, heart, brain stem) during postnatal development. The highest expression of both DLK1 and MEG3 variant 1 (MEG3 var.1) was observed in the brain-stem and muscles, whereas that of MEG3 variant 2 (MEG3var.2) was the most abundant in muscles and the heart. During development (between 60 and 210 days of age) expression of analyzed genes was down-regulated in all the tissues. An exception was the brain- stem, where there was no significant change in MEG3 (both variants) mRNA level, and relatively little decline (2-fold) in that of DLK1 transcription. This may indicate a distinct function of the DLK1 gene in the brain-stem, when compared with other tissues.