Genomic Identification, Evolution, and Expression Analysis of Collagen Genes Family in Water Buffalo during Lactation

Abstract

Collagens, as extracellular matrix proteins, support cells for structural integrity and contribute to support mammary basic structure and development. This study aims to perform the genomic identification, evolution, and expression analyses of the collagen gene family in water buffalo (Bubalus bubalis) during lactation. A total of 128 buffalo collagen protein sequences were deduced from the 45 collagen genes identified in silico from buffalo genome, which classified into six groups based on their phylogenetic relationships, conserved motifs, and gene structure analyses. The identified collagen sequences were unequally distributed on 16 chromosomes. The tandem duplicated genes were found within three chromosomes, while only one segmental event occurred between Chr3 and Chr8. Collinearity analysis revealed that a total of 36 collagen gene pairs were orthologous between buffalo and cattle genomes despite having different chromosome numbers. Comparative transcription analyses revealed that a total of 23 orthologous collagen genes were detected in the milk samples at different lactation periods between the two species. Notably, the duplicated gene pair of COL4A1-COL4A2 during lactation had a higher mRNA expression level than that of cattle, while a higher expression level of COL6A1-COL6A2 pair was found in cattle compared with that of buffalo. The present study provides useful information for investigating the potential functions of the collagen family in buffalo during lactation and helps in the functional characterization of collagen genes in additional research.

Keywords: buffalo; collagen family; evolutionary relationship; gene expression.

Figure 1

Phylogenetic relationship of collagen proteins in six mammals. Line with different colors indicates different groups. Circle of different color also indicates different groups. Buffalo: bbu; Cattle: bta; Goat: chi; Sheep: oar; Horse: eca; Human: has.

Figure 2

Phylogenetic relationships, motif pattern, gene structure, and conserved protein motifs of buffalo collagen gene family. (A) Phylogenetic tree of 45 collagen proteins. (B) Motif pattern of buffalo collagen gene family. Ten putative motifs are indicated in different colored boxes. For details of motifs refer to Table 1. (C) UTR/CDS organization of collagen genes. Yellow box, black line, and red box represent untranslated region (UTR), intron and coding sequencing (CDS), respectively. (D) Distributions of conserved protein motifs in collagen genes.

Figure 3

Gene duplication of buffalo (A) and cattle (B) collagen genes, as well as their collinear analysis (C). The tandem duplicated genes were marked by the green color, and segmentally duplicated genes are indicated by the red line.

Figure 4

Collagen expression analysis of cattle (A) and buffalo (B) in milk at different lactations. Red box indicates early lactation; green box indicates mid-lactation; light blue box indicates late lactation.

Figure 5

Heat map (A) of orthologous collagen genes of cattle and buffalo and validation of selected buffalo collagens by qRT-PCR (B). Distances, representing the relative similarity among genes and tissues, were calculated using Pearson’s correlation coefficients. Color represents the TPM (transcripts per million) values of gene expression after scaling and centering. D7 indicates early lactation, D140 indicates mid-lactation, and D280 indicates late lactation.