Identification of crucial modules and genes associated with backfat tissue development by WGCNA in Ningxiang pigs
Fat deposition is a prominent economic trait and complex process in pigs. Ningxiang pig, one of the four famous indigenous breeds in China, is characterized by high fat content. The underlying gene expression pattern in different developmental periods of backfat tissue remains unclear, and the purpose of this investigation is to explore the potential molecular regulators of backfat tissue development in Ningxiang pigs. Histological analysis of backfat tissue showed that adipocyte area increased and adipocyte number decreased with the development of backfat tissue in different stages (60d, 120d, 180d, 240d, 300d, and 360d). Meanwhile, a total of 1024 differentially expressed genes were identified in five comparison groups (120d vs 60d, 180d vs 120d, 240d vs 180d, 300d vs 240d, and 360d vs 300d). And then strongly co-expressed genes in modules were clustered by Weighted gene co-expression network analysis (WGCNA). Among these modules, turquoise, red, pink, paleturquoise, darkorange, and darkgreen module had close correlation with 60d, 120d, 180d, 240d, 300d, and 360d developmental stage, respectively, while tan, black and turquoise module had intimate relation with backfat thickness, adipocyte area and adipocyte number, respectively. Furthermore, GO and KEGG enrichment were analyzed in each module, the gene co-expression network was constructed, and the hub genes were described. According to the co-expression network, 13 genes (ACSL1, ACOX1, FN1, DCN, CHST13, COL1A1, COL1A2, COL6A3, COL5A1, COL14A1, OAZ3, DNM1, and SELP) were recognized as hub genes. Further analysis revealed that ACSL1 and ACOX1 might perform considerable function in early developmental stage of backfat tissue before 60d. FN1, DCN, COL1A1, COL1A2, COL5A1, COL6A3, and COL14A1 have unignorable position around 120d developmental stage. Besides, the functional relevance associated with SELP and backfat thickness, DNM1 and adipocyte area needs to be elucidated. In addition, QCR analysis validated the strong consistency of genes expressions with RNA-seq data, manifesting the reliability and accuracy of our study. The present research preliminarily depicted the gene co-expression network for each period of backfat developmental stage in Ningxiang pigs, which not only help to understand the integrated mechanism underlying backfat tissue development and lipid metabolism, but also promotes the progress of genetic improvement in pigs. Overall design: Eighteen castrated Ningxiang pigs in six developmental stages (60d, 120d, 180d, 240d, 300d and 360d) were used in this study. Six groups of pigs were half-sibs, and the three samples in each developmental stage were full-sibs. All the experimental pigs were reared under the standard environmental conditions. The backfat tissue at the position of the thickest point in shoulder were collected, and the sample was immediately frozen in liquid nitrogen for the further RNA-sequencing.