Identification of deleterious recessive haplotypes and candidate deleterious recessive mutations in Japanese Black cattle

Abstract

Intensive use of a few elite sires has increased the risk of the manifestation of deleterious recessive traits in cattle. Substantial genotyping data gathered using single-nucleotide polymorphism (SNP) arrays have identified the haplotypes with homozygous deficiency, which may compromise survival. We developed Japanese Black cattle haplotypes (JBHs) using SNP array data (4843 individuals) and identified deleterious recessive haplotypes using exome sequencing of 517 sires. We identified seven JBHs with homozygous deficiency. JBH_10 and JBH_17 were associated with the resuming of estrus after artificial insemination, indicating that these haplotypes carried deleterious mutations affecting embryonic survival. The exome data of 517 Japanese Black sires revealed that AC_000165.1:g.85341291C>G of IARS in JBH_8_2, AC_000174.1:g.74743512G>T of CDC45 in JBH_17, and a copy variation region (CNVR_27) of CLDN16 in JBH_1_1 and JBH_1_2 were the candidate mutations. A novel variant AC_000174.1:g.74743512G>T of CDC45 in JBH_17 was located in a splicing donor site at a distance of 5 bp, affecting pre-mRNA splicing. Mating between heterozygotes of JBH_17 indicated that homozygotes carrying the risk allele died around the blastocyst stage. Analysis of frequency of the CDC45 risk allele revealed that its carriers were widespread throughout the tested Japanese Black cattle population. Our approach can effectively manage the inheritance of recessive risk alleles in a breeding population.

Figure 1

Distribution of the seven haplotypes with homozygous deficiency until an age of 25 months in Japanese Black cattle. (a) Japanese Black haplotype (JBH) _1_1. (b) JBH_1_2. (c) JBH_4. (d) JBH_8_1. (e) JBH_8_2. (f) JBH_10. (g) JBH_17. The overlapping haplotypes with a P value less than 10^–4, calculated using the frequencies of homozygotes and carriers with a binomial test, were identified as the risk haplotype regions. Each red horizontal bar represents a haplotype with homozygosity deficiency. Positions on the X-axis are based on the UMD3.1 assembly of the bovine genome.

Figure 2

Genetic features and quality of whole exome sequences of 517 Japanese Black sires. (a–d) Principal component analysis of 1844 Japanese Black cattle, including 791 cows, 536 steers, and 517 sires, was performed using 593,358 autosomal SNPs. The individuals were plotted in a two-dimensional graph, with the first (x-axis: PC1) and the second (y-axis: PC2) principal components. (d) Data for 791 cows, 536 steers, 517 exome-sequenced sires were merged. (e) Account rate of haplotypes in 25 to 517 sires with the general Japanese Black cattle population. Two consecutive BovineHD SNPs in haplotypes of 517 exome-sequenced sires resulted in an account rate of near 100% with the general Japanese Black cattle population (red plot). The X-axis represents the number of sires, and the Y-axis represents the account rate. (f) Quality of exome sequence data of 517 sires. The genotype concordance rate between the exome-derived genotypes and the Bovine High-Density (HD) BeadChip array-derived genotypes was calculated based on 44,772 autosomal SNVs and plotted. The relationships are presented in a two-dimensional graph, with the average read-depth coverage of the target exome (X-axis) and the genotype concordance rate (%) (Y-axis).

Figure 3

AC_000174.1:g.74743512G>T in JBH_17 Located in the Splicing Donor Site in Intron at a Distance of 5 bp from Exon 14 of CDC45. (a) Sequences with column heights proportional to the information content of 181,495 introns with GT and 102,705 introns with AG from cattle GTF data derived from the Ensembl database. The vertical arrow indicates the position of AC_000174.1:g.74743512G>T. (b) Schematic representation of minigene with exon 14 to exon 15 of CDC45 including an inter-intron with AC_000174.1:g.74743512G>T and CMV promoter for the splicing assay. The vertical arrow indicates the position of AC_000174.1:g.74743512G>T and the horizontal arrows indicate forward (F) and reverse (R) primers for the splicing assay. (c) The minigenes with the reference (G) and risk (T) alleles at AC_000174.1:g.74743512G>T were transfected into Cos-7 cells and amplified using PCR of cDNA with F and R primers shown in (b). Expected spliced and unspliced bands were detected at approximately 136 and 208 bp, respectively. (d) Relative CDC45 expression level in the dermal fibroblasts of homozygotes of the reference allele (G) and heterozygotes of the AC_000174.1:g.74743512G>T allele using quantitative PCR with primers specific to exon 3 and exon 20, respectively. P values in the graph were calculated using a t-test.

Figure 4

Relationships of CNVR_27 of CLDN16 in JBH_1_1 and JBH_1_2. (a) CNV calling of exome data in three ranges included CLDN16 exon 1 to exon 4, 77471171–77559720 bp, 77472102–77491987 bp, and 77472102–77559720 bp was performed using XHMM software. Mean normalized read-depth (X-axis) were calculated using the BAM file and plotted. (b) CNVR_27 of CLDN16 was located on the telomeric side at a distance of approximately 4.4 Mb from JBH_1_1 and 0.6 Mb from JBH_1_2. The r² values (LD) were estimated by the square of the correlation coefficient (%) in 1980 Japanese Black cattle.