Chromaspro v1

Gene fragments sequences were corrected with Chromas Pro v1.33 software (Technelysium) and aligned using either ClustalX [58 (link)] or muscle as implemented in MEGA6 [59 (link)]. Alignments were corrected manually under Genedoc software [60 ] when necessary, and recombination in the datasets was evaluated using the Recombination Detection Program (RDP) v4.35 [61 (link)]. Recombination was inferred as true when at least two programs of RDP (RDP, Geneconv, Bootscan, MaxChi, Chimaera, Siscan or 3Seq) could detect the same event. Single marker phylogenies were built with MEGA6 using either Neighbor-Joining (with Kimura 2 distance correction method [62 (link)] or Maximum likelihood analyses with 1000 bootstrap replicates. A Bayesian phylogenetic tree was built from the concatenate of all 5 gene fragment alignments, using a Markov chain Monte-Carlo (MCMC) analysis. The priors used for the MCMC analysis were based on a GTR+I+G model with 6 types of substitutions, with parameters estimated by maximum likelihood with Modeltest 3.6 [63 (link)].

Six pairs of primers for pig NR1H3 (NM_001101814) were designed using Primer Premier 5.0 software (PREMIER Biosoft International, CA, USA). Amplicons of the primers covered all exons of the gene (Table 1 and Fig. 1). PCR products amplified from 10 samples of each group were pooled and sequenced to identify SNPs using Chromas Pro v.1.33 (Technelysium Pty Ltd, Helensvale, Australia) and DNAMAN6.0 software (Lynnon, Pointe-Claire, QC, Canada).Table 1

Six pairs of primers used for SNP identification in pig NR1H3

Name	Sequences (5′ to 3′)	Amplicon region	Amplicon size (bp)	annealing temperature
NR1H3-1	F: TCCCACTCTGAGGTTCTTTTT	Exon 1	235	58 °C
	R: CTTACGGACCTGACACTGGA
NR1H3-2	F:GCCAGGAAAGCCTTAGCACA	Exon 2	361	60 °C
	R: AGGAGGCAAGCAACAGCAAG
NR1H3-3	F:CTGAGACCCCCCCTGTGC	Exon 3	259	59 °C
	R: GCCCCTACCTCCTCCAAATC
NR1H3-4	F: GAACATTAAGCCTCTTCCAT	Exon 4	347	54 °C
	R: TTCCCTCTTTCCTATCAGC
NR1H3-5	F: ATCTCTTCCTTGTCTTTACCC	Exon 5, exon 6 and exon 7	889	56 °C
	R: CAATCCCTTTGTGATCTCAG
NR1H3-6	F: AGCAGTTTCCTCAGTTGAGC	Exon 8, exon 9 and exon 10	1029	56 °C
	R: AGGGTCAGTACCGTCTTCAC

Fig. 1

Structure of the pig NR1H3 gene and the positions of primers used for SNP identification. The thick black lines represent introns; the grey blocks represent exons of the NR1H3 gene; the thin black lines represent positions of amplicons. Pig total DNA was used as PCR templates for the NR1H3-1, NR1H3-2, NR1H3-3, NR1H3-4, NR1H3-5 and NR1H3-6 primers

All bacterial isolates were identified by the Sanger sequencing of the 16S rRNA gene. DNA was obtained either by simple freeze-and-thaw procedure as described earlier [36 (link)] or mechanical cell lysis in nuclease-free water with a Retsch mill model MM200 (Retsch GmbH, Haan, Germany). If both methods failed, the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) was used following the manufacturer’s guidelines. The 16S rRNA genes of the obtained DNA were amplified by PCR using primers Eub27f and 1387r in a previously published cycling protocol [90 (link)]. PCR products with correct fragment length were sent for Sanger sequencing at LGC Genomics (Berlin, Germany) using the primer 1387R. Resulting sequences were analysed and trimmed with ChromasPro V1.33 (Technelysium Pty Ltd., South Brisbane, Australia) and compared to the National Center for Biotechnology Information (NCBI) nucleotide database using the nucleotide BLAST function [91 (link)]. All sequences were submitted to NCBI Genbank. Isolates returning identical closest relatives after nucleotide BLAST comparison with (i) the full database and (ii) type strains were excluded from further analysis, resulting in a selection of 16 different isolates for further analysis.