A dual targeted β-defensin and exome sequencing approach to identify, validate and functionally characterise genes associated with bull fertility

Abstract

Bovine fertility remains a critical issue underpinning the sustainability of the agricultural sector. Phenotypic records collected on >7,000 bulls used in artificial insemination (AI) were used to identify 160 reliable and divergently fertile bulls for a dual strategy of targeted sequencing (TS) of fertility-related β-defensin genes and whole exome sequencing (WES). A haplotype spanning multiple β-defensin genes and containing 94 SNPs was significantly associated with fertility and functional analysis confirmed that sperm from bulls possessing the haplotype showed significantly enhanced binding to oviductal epithelium. WES of all exons in the genome in 24 bulls of high and low fertility identified 484 additional SNPs significantly associated with fertility. After validation, the most significantly associated SNP was located in the FOXJ3 gene, a transcription factor which regulates sperm function in mice. This study represents the first comprehensive characterisation of genetic variation in bovine β-defensin genes and functional analysis supports a role for β-defensins in regulating bull sperm function. This first application of WES in AI bulls with divergent fertility phenotypes has identified a novel role for the transcription factor FOXJ3 in the regulation of bull fertility. Validated genetic variants associated with bull fertility could prove useful for improving reproductive outcomes in cattle.

Figure 1

Range in fertility phenotype for all AI bulls assessed in this study. Adjusted animal model (AAM) bull fertility phenotypes calculated for all 7,000 sires (white) and 602 sires used for >1,000 inseminations with <25% being in 2013 (black). Bulls for sequencing were selected from the extremes of high and low fertility, defined as greater than one standard deviation from the mean, >0.052 or <−0.017, (outside region denoted by red lines). Restricting the samples to sires which have been used in >1,000 inseminations increased the reliability of the fertility phenotypes but removed sires identified as having very extreme pregnancy rates based on small numbers of inseminations.

Figure 2

Genomic location of β-defensin genetic variants detected in bulls used in artificial insemination. Pie chart of percent of total SNPs identified in 160 bulls selected targeted sequencing of β-defensin genes. Genomic features where SNPs are located are identified in the key: intron (non-coding sections of DNA), upstream (5 k bp 5′UTR of a gene), intergenic region, downstream (5 k bp 3′UTR of a gene), exon (coding region), other (non-classified variants). Because the genes are found in four clusters a single SNP can have multiple annotations; for example, one may be downstream of one gene, upstream of a second and intergenic.

Figure 3

Allele frequencies of the SNPs identified in the targeted sequencing of β-defensin genes in bulls of high and low fertility. The frequency of the alternate allele in bulls of high fertility is shown on the x axis and the alternate allele frequency in bulls of low fertility on the y axis. SNPs which have a difference in SNP frequency of >20% between bulls of high and low fertility are highlighted in red. SNPs with a SNP frequency difference over 20% between groups are more likely to be under selection pressures, than SNPs with low SNP frequency differences.

Figure 4

Significant association detected between β-defensin haplotype and bull fertility. (A) Association analysis of high quality SNPs and the adjusted animal model phenotype of fertility, -log₁₀(P-value) is shown for the SNPs in the four β-defensin gene clusters on BTA8, BTA13, BTA23 and BTA27. The most significantly associated SNPs are all located on BTA13. Level of significance: unadjusted P-value < 0.01, indicated by red line. (B) –log₁₀(P-value for all SNPs in the region on BTA13 containing the 98 SNPs found in a heterozygous haplotype in 9 sires (shown in red). The region contains 8 β-defensin genes, including BBD126.

Figure 5

Genomic location of genetic variants detected in bulls used in artificial insemination using WES. Pie chart of percent of total SNPs identified in 24 bulls selected for whole-exome sequencing located in various genomic features. Genomic features where SNPs are located are identified in the key: Exon (coding region of DNA), intron (non-coding region of DNA), upstream (non-coding region 5 k bases 5′ of a gene), UTR 3′ (untranslated region 5 k bases 3′ of a gene), downstream (3′UTR of a gene), intergenic region, splice site (exon boundaries), UTR5′ (Untranslated region containing transcription factors).

Figure 6

SNP Association of genotypes and fertility for whole-exome sequence data. Manhattan plot shows the association of variants identified by whole-exome sequencing and their associated P-value with the adjusted animal model fertility phenotype. The P-value for each variant association is on the y-axis. The chromosomal position of each variant is on the x-axis. In total, there are 144,178 variants after quality control shown. Red box indicates cluster of associated SNPs with fertility located on chromosome 13, located near the β-defensin gene cluster (16 out of 38 SNPs in this region are within the β-defensin gene region).

Figure 7

Validated SNP frequencies in independent population of AI bulls. Scatterplot of SNP frequencies of validated SNPs (n = 42) with x-axis = low-fertility bulls SNP frequencies and y-axis = high-fertility bulls SNP frequencies (n = 123 bulls). Red line = line of regression. R² = Pearson’s correlation coefficient. The most significantly associated SNP in the validation dataset (located in FOXJ3) is highlighted in red (SNP frequency difference = 21% (48–69%)).

Figure 8

Significant effect of β-defensin haplotype on sperm binding to oviductal epithelium. Binding density of sperm from bulls of varying β-defensin haplotype (high fertility with β-defensin haplotype, H + ive; High fertility without β-defensin haplotype, H-ive; and low fertility without β-defensin haplotype, L − ive) to bovine oviductal epithelial cell explants. n = 4 biological and a further 3 technical replicates per group. Twelve straws per haplotype were assessed. Vertical error bars represent s.e.m. Different superscripts refer to statistically significant differences (P < 0.05).