Journal of Applied Genetics

, Volume 59, Issue 2, pp 231–241 | Cite as

Linkage disequilibrium and functional analysis of PRE1 insertion together with SNPs in the promoter region of IGFBP7 gene in different pig breeds

  • Qingyan Wu
  • Hao Yu
  • Wenzhen Wei
  • Yunyun Cheng
  • Shan Huang
  • Hongyu Shi
  • Songcai Liu
  • Jichao Xia
  • Hongyao JiaEmail author
  • Linlin HaoEmail author
Animal Genetics • Original Paper


Polymorphisms in regions upstream of transcription initiation site may modify the transcriptional activity of target genes by changing promoter activity. This study aims to determine whether or not polymorphisms at porcine IGFBP7 promoter region affect gene expression. In this study, eight SNPs and one PRE1 insertion in this region were first confirmed. The PRE1 insertion was widespread in 20 Chinese indigenous breeds, but was not observed in three commercial breeds. A perfect linkage disequilibrium, consisting of six of those SNPs and a PRE1, was observed with two haplotypes (h1 and h2) in five pig breeds. The h1 haplotype had an overwhelming superiority distribution in Large White, Landrace, and Bama mini-pig; in turn, the h2 only existed in the PRE1 presence breeds. As the haplotypes and PRE1 were located at gene promoter regions, we further investigated the transfection of plasmids with three different fragments of IGFBP-7 promoter region (H1, H2, RF). The CMV promoter of the pEGFP-N1 was substituted by these three different fragments, respectively. Different transcriptional and translational activities of EGFP in PK-15 cells were observed in these three recombinant plasmids by quantitative real-time PCR and flow cytometric analysis. The results indicated that H1 had the higher transcriptional and translational activities of EGFP as compared to the H2 (P < 0.05, P < 0.05). As compared to the RF group, EGFP mRNA expression level was significantly higher in H1 groups (P < 0.05). The IGFBP-7 promoter polymorphisms detected in this study may be important functional variants and potential genetic markers for pig population genetic study.


PRE1 SNP LD Haplotype Promoter activity 



This work was supported by the National Natural Science Foundations of China (31672514 and 31772699) and Science and Technology Development Plan of Jilin Province (20170101024JC).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

13353_2018_430_Fig6_ESM.gif (61 kb)
Fig. S1

Schematic representations of recombinant plasmids and fragments. (A) The CMV promoter of pEGFP-N1 was removed by using Ase I and Xoh I restriction enzymes and replaced by the different porcine IGFBP7 promoter haplotypes or recombinant fragments, respectively. The difference between H1/H2 haplotype and h1/h2 haplotype was that the H1 and H2 haplotypes contained the SNP of g.-318 A > G, this is because the genotype of g.-318 A > G was GG in all the PRE1 presence individuals, while only AA and AG genotypes were found in the PRE1 absence individuals as well as the AA genotype was the dominant haplotype. (B) Over lapextension PCR amplification of recombinant fragment (RF). (GIF 60 kb)

13353_2018_430_MOESM1_ESM.tif (12.4 mb)
High resolution image (TIFF 12707 kb)
13353_2018_430_Fig7_ESM.gif (542 kb)
Fig. S2

The distribution of the PRE1 insertion +/− polymorphism in the promoter region of IGFBP7 in individuals from eight pig breeds. (Lane M: 2000 bp DNA marker, Lane 1–3: Tibetan pig, Lane 4–6: Northeast wild boar, Lane 7–9: Jinhua pig, Lane 10: Bama mini-pig, Lane 11: Luchuan pig . Lane 12: Large White, Lane 13: landrace, Lane 14: Duroc, −/− homozygous for the absence of the insertion (1057 bp); +/+ homozygous for the presence of the insertion (1356 bp); +/− heterozygous for the presence/absence of the insertion (1356 bp and 1057 bp). (GIF 541 kb)

13353_2018_430_MOESM2_ESM.tif (53.1 mb)
High resolution image (TIFF 54369 kb)
13353_2018_430_Fig8_ESM.gif (167 kb)
Fig. S3

Inserting the PRE1 SINE into the porcine IGFBP-7 promoter region generates a novel CpG island. Islands highlighted by light blue background were identified by the MethPrimer software. Red bars show CpG dinucleotides. (A) The CpG island prediction of haplotype H1. CpG island (208 bp): nucleotides −264 to −57. (B) The CpG island prediction of haplotype H2. CpG island (277 bp): nucleotides −332 to −56; CpG island (275 bp): nucleotides −911 to −637. The inverted insertion of a PRE1 SINE located in the nucleotide position from −985 to −687. The flanking set of 15 bp short intact direct repeats, AAAAGCCCGTTATTC, one being part of the PRE1 insertion, the other being adjacent to the PRE1 insertion, is displayed by gray arrows. (GIF 166 kb)

13353_2018_430_MOESM3_ESM.tif (16.8 mb)
High resolution image (TIFF 17202 kb)
13353_2018_430_MOESM4_ESM.docx (21 kb)
ESM 1 (DOCX 21 kb)


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Copyright information

© Institute of Plant Genetics, Polish Academy of Sciences, Poznan 2018

Authors and Affiliations

  • Qingyan Wu
    • 1
  • Hao Yu
    • 1
  • Wenzhen Wei
    • 1
  • Yunyun Cheng
    • 1
  • Shan Huang
    • 1
  • Hongyu Shi
    • 1
  • Songcai Liu
    • 1
    • 2
  • Jichao Xia
    • 3
  • Hongyao Jia
    • 4
    Email author
  • Linlin Hao
    • 1
    Email author
  1. 1.College of Animal ScienceJilin UniversityChangchunChina
  2. 2.Five-Star Animal Health Pharmaceutical FactoryChangchunChina
  3. 3.Melbourne School of Population and Global HealthThe University of MelbourneCarltonAustralia
  4. 4.First hospital of Jilin UniversityChangchunChina

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