The genetic diversity of the GH/IGF axis genes and their association with the variation of gene expression and phenotypic traits, principally represented by SNPs, have been extensively reported. Nevertheless, the impact of retrotransposon insertion polymorphisms (RIPs) on the GH/IGF axis gene activity has not been reported. In the present study, bioinformatic prediction and PCR verification were performed to screen RIPs in four GH/IGF axis genes (GH, GHR, IGF1 and IGF1R). In total, five RIPs, including one SINE RIP in intron 3 of IGF1, one L1 RIP in intron 7 of GHR, and three SINE RIPs in intron 1, intron 5 and intron 9 of GHR, were confirmed by PCR, displaying polymorphisms in diverse breeds. Dual luciferase reporter assay revealed that the SINE insertion in intron 1 of GHR significantly repressed the GHR promoter activity in PK15, Hela, C2C12 and 3T3-L1 cells. Furthermore, qPCR results confirmed that this SINE insertion was associated with a decreased expression of GHR in the leg muscle and longissimus dorsi, indicating that it may act as a repressor involved in the regulation of GHR expression. In summary, our data revealed that RIPs contribute to the genetic variation of GH/IGF axis genes, whereby one SINE RIP in the intron 1 of GHR may decrease the expression of GHR by acting as a repressor.
Short interspersed nuclear elements (SINEs), one type of retrotransposon, are considered to be ideal molecular markers due to their wide distribution in the genome, high copy number, and high polymorphism. Preliminary studies have identified more than 35,000 SINE-retrotransposon insertion polymorphisms (RIPs) in the pig genome. In this study, 18 SINE-RIPs were used to evaluate the genetic variation and population structure of seven native pig populations and two crossbreeds in the Jiangsu Province of China. Two commercial pig breeds (Duroc and Large White) and one Italian native breed (Sicilian Black pig) were selected as the control. The results showed that all 18 SINE-RIPs were polymorphic among these pigs. The Jiangsu native pig populations (Erhualian, Fengjing, Middle Meishan, Mi, Shawutou, Small Meishan, and Huai) were shown to be more polymorphic than the crossbreeds (Sushan and Sujiang) and external breeds (Sicilian Black pig, Large White, and Duroc) based on the expected heterozygosity and polymorphic information content values. Some native pigs, including Small Meishan, Mi, Middle Meishan, and Erhualian, had a higher degree of inbreeding according to the FIS values. Based on the neighbor-joining tree, all of the Jiangsu native pig populations formed one branch, while the three external pig breeds formed the other branches, with the two crossbreeds containing more than 50% external pig ancestry. The Huai pigs were independent of the other Jiangsu native pigs but shared a common ancestor with Sujiang and Mi. The results provide a new perspective on the population structure of these native pig breeds and will assist with the conservation and utilization of Chinese native pigs.
Retrotransposon is an important component of the mammalian genome. Previous studies have shown that the expression of protein-coding genes was affected by the insertion of retrotransposon into the proximal genes, and the phenotype variations would be related to the retrotransposon insertion polymorphisms (RIPs). In this study, leptin (LEP), leptin receptor (LEPR), and leptin receptor overlapping transcript (LEPROT), which play important roles in the regulation of fat synthesis and body weight, were screened to search for the RIPs and their effect on phenotype and gene expression, as well as to further study the function of the insertion. The results showed that three RIPs located in intron 1 of LEPROT and intron 2 and 21 of LEPR were identified, and they were all SINEA1, which was one type of retrotransposon. The SINE insertion at the LEPROT was the dominant allele in native pig breeds. The age of 100 kg body weight of SINE+/+ Large White individuals was significantly higher than those of SINE+/− and SINE−/− individuals (p < 0.05). The LEPROT gene expression in the liver and suet of 30-day-old SINE−/− Sujiang piglets were significantly higher than those of SINE+/+ and SINE+/− piglets (p < 0.01). The dual-luciferase reporter gene assay showed that SINE insertion in PK15 and 3T3-L1 cells significantly reduced the promoter activity of the LEPROT gene (p < 0.01). Therefore, SINE insertion can be a repressor to reduce the expression of LEPROT and could be a useful molecular marker for assisted selection of growth traits in pig breeding.
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