RIP screen
Ten TLR genic and their flanking sequences (5 kb 5’ flank and 3 kb 3’ flank) were obtained from fifteen assembled non-reference genomes (Landrace, Yorkshire, Pietrain, Berkshire, Hampshire, Cross-breed of Yorkshire/Landrace/Duroc, Wuzhishan, Tibetan, Rongchang, Meishan, Bamei, Bama, and Jinhua, Goettingen, and Ellegaard Gottingen minipigs) and one reference genome (Duroc) deposited in NCBI database (https://www.ncbi.nlm.nih.gov/)to screen the structural variations by alignment with ClustalX program. Large structural variations (more than 50 bp) were remained for further analysis. Retrotransposon (SINE, LINE, and ERV) insertions were annotated by RepeatMasker (http://www.repeatmasker.org/) with a customer constructed library[51]. Promoters were predicted in http://www.cbs.dtu.dk/services/Promoter/.Transcription factor binding sites were searched at https://bip.weizmann.ac.il/toolbox/seq_analysis/promoters.html#databases and CPGs islands were recognized in https://www.novopro.cn/tools/cpg_islands.html?tdsourcetag=s_pctim_aiomsg. The predicted large structural variations (more than 50 bp) overlapping with retrotransposon (SINE, LINE, and ERV) insertions were designated as RIPs. These RIPs were further evaluated in seven Chinese native pig breeds (Diannan small-ear Pigs, Erhualian, Wuzhishan, Bama, Tibet, Meishan, Fengjing Pigs ), three commercial pig breeds(Duroc, Landrace, Yorkshire), one cross breed (Sujiang) and wild boars (from Anhui province,Fujian province and Heilongjiang province) by PCR amplification(Vazyme, Nanjing, China). Fort each breed, two pooled DNA samples, each contained at least three animal samples, were used. Origins of pigs and primers used for RIP evaluation was listed in Table S2 and Table S3. All obtained RIPs were further confirmed by TA cloning (Tiangen, Beijing, China)following the manufacturer's instructions and sequencing.
RIP Genotyping
Totally, twelve breeds (Duroc, Landrace, Yorkshire, Erhualian, Meishan, Fengjing, Bama, Tibetan, Wuzhishan, Diannan small-ear, Sujiang, and Sicilian black) were used to genotype the RIP distribution. Among these breeds, Duroc, Landrace, and Yorkshire are three lean type breeds, Sicilian black, Erhualian, Meishan, and Fengjing are five fat type pigs and Bama, Diannan small-ear, Wuzhishan, and Tibetan are four miniature pigs. Erhualian, Meishan, Fengjing, Bama, Tibetan, Diannan small-ear, Wuzhishan, are Chinese native breeds, while Sujing is a middle-type pigs, which is a new cross breed with 62.5% Duroc, 18.75% Jiangquhia, and 18.75% Fengjing bloods. Sicilian black pigs are Italian native breeds. The genotype and the allele frequencies were calculated, and Hardy-Weinberg equilibrium were tested using X2. Polymorphic information content(PIC)was calculated by the formula:
Dual luciferase reporter assay
The predicted core promoter regions (NC_010450.4, 30171883-30172870) of TLR6 with and without the ERV insertion was cloned from the Sujiang genomic DNA (primers were listed in Table S2), and verified by sequencing. Then the clones were inserted into the pGL3-Basic vectors (Ambion, Austin, American) to construct TLR6ERV+-Luc(En) vector and TLR6ERV-Luc(En) Vector. In addition, β-globin and Oct4 mini-promoter were cloned from pEDV-β-globin-GFP and pTol2-Oct4-mCherry vector, respectively[63] and inserted into the pGL3-Basic vectors with or without the 192bp ERV for enhancer activity evaluation. A total of 2×104 PK15 and Hela cells were plated in a 24-well plates and transfected with the plasmids by using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, American). After 48 hours, cells were collected for luciferase activity evaluation by using the dual luciferase reporter system (Promega, Madison, American) according to the manufacturer’s protocol.
Expression analysis
The Sujing piglets were genotyped and five piglets for each genotype (ERV+/+, ERV+/-, and ERV-/-) were selected and slaughtered to collect tissue samples at 30 days. The mRNAs were extracted and cDNAs were prepared according to the manufacturer’s protocol by using TAKARA Kit(Takara,Tokyo,Japan). Then, the mRNA expressions of TLR6, TLR1, MyD88, RAC1, TOLLip, TIRAP, TNFα, IL-6, and IL-8 mRNA were evaluated by quantitative real-time PCR (qPCR) using the 7900HT Fast Real-Time PCR System (Applied Biosystems, new York, American) in a total volume of 20 μl containing SYBR mix (10 μl), primers (4 ng), and cDNA sample (50 ng) according to the manufacturer’s instructions (Takara,Tokyo, Japan). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control to normalize the target gene expression in four different tissues including liver, lung, kidney, and spleen. All gene expression was measured using the 2− ΔΔCt method. PCR products were run on 1.5% ethidium bromide-stained agarose gels and confirmed using melting curve analyses to assess PCR product quality.
Measurement of serum TNFα, IL-6, and IL8 by enzyme linked immunosorbent assay (ELISA)
TNFα, IL-6, IL8 concentration in serum of 30-day Sujian piglets were measured using the Pig TNFα, IL-6, and IL8 ELISA Kit (Solarbio Science, Beijing, China) by following the manufacturer’s protocol. All measurements were performed in 3 replicates, and mean values were used for statistical analysis.
Statistical analysis
Experimental results were processed by statistical SPSS17.0 software package (SPSS, Chicago, USA) using one-way analysis of variance with Tukey's post hoc test, and the data were expressed as mean ± S.D.
Animal welfare
All treatments and protocols involving animals in this study was been strictly done in accordance with the guidelines of the Animal Experiment Ethics Committee of Yangzhou University (approval number: YZUDWSY2018-12).