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Background Epigenetic variation may result from selection for complex traits related to metabolic processes or appear in the course of adaptation to mediate responses to exogenous stressors. Moreover epigenetic marks, in particular the DNA methylation state, of specific loci are driven by genetic variation. In this sense, polymorphism with major gene effects on metabolic and cell signaling processes, like the variation of the ryanodine receptors in skeletal muscle, may affect DNA methylation. Methods DNA-Methylation profiles were generated applying Reduced Representation Bisulfite Sequencing (RRBS) on 17 Musculus longissimus dorsi samples. We examined DNA methylation in skeletal muscle of pig breeds differing in metabolic type, Duroc and Pietrain. We also included F2 crosses of these breeds to get a first clue to DNA methylation sites that may contribute to breed differences. Moreover, we compared DNA methylation in muscle tissue of Pietrain pigs differing in genotypes at the gene encoding the Ca2+ release channel (RYR1) that largely affects muscle physiology. Results More than 2000 differently methylated sites were found between breeds including changes in methylation profiles of METRNL, IDH3B, COMMD6, and SLC22A18, genes involved in lipid metabolism. Depending on RYR1 genotype there were 1060 differently methylated sites including some functionally related genes, such as CABP2 and EHD, which play a role in buffering free cytosolic Ca2+ or interact with the Na+/Ca2+ exchanger. Conclusions The change in the level of methylation between the breeds is probably the result of the long-term selection process for quantitative traits involving an infinite number of genes, or it may be the result of a major gene mutation that plays an important role in muscle metabolism and triggers extensive compensatory processes. Electronic supplementary material The online version of this article (10.1186/s12864-019-5880-1) contains supplementary material, which is available to authorized users.

Lists of IPA canonical pathways significantly enriched for differentially expressed genes in non-depleted (0) and depleted RNA samples (protocol variants A and B) from pathogen-challenged and non-challenged udder quarters. Pathways are classified for being observed across all protocols (0, A or B) or shared between two or all three protocols. (PDF 214 kb)

Distribution of gene expression level (FPKM) for the depleted and non-depleted RNA samples from non-challenged udder samples. 0, A, and B represent the non-depleted or the depleted RNA samples (protocol variant A and B), respectively. (PDF 283 kb)

IGV screen shot of the read distribution across the PAEP gene for the E. coli challenged udder sample from cow 2. O, A and B represent the non-depleted or the depleted RNA samples (protocol variant A and B), respectively. Exon 6 is zoomed in for better demonstration of the position of the antisense capture oligonucleotide (see red line). (PDF 235 kb)

Commercial and customized microarrays are valuable tools for the analysis of holistic expression patterns, but require the integration of the latest genomic information. This study provides a comprehensive workflow implemented in an R package (rePROBE) to assign the entire probes and to annotate the probe sets based on up-to-date genomic and transcriptomic information. The rePROBE R package is freely available at https://github.com/friederhadlich/rePROBE. It can be applied to available gene expression microarray platforms and addresses both public and custom databases. The revised probe assignment and updated probe-set annotation were applied to commercial microarrays available for different livestock species, i.e. ChiGene-1_0-st (Gallus gallus, 443,579 probes; 18,530 probe sets), PorGene-1_1-st (Sus scrofa, 592,005; 25,779) and BovGene-1_0-st (Bos taurus, 530,717; 24,759) as well as human (Homo sapiens, HuGene-1_0-st) and mouse (Mus musculus, HT_MG-430_PM) microarrays. Using current specie-specific transcriptomic information (RefSeq, Ensembl and partially non-redundant nucleotide sequences) and genomic information, the applied workflow revealed 297,574 probes for chickens (pig: 384,715; cattle: 363,077; human: 481,168; mouse: 324,942) assigned to 15,689 probe sets (pig: 21,673; cattle: 21,238; human: 23,495; mouse: 32,494). These are representative of 12,641 unique genes that were both annotated and positioned (pig: 15,758; cattle: 18,046; human: 20,167; mouse: 16,335). Additionally, the workflow collects information on the number of single nucleotide polymorphisms (SNPs) within respective targeted genomic regions and thus provides a detailed basis for comprehensive analyses such as quantitative trait locus (eQTL) expression studies to identify quantitative and functional traits.

Definitions of traits, number of samples, means, standard deviations for each of the haematological and biochemical traits.

Additional file 7: Table S2. Genes in the region between 10 and 25 Mb on BTA5 that were expressed in the pigmented skin transcriptome of RTS-affected and wild type animals.

Non-classical MHC class I alleles: multiple alignment of amino acids. Alignment of the predicted amino acid sequences for classical MHC class I derived from the nucleotide sequence of alleles expressed in the data set. Dots: indicate identity to the first allele in the list; dashes: represent gaps compared to the first allele in the list; asterisk: denotes a stop codon. (DOCX 33 kb)

Additional file 1: Figure S1. Phenotype scoring of RTS associated hair conformation and pigmentation traits. Phenotypes and corresponding scores for HS: HS 1: wild type, straight hair, HS 2: hair with fluffy, smooth structure, HS 3: curly hair with a mildly frizzy/wiry structure, HS 4: coarse, frizzy/wiry hair, HS 5: very coarse/wiry hair, HS 6: extremely coarse/wiry HS. Phenotypes and corresponding scores for hair density (HD): HD 1: wild type, full, thick, long hair covering the whole body as well as in the ears and around the eyes, HD 2: scarce hair at the trunk and/or few, short hair in the ears and around the eyes, HD 3: short and sparse hair in the ears and around the eyes as well as thin coat, HD 4: without eye lashes and/or hair in the ears and with very thin hair coat and/or bald areas. Phenotypes and corresponding scores for hair length variations between pigmented and unpigmented areas of spotted animals (HLV): HLV 1: wild type, equally long hair in pigmented and unpigmented sections. HLV 2: pigmented hair mildly shorter than unpigmented hair, HLV 3: pigmented hair moderately shorter than unpigmented hair, HLV 4: pigmented hair extremely shorter than unpigmented hair. Animals without white spotting marks were scored according to their phenotype for RT. Phenotypes and corresponding scores for RTS classification phenotype: RT 1: wild type without any RTS-associated hair conformation malformation, RT 2: mild HLV and/or short hair in ears and short, thin eye lashes, reduced tail switch. RT 3: sparse, coarse hair, moderate HLV, few hairs in ears/around eyes and/or few hair in eye lashes, tail switch only rudimentary. RT 4: very sparse coat, extreme HLV, scarce/wiry hair in pigmented areas, no hair in ears, barely/no eyelashes, extremely visible skin folds at face/throat, bald areas surrounding the eyes/in the ears, missing tail switch. Status of tail switch was only informative, if the end of the tail was pigmented. Phenotypes and corresponding scores for coat colour dilution (Dilu) in pigmented coat sections on a eumelanic (black, brown) and pheomelanic (red, yellow) colour background. Dilu 1: undiluted coat colour, black or red-brown; Dilu 2: mild colour dilution with dark grey colour for the black/brown background and orange/caramel for the red background, Dilu 3: mouse grey/bright grey (black background) or yellow/beige (red background), Dilu 4: extremely diluted, with crème and nearly white pigmentation.

Additional file 4: Figure S4. Test statistic of the linkage analysis on BTA20 for the half-sib family sire 2. Dilu: level of coat colour dilution in pigmented areas, HLV: level of hair lengths variation between pigmented and unpigmented coat, HS: hair structure, HD: hair density, RT: RTS classification phenotype.

Classical MHC class I alleles: multiple alignment of amino acids. Alignment of the predicted amino acid sequences for classical MHC class I derived from the nucleotide sequence of alleles expressed in the data set. Dots: indicate identity to the first allele in the list; dashes: represent gaps compared to the first allele in the list; asterisk: denotes a stop codon; yellow labelled amino acids: uniquely occurring amino acid in an allele at a specific amino acid position in comparison to the other alleles. (DOCX 34 kb)

Examples of read coverage for single MHC class I alleles in individual cows. A: Read coverage of the allele BoLA-2*1601 in the full-sibs SEG11 and SEG31. SEG11 shows complete coverage of reads with the option of zero mismatches (0mm) per read. SEG31 displays incomplete coverage of reads even with the option of three mismatches (3mm) per read indicating that the allele BoLA-2*1601 is not expressed in this animal. B: Read coverage of the previously published allele BoLA-2*00601 in cow SEG12. Alignment allowing for three (3mm) or zero mismatches (0mm) per read in order to identify novel variant alleles. (PDF 220 kb)

Additional file 2: Figure S2. Test statistic for hair conformation and pigmentation traits on BTA5 from the F2 full sib design fitting sex as fixed effect in an additive-dominant model. HLV: level of hair length variation between pigmented and unpigmented body areas, HS: hair structure, HD: hair density, RT: RTS classification phenotype. The test statistic for Dilu is not shown due to excessively high F values (>500), which exceed the scale of the y axis.

Allele specific primers used for experimental MHC class I allele confirmation by Sanger sequencing. The table lists the oligonucleotide primers used for amplification and sequencing of MHC class I sequences. (DOCX 16 kb)

MHC class I and SNP haplotype tracking in a F2 full-sib family. Detection of paternally and maternally inherited MHC class I haplotypes. Bluish strands: paternal chromatids; reddish strands: maternal chromatids; rs-number: Reference SNP cluster ID; green boxes: SNP alleles; yellow boxes: classical MHC class I alleles; grey boxes: non-classical MHC class I alleles; alleles deduced from genotypes of progeny are indicated in italic. (PDF 286 kb)

Positions of primers used for MHC class I allele amplification and sequencing. For primer sequences see Additional file 4 (Table S2). (PDF 121 kb)

Additional file 3: Figure S3. Contour plot showing the test statistic for coat colour dilution (Dilu) on BTA5 for a 2-QTL model in a F2 full sib design. The x and y axes represent chromosomal positions for QTL1 and QTL2, respectively. The z axis indicates the F value of the QTL test statistic for a 2-QTL model.

Non-classical MHC class I alleles: multiple alignment of amino acids. Alignment of the predicted amino acid sequences for classical MHC class I derived from the nucleotide sequence of alleles expressed in the data set. Dots: indicate identity to the first allele in the list; dashes: represent gaps compared to the first allele in the list; asterisk: denotes a stop codon. (DOCX 33 kb)

Examples of read coverage for single MHC class I alleles in individual cows. A: Read coverage of the allele BoLA-2*1601 in the full-sibs SEG11 and SEG31. SEG11 shows complete coverage of reads with the option of zero mismatches (0mm) per read. SEG31 displays incomplete coverage of reads even with the option of three mismatches (3mm) per read indicating that the allele BoLA-2*1601 is not expressed in this animal. B: Read coverage of the previously published allele BoLA-2*00601 in cow SEG12. Alignment allowing for three (3mm) or zero mismatches (0mm) per read in order to identify novel variant alleles. (PDF 220 kb)

Positions of primers used for MHC class I allele amplification and sequencing. For primer sequences see Additional file 4 (Table S2). (PDF 121 kb)

Allele specific primers used for experimental MHC class I allele confirmation by Sanger sequencing. The table lists the oligonucleotide primers used for amplification and sequencing of MHC class I sequences. (DOCX 16 kb)

Overview of samples included in the analysis regarding BNP status and statistics of read alignments. (DOCX 19 kb)

Additional file 6: Table S1. Genotypes of wild type and RTS-affected F2 individuals for SNP BTA-74302-no-rs. F2_wt: F2 individual with wild type phenotype and F2_RT: F2 individual with RTS phenotype.

Additional file 5: Figure S5. Manhattan plots of a whole-genome association study for the traits: hair length variation (HLV), hair structure (HS), hair density (HD) and coat colour dilution (Dilu) in the linear mixed model and for coat colour dilution (Dilu) and hair length variation (HLV) in the case–control design. The upper horizontal line represents the genome-wide significance threshold q = 0.05, the lower horizontal line represents the nominal p value = 0.05. Only F1, F2 and BC individuals with a E D/* and Dc/dc + genotype at the extension and the dilution locus were included.

MHC class I and SNP haplotype tracking in a F2 full-sib family. Detection of paternally and maternally inherited MHC class I haplotypes. Bluish strands: paternal chromatids; reddish strands: maternal chromatids; rs-number: Reference SNP cluster ID; green boxes: SNP alleles; yellow boxes: classical MHC class I alleles; grey boxes: non-classical MHC class I alleles; alleles deduced from genotypes of progeny are indicated in italic. (PDF 286 kb)