[논문]Whole-genome sequence association study identifies cyclin dependent kinase 8 as a key gene for the number of mummified piglets

Pingxian, Wu; Dejuan, Chen; Kai, Wang; Shujie, Wang; Yihui, Liu; Anan, Jiang; Weihang, Xiao; Yanzhi, Jiang; Li, Zhu; Xu, Xu; Xiaotian, Qiu; Xuewei, Li; Guoqing, Tang

doi:10.5713/ab.22.0115

Whole-genome sequence association study identifies cyclin dependent kinase 8 as a key gene for the number of mummified piglets 원문보기

Animal bioscience, v.36 no.1, 2023년, pp.29 - 42

Pingxian, Wu (Chongqing Academy of Animal Sciences) , Dejuan, Chen (Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University) , Kai, Wang (Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University) , Shujie, Wang (Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University) , Yihui, Liu (Sichuan Animal Husbandry Station) , Anan, Jiang (Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University) , Weihang, Xiao (Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University) , Yanzhi, Jiang (College of Life Science, Sichuan Agricultural University) , Li, Zhu (Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University) , Xu, Xu (Sichuan Animal Husbandry Station) , Xiaotian, Qiu (National Animal Husbandry Service) , Xuewei, Li (Farm Animal Genetic Resources Expl) , Guoqing, Tang

Abstract ▼ AI-Helper

Objective: Pigs, an ideal biomedical model for human diseases, suffer from about 50% early embryonic and fetal death, a major cause of fertility loss worldwide. However, identifying the causal variant remains a huge challenge. This study aimed to detect single nucleotide polymorphisms (SNPs) and candidate genes for the number of mummified (NM) piglets using the imputed whole-genome sequence (WGS) and validate the potential candidate genes. Methods: The imputed WGS was introduced from genotyping-by-sequencing (GBS) using a multi-breed reference population. We performed genome-wide association studies (GWAS) for NM piglets at birth from a Landrace pig populatiGWAS peak located on SSC11: 0.10 to 7.11 Mbp (Top SNP, SSC11:1,889,658 bp; p = 9.98E-13) was identified in cyclin dependent kinase on. A total of 300 Landrace pigs were genotyped by GBS. The whole-genome variants were imputed, and 4,252,858 SNPs were obtained. Various molecular experiments were conducted to determine how the genes affected NM in pigs. Results: A strong GWAS peak located on SSC11: 0.10 to 7.11 Mbp (Top SNP, SSC11:1,889,658 bp; p = 9.98E-13) was identified in cyclin dependent kinase 8 (CDK8) gene, which plays a crucial role in embryonic retardation and lethality. Based on the molecular experiments, we found that Y-box binding protein 1 (YBX1) was a crucial transcription factor for CDK8, which mediated the effect of CDK8 in the proliferation of porcine ovarian granulosa cells via transforming growth factor beta/small mother against decapentaplegic signaling pathway, and, as a consequence, affected embryo quality, indicating that this pathway may be contributing to mummified fetal in pigs. Conclusion: A powerful imputation-based association study was performed to identify genes associated with NM in pigs. CDK8 was suggested as a functional gene for the proliferation of porcine ovarian granulosa cells, but further studies are required to determine causative mutations and the effect of loci on NM in pigs.

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표/그림 (9)

표 Table 1. Summary statistics of the analyzed phenotypes in Landrace pigs
그림 Figure 1. Average imputation accuracy against minor allele frequency (MAF). The x-axis is the MAF range from 0 to 0.5, and the y-axis is average imputation accuracy denoted by the Beagle R2. Single nucleotide polymorphisms (SNPs) were divided into bins of SNPs with common MAF. MAF, minor allele frequency; SNPs, single nucleotide polymorphisms.
그림 Figure 2. Genome-wide association studies (GWAS) results for the number of mummified (NM) in Landrace pigs. Each dot on this figure corresponds to an SNP within the dataset, while the horizontal red and blue lines denote the genome-wide significance (1.18E-08) and suggestive significance threshold (2.35E-07), respectively. GWAS, genome-wide association studies; NM, the number of mummified; SNP, single nucleotide polymorphisms.
그림 Figure 3. Identification and characterization of CDK8 gene. (a) Tissue expression profile of CDK8 gene. Each group contained at least 3 biologic replicate samples and results are shown as mean±standard deviation. (b) Localization of CDK8 gene in ovarian tissue (40×, 200×). CDK8, cyclin dependent kinase 8.
그림 Figure 4. Identifying informative transcription factors of CDK8 gene. (a) Wayne diagram of the identified transcription factors. (b) Histogram of transcription factor iBAQ value. (c) Histogram of transcription factor score. CDK8, cyclin dependent kinase 8; iBAQ, intensity based absolute quantification.
그림 Figure 5. Exploring the biological functions for YBX1 gene. (a) The interaction map of 51 interacting genes, (b) GO analysis for 51 interacting genes, (c) Pathway analysis for 51 interacting genes. YBX1, Y-box binding protein 1; GO, gene ontology.
그림 Figure 6. The mRNA expression level after YBX1 treated with overexpression and inhibitor in porcine ovarian granulosa cells. (a) Calculation of porcine ovarian granulocyte YBX1 overexpression and inhibition efficiency. (b-f) The mRNA expression level of YBX1, CDK8, BCL-2, Bax, and BCL-2/Bax gene in overexpression and inhibitor groups. Each group contained at least 3 biologic replicate samples and results are shown as mean±standard deviation. YBX1, Y-box binding protein 1; CDK8, cyclin dependent kinase 8; BCL-2, BCL2 apoptosis regulator; Bax, BCL2 associated X, apoptosis regulator; BCL-2/Bax, BCL2 apoptosis regulator/BCL2 associated X, apoptosis regulator. * Indicates p<0.05, ** indicates p<0.01.
그림 Figure 7. Effect of YBX1 on apoptosis porcine ovarian granulosa cells after interfered and or overexpression. Representative of three experimental replicates with porcine ovarian granulosa cells. Flow cytometry was formed using fluorescer conjugated Annexin V-FITC and PI double staining. YBX1, Y-box binding protein 1; V-FITC, fluorescently labeled Annexin V; PI, propidium iodide.
그림 Figure 8. Effect of YBX1 on the protein expression levels of SMAD3, P-SMAD3, and β-actin after interfered and overexpression treatment. (a-b) The gray degree values analysis of SMAD3 and P-SMAD3 in Western blot. (c) Western blot analysis showed the level of SMAD3, P-SMAD3, and β-actin protein abundance. Each group contained at least 3 biologic replicate samples and results are shown as mean±standard deviation. YBX1, Y-box binding protein 1; SMAD3, small mother against decapentaplegic 3; P-SMAD3, phospho small mother against decapentaplegic 3. * Indicate p<0.05, ** indicate p<0.01.

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