Analysis of the differentially expressed genes in the combs and testes of Qingyuan partridge roosters at different developmental stages

Abstract

Background: The sexual maturity of chickens is an important economic trait, and the breeding of precocious and delayed puberty roosters is an important selection strategy for broilers. The comb serves as an important secondary sexual characteristic of roosters and determines their sexual precocity. Moreover, comb development is closely associated with gonad development in roosters. However, the underlying molecular mechanism regulating the sexual maturity of roosters has not yet been fully explored.

Results: In order to identify the genes related to precocious puberty in Qingyuan partridge roosters, and based on the synchrony of testis and combs development, combined with histological observation and RNA-seq method, the developmental status and gene expression profile of combs and testis were obtained. The results showed that during the early growth and development period (77 days of age), the development of combs and testis was significant in the high comb (H) group versus the low comb (L) group (p < 0.05); however, the morphological characteristic of the comb and testicular tissues converged during the late growth and development period (112 days of age) in the H and L groups. Based on these results, RNA-sequencing analysis was performed on the comb and testis tissues of the 77 and 112 days old Qingyuan Partridge roosters with different comb height traits. GO and KEGG analysis enrichment analysis showed that the differentially expressed genes were primarily enriched in MAPK signaling, VEGF signaling, and retinol metabolism pathways. Moreover, weighted correlation network analysis and module co-expression network analysis identified WNT6, AMH, IHH, STT3A, PEX16, KPNA7, CATHL2, ROR2, PAMR1, WISP2, IL17REL, NDRG4, CYP26B1, and CRHBP as the key genes associated with the regulation of precocity and delayed puberty in Qingyuan Partridge roosters.

Conclusions: In summary, we identified the key regulatory genes of sexual precocity in roosters, which provide a theoretical basis for understanding the developmental differences between precocious and delayed puberty in roosters.

Keywords: Chicken; Comb; Sexual maturity; Testis; Weighted correlation network analysis (WGCNA).

Fig. 1

Morphological differences in the comb and testis tissues of the Qingyuan partridge roosters at different developmental stages. (A and B) Morphological analysis of the comb (A) and testis (B) tissues of the high-and low-comb groups at different developmental stages using hematoxylin and eosin staining. Original magnification: a-f in Figure A and B: 20x, scale bar = 100 μm and g-l in Figure A and B: 40x, scale bar = 50 μm. In Figure B, ▲ indicates spermatogonium and → indicates sperm cells

Fig. 2

Differentially expressed genes (DEGs) in the comb and testis tissues of the high-and low-comb groups at the same developmental stage. (A, C) PCA analysis of RNA-Seq data in the comb (A) and testis (C) tissues; (B, D) The Wayne map of differentially expressed genes in the comb (B) and testis (D) tissues; (E and F) The volcano maps of the DEGs in the comb tissues of the 77 (E) and 112 (F) day-old high- and low-comb groups and (G and H) the volcano maps of DEGs in the testis tissues of the 77 (G) and 112 (H) day-old high- and low-comb groups

Fig. 3

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of DEGs in the comb and testes tissues of the high- and low-comb groups at the same developmental stage. (A and B) GO enrichment analysis of the DEGs in the comb (A) and testes (B) tissues in the molecular function, cellular composition, and biological processes categories; (C and D) KEGG enrichment analysis of the DEGs in the comb tissues of the 77 (C) and 112 (D) day-old high-and low-comb groups; (E and F) KEGG enrichment analysis of the DEGs in the testes tissues of the 77 (E) and 112 (F) day-old high-and low-comb groups

Fig. 4

DEGs in the comb and testis tissues of the high-or low-comb height groups at different developmental stages. (A and B) The volcano map (A) and heat map (B) of the DEGs in the comb tissues of the H group at 77 and 112 days of age; (C and D) the volcano map (C) and heat map (D) of the DEGs in the comb tissues of the L group at 77 and 112 days of age; (E and F) the volcano map (E) and heat map (F) of DEGs in the testes tissues of the H group at 77 and 112 days of age; and (G and H) the volcano maps (G) and heat map (H) of the DEGs in the testes tissues of the L group at 77 and 112 days of age

Fig. 5

GO and KEGG analysis of DEGs in the comb and testis tissues of the high- or low-comb groups at different developmental stages. (A and B) GO enrichment analysis of the DEGs in the comb (A) and testes (B) tissues in the molecular function, cellular composition, and biological processes categories; (C and D) KEGG enrichment analysis of the DEGs in the comb tissues of the high- (C) and low-comb groups (D) at 77 and 112 days of age; (E and F) KEGG enrichment analysis of the DEGs in the testes tissues of the high- (E) and low-comb groups (F) at 77 and 112 days of age

Fig. 6

Weighted correlation network analysis of the DEGs in the comb and testes tissues. (A) Analysis of the scale-free fitting index of various soft-thresholding powers (left) and the average connectivity of various soft-thresholding powers (right); (B) sample tree obtained from hierarchical clustering; (C) gene dynamic shearing clustering tree, each color represents a module; (D) classification of the DEGs into 21 modules according to the gene co-expression patterns; (E) the heat map of gene expression patterns of different phenotypic tissues at different developmental stages

Fig. 7

GO and KEGG analysis of the high-expression modules. (A-D) GO enrichment analysis of the blue (A), darkolivegreen (B), sienna3 (C), and plum1 (D) modules in the molecular function, cellular composition, and biological processes categories; (E-H) KEGG enrichment analysis of the blue (E), darkolivegreen (F), sienna3 (G), and plum1 (H) modules

Fig. 8

Construction of the co-expression subnetwork and screening of the hub genes. (A-D) Gene co-expression network of the blue (A), darkolivegreen (B), sienna3 (C), and plum1 (D) modules