TCF21 is related to testis growth and development in broiler chickens

Abstract

Background: Large amounts of fat deposition often lead to loss of reproductive efficiency in humans and animals. We used broiler chickens as a model species to conduct a two-directional selection for and against abdominal fat over 19 generations, which resulted in a lean and a fat line. Direct selection for abdominal fat content also indirectly resulted in significant differences (P < 0.05) in testis weight (TeW) and in TeW as a percentage of total body weight (TeP) between the lean and fat lines.

Results: A total of 475 individuals from the generation 11 (G₁₁) were genotyped. Genome-wide association studies revealed two regions on chicken chromosomes 3 and 10 that were associated with TeW and TeP. Forty G₁₆ individuals (20 from each line), were further profiled by focusing on these two chromosomal regions, to identify candidate genes with functions that may be potentially related to testis growth and development. Of the nine candidate genes identified with database mining, a significant association was confirmed for one gene, TCF21, based on mRNA expression analysis. Gene expression analysis of the TCF21 gene was conducted again across 30 G₁₉ individuals (15 individuals from each line) and the results confirmed the findings on the G₁₆ animals.

Conclusions: This study revealed that the TCF21 gene is related to testis growth and development in male broilers. This finding will be useful to guide future studies to understand the genetic mechanisms that underlie reproductive efficiency.

Fig. 1

Observed phenotypic means of male broiler chickens from the lean and fat lines that were selected for abdominal fat over 19 generations. The two lines originated from the same population at generation 1. Starting from generation G₄, significant differences (P < 0.05) in abdominal fat percentage (AFP) were observed at 7 weeks of age (a). A sevenfold difference was found between the two lines in the last generation (G₁₉); however, body weight at the same age (BW7) remained similar between the fat and lean lines across all generations (b). Testis weight (TeW) was recorded (c) and testis percentage (TeP) was calculated (d). Both TeW and TeP exhibited significant differences between the lean and fat lines from G₇ forward

Fig. 2

Characteristics of the genotyped SNPs and population structure. 475 individuals (203 individuals from the lean line and 272 from the fat line) were randomly selected from G₁₁ for genotyping. Genotyping was performed with the Chicken-60 K Illumina chip with 80% of the intervals between adjacent SNPs <30 kb (a). The minor allele frequency (MAF) of the SNPs in the lean and fat lines respectively, used in the GWAS study (b). Principal component analysis based on these SNPs shows the separation between the lean and fat lines in a two-dimension plot (c) and a three-dimension plot (d)

Fig. 3

Results of genome-wide association studies using PLINK. The results are presented as Manhattan plots in the top panels (a, b) and Q–Q plots in the bottom panels (c, d). The left panels show the results for testis weight (TeW) and the right panels for testis percentage of body weight (TeP). The solid line indicates the Bonferroni threshold for multiple test correction with a type I error of 5% (P value <5.48 × 10⁻⁷)

Fig. 4

mRNA expression analysis of the nine candidate genes (identified by GWAS) in male broiler testis tissue. a C _t values for the nine candidate genes and four housekeeping genes. b –log P value of the association between the expression levels of the nine genes and TeW and TeP of birds from the G₁₆ population, using two different housekeeping genes as an internal reference. c –log P value of the difference in expression levels across the nine candidate genes between the lean and fat lines of G₁₆, using two different housekeeping genes as an internal reference. d Difference in TCF21 expression level ($2^{-\mathrm{\Delta }C\text{t}}$ + SE) between the lean and fat lines at G₁₉, using two different housekeeping genes as an internal reference. *P < 0.05, TeW testis weight, TeP testis percentage

Fig. 5

Allele frequencies of the significant SNPs that are linked to the TCF21 gene. Allele frequencies of the significant SNPs within the regions of interest on chromosomes 3 and 10 were calculated for both lean and fat lines. When plotted against each other, SNPs near the diagonal demonstrated similar frequencies between the two lines. The SNP associated with TCF21 is one of the SNPs that deviates from the diagonal (a). When the individuals were characterized by the genotypes associated with the TCF21 SNP (Gga_rs14364352), individuals with genotypes AG (170 birds) and GG (52 birds) appear to have larger values for both TeW and TeP than those with AA (253 birds) genotype (b). The individuals with AG and GG genotypes have higher values than individuals with AA genotypes for both TeW (c) and TeP (d). This trend was the same in both lean and fat lines. The individuals with AG and GG genotypes also have larger variations in trait values than individuals with AA genotypes. Standard error of the mean is indicated by the size of the circles