Identifying molecular pathways and candidate genes associated with knob traits by transcriptome analysis in the goose (Anser cygnoides)

Abstract

Anser cygnoides has a spherical crest on the beak roof, which is described as knob. However, the mechanisms affecting knob morphology are unclear. Here, we investigated the phenotypic characteristics and molecular basis of knob-size differences in Yangzhou geese. Anatomically, the knob was identified as frontal hump in the frontal area of the skull, rather than hump of upper beak. Although the frontal hump length, and height varied greatly in geese with different knob phenotypes, little was changed in the width. Histologically, knob skin in large-size knobs geese have a greater length in the stratum corneum, stratum spinosum, and stratum reticular than that in small-size knobs geese. Moveover, the 415 differentially expressed genes were found between the large knobs and small ones through transcriptome profiling. In addition, GO enrichment and KEGG pathway analysis revealed 455 significant GO terms and 210 KEGG pathways were enriched, respectively. Among these, TGF-β signaling and thyroid hormone synthesis-signaling pathways were identified to determine knob-size phenotype. Furthermore, BMP5, DCN, TSHR and ADCY3 were recognized to involve in the growth and development of knob. Our data provide comprehensive molecular determinants of knob size phenotype, which can potentially promote the genetic improvement of goose knobs.

Figure 1

Measurements of the morphometric in goose knob and anatomical measurements made on the goose frontal hump. (A) Beak area: blue area; frontal area: red area; cranial area: orange lines; integumentary outgrowth: yellow area; fronto-nasal junction: black line. (B) Knob length: red line; knob width: green line; knob height: blue line; the phenotypic boundary of knob: yellow line; fronto-nasal junction: black line. (C) From top to bottom: lateral view, dorsal view, and cranial view, respectively.

Figure 2

Phenotypes of knobs with different sizes in Yangzhou geese. (A,B) Differences in knob sizes were observed at 380 days of age between geese with large (L) knobs (A) and geese with small (S) knobs (B). (C) Comparison of knob sizes in the L and S groups. Significant differences are indicated with two asterisks (P < 0.01). Kl knob length, Kw knob width, Kh knob height.

Figure 3

Frontal humps of Yangzhou geese with different anatomical knob sizes. (A,B) Differences were observed in the frontal hump size at 380 days of age between geese in the L (A) and S (B) groups. (C) Comparison of frontal humps of geese in the L and S groups. Significant differences are indicated with two asterisks (P < 0.01). Fhl frontal hump length, Fhw frontal hump width, Fhh frontal hump height.

Figure 4

Histological characteristics of knobs with different sizes in Yangzhou geese. (A,B) Histological analysis of skin from geese with large knobs (A) and small knobs (B), based on HE staining. Scn stratum corneum, Ss stratum spinosum, Se stratum epidermis, Sr stratum reticular, Sci stratum corium. (C) Comparing the thicknesses of five layers of skin from geese in the L and S groups. Significant differences are indicated with two asterisks (P < 0.01).

Figure 5

Scatter plot, volcano plot, and heat-map of differentially expressed genes (DEGs). (A) The scatter plot was used to assess variations in gene-expression levels between small and large knobs in geese. (B) Volcano plot showing DEGs. The X-axis, log₂FC represents the logarithm of the expression ratio (fold change), and the Y-axis represents adjusted P (P adjust) value. Each dot represents a specific gene. Red dots represent genes expressed at significantly higher levels in geese with large knobs, and green dots represent genes with significantly lower relative expression. Genes showing no significant differences in expression levels are represented with gray dots. (C) Hierarchical-clustering analysis for the transcriptome profiles of knobs in the S versus L groups. The heat-map presents the mean relative abundances of genes with a color scale. FC fold-change.

Figure 6

GO analysis of the diferentially expressed genes between the Large knob (L) and small knob (S) groups. The X-axis represents the name of GO terms. The Y-axis represents the numbers of enriched genes in each GO term. The three colors represent the three categories of biological processes (BP), cellular components (CC), and molecular functions (MF), respectively (P adjust < 0.05).

Figure 7

KEGG pathway analysis of the diferentially expressed genes (DEGs) between the large knob (L) and small knob (S) groups. The X-axis represents the larger the − log₁₀ P value is, the more significant of enrichment of the DEGs in this pathway is. The Y-axis represents the name of pathway. The size and color of each bubble represent the amount of DEGs enriched in the pathway and enrichment signifcance, respectively (P adjust < 0.05).

Figure 8

Protein–protein interaction network for the DEGs identified in this study. Nodes represent proteins, and edges represent interaction between proteins. The size of a node is proportional to its degree (degree is defined as the number of proteins that interact with the node). The color of a node is used to distinguish nodes with different degrees.

Figure 9

mRNA expression of the key 13 DEGs in large-knob geese and small-knob ones. Gene expression was characterized using RT-qPCR and was represented relative to β-actin. Mean △Ct values of the small-knob group served as calibrators. Vertical bars represent the mean ± SD (n = 3). Significant differences relative to controls are indicated with one (P < 0.05) or two (P < 0.01) asterisks. The black and gray bar were large-knob geese and small-knob geese, respectively.