De Novo Genome Assembly of Japanese Black Cattle as Model of an Economically Relevant Animal.

A genetic analysis of Japanese Black cattle using short reads and guided by the reference genome from Western breeds would miss the structural variation and/or other unique characteristics of Japanese Black cattle. To overcome this difficulty, a de novo genome assembly independent from the reference genome is required. This chapter describes the technical developments, with respect to both experimental and bioinformatics procedures, including the use of short and long reads, required for de novo genome assembly of Japanese Black cattle.

Comprehensive analysis of 124 transcriptomes from 31 tissues in developing, juvenile, and adult Japanese Black cattle.

Omic analyses of economically important animals, including Japanese Black cattle, are currently underway worldwide. In particular, tissue and developmental stage-specific transcriptome characterization is essential for understanding the molecular mechanisms underlying the phenotypic expression of genetic disorders and economic traits. Here, we conducted a comprehensive analysis of 124 transcriptomes across 31 major tissues from fetuses, juvenile calves, and adult Japanese Black cattle using short-read sequencing. We found that genes exhibiting high tissue-specific expression tended to increase after 60 days from fertilization and significantly reflected tissue-relevant biology. Based on gene expression variation and inflection points during development, we categorized gene expression patterns as stable, increased, decreased, temporary, or complex in each tissue. We also analysed the expression profiles of causative genes (e.g. SLC12A1, ANXA10, and MYH6) for genetic disorders in cattle, revealing disease-relevant expression patterns. In addition, to directly analyse the structure of full-length transcripts without transcript reconstruction, we performed RNA sequencing analysis of 22 tissues using long-read sequencing and identified 232 novel non-RefSeq isoforms. Collectively, our comprehensive transcriptomic analysis can serve as an important resource for the biological and functional interpretation of gene expression and enable the mechanistic interpretation of genetic disorders and economic traits in Japanese Black cattle.

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

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.

Genetic diversity and genomic inbreeding in Japanese Black cows in the islands of Okinawa Prefecture evaluated using single-nucleotide polymorphism array.

Maintaining genetic diversity and inbreeding control are important in Japanese Black cattle production, especially in remote areas such as the islands of Okinawa Prefecture. Using a single-nucleotide polymorphism (SNP) array, we evaluated the genetic diversity and genomic inbreeding in Japanese Black cows from the islands of Okinawa Prefecture and compared them to those from other locations across Japan. Linkage disequilibrium decay was slower in cows in the islands of Okinawa Prefecture. The estimated effective population size declined over time in both populations. The genomic inbreeding coefficient (FROH ) was estimated using long stretches of consecutive homozygous SNPs (runs of homozygosity; ROH). FROH was higher in the cows on the islands of Okinawa Prefecture than on other locations. In total, 818 ROH fragments, including those containing NCAPG and PLAG1, which are major quantitative trait loci for carcass weight in Japanese Black cattle, were present at significantly higher frequencies in cows in the islands of Okinawa Prefecture. This suggests that the ROH fragments are under strong selection and that cows in the islands of Okinawa Prefecture have low genetic diversity and high genomic inbreeding relative to those at other locations. SNP arrays are useful tools for evaluating genetic diversity and genomic inbreeding in cattle.

A 44-kb deleted-type copy number variation is associated with decreasing complement component activity and calf mortality in Japanese Black cattle.

BACKGROUND: Calf mortality generally occurs in calves prior to weaning, which is a serious problem in cattle breeding. Several causative variants of monogenic Mendelian disorders in calf mortality have been identified, whereas genetic factors affecting the susceptibility of calves to death are not well known. To identify variants associated with calf mortality in Japanese Black cattle, we evaluated calf mortality as a categorical trait with a threshold model and performed a genome-wide copy number variation (CNV) association study on calf mortality. RESULTS: We identified a 44-kb deleted-type CNV ranging from 103,317,687 to 103,361,802 bp on chromosome 5, which was associated with the mortality of 1-180-day-old calves. The CNV harbored C1RL, a pseudogene, and an IncRNA localized in the C1R and C1S gene cluster, which is a component of the classical complement activation pathway for immune complexes for infectious pathogens. The average complement activity in CNVR_221 homozygotes at postnatal day 7 was significantly lower than that of wild-type animals and heterozygotes. The frequency of the risk allele in dead calves suffering from diarrhea and pneumonia and in healthy cows was 0.35 and 0.28, respectively (odds ratio = 2.2, P = 0.016), suggesting that CNVR_221 was associated with the mortality of Japanese Black calves suffering from an infectious disease. CONCLUSIONS: This study identified a deleted-type CNV associated with the mortality of 1-180-day-old calves. The complement activity in CNVR_221 homozygotes was significantly lower than that in heterozygotes and wild type animals. The frequency of the risk allele was higher in dead calves suffering from an infectious disease than in healthy cows. These results suggest that the existence of CNVR_221 in calves could be attributed to a reduction in complement activity, which in turn leads to susceptibility to infections. Thus, the risk allele could serve as a useful marker to reduce the mortality of infected Japanese Black calves.

A long-term study of the effects of SLC12A1 homozygous mutation (g.62382825G>A, p.Pro372Leu) in Japanese Black cattle.

Recessive missense mutation in the solute carrier family 12, member 1 (SLC12A1) gene (g.62382825G>A) is associated with hydrallantois, which is the accumulation of fluid in the allantoic cavity of a pregnant animal, and usually causes fetal death in Japanese Black cattle. However, the symptoms of a homozygote with this mutation that do not result in fetal death have not previously been tracked and evaluated. In the present study, we observed a homozygote with the SLC12A1 risk allele over a long-term period. The calf did not show any obvious clinical symptoms, although it did exhibit a slight growth retardation that accompanied mild calciuria. At 28 months of age, the homozygote showed renal dysfunction, which in turn resulted in hydronephrosis. The time course of the symptoms was consistent with the phenotype of Bartter syndrome in humans. Additionally, the risk heterozygous genotype did not any effects on carcass traits, which indicates that eliminating the risk allele would not have any unfavorable effects. Therefore, we emphasize that both the fetal- and late-stage symptoms associated with the SLC12A1 risk allele compromise animal welfare, and consequently may result in severe economic losses for individual farmers if the SLC12A1 risk allele is not eliminated from the population.

Genotypes and allele frequencies of buried SNPs in a bovine single-nucleotide polymorphism array in Japanese Black cattle.

Single nucleotide polymorphism (SNP) arrays are widely used for genetic and genomic analyses in cattle breeding; thus, data derived from SNP arrays have accumulated on a large scale nationwide. Commercial SNP arrays contain a considerable number of unassigned SNPs on the chromosome/position on the genome; these SNPs are excluded in subsequent analyses. Notably, the position-unassigned SNPs, or "buried SNPs" include some of the markers associated with genetic disease. In this study, we identified the position of buried SNPs using the Basic Local Alignment Search Tool against the surrounding sequences and characterized the relationship between SNPs and genetic diseases in Online Mendelian Inheritance in Animals based on the genomic position. We determined the position of 285 buried SNPs on the genome and surveyed the genotype and allele frequencies of these SNPs in 5,955 individual Japanese Black cattle. Eleven SNPs associated with genetic disease, which contained five buried SNPs, were found in the population with the risk allele frequency ranging from 0.00008396 to 0.46. These results indicate that buried SNPs in the bovine SNP array can be utilized to identify associations with genetic disorders from large scale accumulated SNP genotype data in Japanese Black cattle.

Relationship between call rate per individual and genotyping accuracy of bovine single-nucleotide polymorphism array using deoxyribonucleic acid of various qualities.

Single nucleotide polymorphism (SNP) arrays are widely used for genetic and genomic analyses in cattle breeding. However, the relationship among sample genotyping efficiency (call rate per individual), accuracy of SNP genotypes, and DNA quality (integrity, concentration, and mixture of DNA, i.e., chimerism) remains unknown. We determined the effect of DNA quality on call rate per individual and accuracy of SNP genotypes using artificial DNA samples of various qualities. Integrity and concentration of DNA were less sensitive to call rate per individual and accuracy of genotyping in the SNP array. Chimerism strongly affected call rate per individual and accuracy of SNP genotypes. Artificial chimerism experiments showed that relative to unmixed DNA, the genotypic matching error (%) of mixed DNAs linearly increased with mix ratio, whereas the call rate per individual in some samples at 50% mix ratio was >0.95. However, individuals with higher chimerism were readily identified based on standard deviation of B-allele frequency (BAF) and BAF distribution across the genome from SNP array data. Thus, we effectively managed the balance by maximizing genotyping accuracy and minimizing the number of samples for re-genotyping by using quality control for combining call rate per individual with BAF.

Genome-wide association studies identified variants for taurine concentration in Japanese Black beef.

We performed genome-wide association studies (GWAS) using the BovineSNP50 array to detect significant single nucleotide polymorphisms (SNPs) that may affect the concentration of 22 free amino acids and three peptides in Japanese Black beef cattle. A total of 574 Japanese Black cattle and 40,657 SNPs from the array were used for this study. Genome-wide significant SNPs were detected for ß-alanine (three SNPs on chromosomes 22 and 29) and taurine (26 SNPs on chromosome 22). Importantly, the top two SNPs for taurine were highly significant (p = 6.2 × 10-21 ), and the frequency of the increase-concentration allele (Q) for taurine was found to be 0.73. The Q allele frequency of this population was similar to that of the other unrelated Japanese Black cattle, but different from that of the other breeds. In addition, the significant SNPs were not associated with carcass traits or fatty acid compositions. Interestingly, the top three of the four most significant SNPs for taurine were located near solute carrier family 6, member 6 (SLC6A6), which is a membrane transporter for taurine. We also found two associated variants in the 5'-upstream region of SLC6A6; however, they were less significantly associated than the SNPs from the BovineSNP50 array.

Great Saphenous Vein Flow Pattern as a Simple Ultrasonographic Sign of Early Recanalization of Deep Vein Thrombosis: A Case Series Report.

We retrospectively examined patients with ultrasonographically occlusive acute proximal deep vein thrombosis (DVT). All patients were categorized into two groups on the basis of whether great saphenous vein (GSV) flow toward the common femoral vein was detected (flow [+]; n=10) or undetected (flow [-]; n=10). We investigated the relationship between the GSV flow pattern and DVT recanalization. Thrombus recanalization, which is defined as diameter reduction to lower than 40% of the vessel diameter, was confirmed in seven of the flow (+), and none of the flow (-). This study proposes that the GSV flow pattern may be a simple marker for the recanalization of proximal occlusive DVT.

Maternal variant in the upstream of FOXP3 gene on the X chromosome is associated with recurrent infertility in Japanese Black cattle.

BACKGROUND: Repeat breeding, which is defined as cattle failure to conceive after three or more inseminations in the absence of clinical abnormalities, is a substantial problem in cattle breeding. To identify maternal genetic variants of repeat breeding in Japanese Black cattle, we selected 29 repeat-breeding heifers that failed to conceive following embryo transfer (ET) and conducted a genome-wide association study (GWAS) using the traits. RESULTS: We found that a single-nucleotide polymorphism (SNP; g.92,377,635A > G) in the upstream region of the FOXP3 gene on the X chromosome was highly associated with repeat breeding and failure to conceive following ET (P = 1.51 × 10-14). FOXP3 is a master gene for differentiation of regulatory T (Treg) cells that function in pregnancy maintenance. Reporter assay results revealed that the activity of the FOXP3 promoter was lower in reporter constructs with the risk-allele than in those with the non-risk-allele by approximately 0.68 fold. These findings suggest that the variant in the upstream region of FOXP3 with the risk-allele decreased FOXP3 transcription, which in turn, could reduce the number of maternal Treg cells and lead to infertility. The frequency of the risk-allele in repeat-breeding heifers is more than that in cows, suggesting that the risk-allele could be associated with infertility in repeat-breeding heifers. CONCLUSIONS: This GWAS identified a maternal variant in the upstream region of FOXP3 that was associated with infertility in repeat-breeding Japanese Black cattle that failed to conceive using ET. The variant affected the level of FOXP3 mRNA expression. Thus, the results suggest that the risk-allele could serve as a useful marker to reduce and eliminate animals with inferior fertility in Japanese Black cattle.

Effect of two non-synonymous ecto-5'-nucleotidase variants on the genetic architecture of inosine 5'-monophosphate (IMP) and its degradation products in Japanese Black beef.

BACKGROUND: Umami is a Japanese term for the fifth basic taste and is an important sensory property of beef palatability. Inosine 5'-monophosphate (IMP) contributes to umami taste in beef. Thus, the overall change in concentration of IMP and its degradation products can potentially affect the beef palatability. In this study, we investigated the genetic architecture of IMP and its degradation products in Japanese Black beef. First, we performed genome-wide association study (GWAS), candidate gene analysis, and functional analysis to detect the causal variants that affect IMP, inosine, and hypoxanthine. Second, we evaluated the allele frequencies in the different breeds, the contribution of genetic variance, and the effect on other economical traits using the detected variants. RESULTS: A total of 574 Japanese Black cattle were genotyped using the Illumina BovineSNP50 BeadChip and were then used for GWAS. The results of GWAS showed that the genome-wide significant single nucleotide polymorphisms (SNPs) on BTA9 were detected for IMP, inosine, and hypoxanthine. The ecto-5'-nucleotidase (NT5E) gene, which encodes the enzyme NT5E for the extracellular degradation of IMP to inosine, was located near the significant region on BTA9. The results of candidate gene analysis and functional analysis showed that two non-synonymous SNPs (c.1318C > T and c.1475 T > A) in NT5E affected the amount of IMP and its degradation products in beef by regulating the enzymatic activity of NT5E. The Q haplotype showed a positive effect on IMP and a negative effect on the enzymatic activity of NT5E in IMP degradation. The two SNPs were under perfect linkage disequilibrium in five different breeds, and different haplotype frequencies were seen among breeds. The two SNPs contribute to about half of the total genetic variance in IMP, and the results of genetic relationship between IMP and its degradation products showed that NT5E affected the overall concentration balance of IMP and its degradation products. In addition, the SNPs in NT5E did not have an unfavorable effect on the other economical traits. CONCLUSION: Based on all the above findings taken together, two non-synonymous SNPs in NT5E would be useful for improving IMP and its degradation products by marker-assisted selection in Japanese Black cattle.

Single nucleotide polymorphisms in the bovine MHC region of Japanese Black cattle are associated with bovine leukemia virus proviral load.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, a malignant B cell lymphoma that has spread worldwide and causes serious problems for the cattle industry. The BLV proviral load, which represents the BLV genome integrated into host genome, is a useful index for estimating disease progression and transmission risk. Here, we conducted a genome-wide association study to identify single nucleotide polymorphisms (SNPs) associated with BLV proviral load in Japanese Black cattle. The study examined 93 cattle with a high proviral load and 266 with a low proviral load. Three SNPs showed a significant association with proviral load. One SNP was detected in the CNTN3 gene on chromosome 22, and two (which were not in linkage disequilibrium) were detected in the bovine major histocompatibility complex region on chromosome 23. These results suggest that polymorphisms in the major histocompatibility complex region affect proviral load. This is the first report to detect SNPs associated with BLV proviral load in Japanese Black cattle using whole genome association study, and understanding host factors may provide important clues for controlling the spread of BLV in Japanese Black cattle.

Furosemide loading test in a case of homozygous solute carrier family 12, member 1 (SLC12A1) mutation (g.62382825G>A, p.Pro372Leu) in Japanese Black cattle.

Hydrallantois is the excessive accumulation of fluid in the allantoic cavity in a pregnant animal and is associated with fetal death. We recently identified a recessive missense mutation in the solute carrier family 12, member 1 (SLC12A1) gene (g.62382825G>A, p.Pro372Leu) that is associated with hydrallantois in Japanese Black cattle. Unexpectedly, we found a case of the homozygous risk-allele for SLC12A1 in a calf, using a PCR-based direct DNA sequencing test. The homozygote was outwardly healthy up to 3 months of age and the mother did not exhibit any clinical symptoms of hydrallantois. In order to validate these observations, we performed confirmation tests for the genotype and a diuretic loading test using furosemide, which inhibits the transporter activity of the SLC12A1 protein. The results showed that the calf was really homozygous for the risk-allele. In the homozygous calf, administration of furosemide did not alter urinary Na+ or Cl- levels, in contrast to the heterozygote and wild-type calves in which these were significantly increased. These results demonstrate that the SLC12A1 (g.62382825G>A, p.Pro372Leu) is a hypomorphic or loss-of-function mutation and the hydrallantois with this mutation shows incomplete penetrance in Japanese Black cattle.

Loss of maternal ANNEXIN A10 via a 34-kb deleted-type copy number variation is associated with embryonic mortality in Japanese Black cattle.

BACKGROUND: Conception is a fundamental trait for successful cattle reproduction. However, conception rates in Japanese Black cattle have been gradually declining over the last two decades. Although conception failures are mainly caused by embryonic mortality, the role of maternal genetic factors in the process remains unknown. Copy number variation (CNV), defined as large-scale genomic structural variants, contributes to several genetic disorders. To identify CNV associated with embryonic mortality in Japanese Black cattle, we evaluated embryonic mortality as a categorical trait with a threshold model and conducted a genome-wide CNV association study for embryonic mortality using 791 animals. RESULTS: We identified a deleted-type CNV ranging from 378,127 to 412,061 bp on bovine chromosome 8, which was associated with embryonic mortality at 30-60 days after artificial insemination (AI). The CNV harbors exon 2 to 6 of ANNEXIN A10 (ANXA10). Analysis of sequence traces from the CNV identified that 63 bp reads bridging the breakpoint were present on both sides of the CNV, indicating that the CNV was generated by non-allelic homologous recombination using the 63 bp homologous sequences. Western blot analysis showed that the CNV results in a null allele of ANXA10. This association was replicated using a sample population size of 2552 animals. To elucidate the function of ANXA10 in vivo, we generated Anxa10 null mice using the CRISPR/Cas9 system. Crossbreeding experiments showed that litter size from crosses of both Anxa10 -/- and Anxa10 +/- females had fewer pups than did Anxa10 +/+ females, and embryos of Anxa10 -/- females died between implantation stages E4.5 and E12.5. These results indicate that loss of maternal Anxa10 causes embryonic mortality. CONCLUSIONS: This study identified a deleted-type CNV encompassing ANXA10 in cows that was associated with embryonic mortality at 30-60 days after AI. Using a mouse model, we confirmed that litter sizes were smaller in crosses of both Anxa10 -/- and Anxa10 +/- females relative to those of wild females. These results indicate that ANXA10 is a maternal factor that is critical for embryo development.

A missense mutation in solute carrier family 12, member 1 (SLC12A1) causes hydrallantois in Japanese Black cattle.

BACKGROUND: Hydrallantois is the excessive accumulation of fluid within the allantoic cavity in pregnant animals and is associated with fetal mortality. Although the incidence of hydrallantois is very low in artificial insemination breeding programs in cattle, recently 38 cows with the phenotypic appearance of hydrallantois were reported in a local subpopulation of Japanese Black cattle. Of these, 33 were traced back to the same sire; however, both their parents were reported healthy, suggesting that hydrallantois is a recessive inherited disorder. To identify autozygous chromosome segments shared by individuals with hydrallantois and the causative mutation in Japanese Black cattle, we performed autozygosity mapping using single-nucleotide polymorphism (SNP) array and exome sequencing. RESULTS: Shared haplotypes of the affected fetuses spanned 3.52 Mb on bovine chromosome 10. Exome sequencing identified a SNP (g.62382825G > A, p.Pro372Leu) in exon 10 of solute carrier family 12, member 1 (SLC12A1), the genotype of which was compatible with recessive inheritance. SLC12A1 serves as a reabsorption molecule of Na(+)-K(+)-2Cl(-) in the apical membrane of the thick ascending limb of the loop of Henle in the kidney. We observed that the concentration of Na(+)-Cl(-) increased in allantoic fluid of homozygous SLC12A1 (g.62382825G > A) in a hydrallantois individual. In addition, SLC12A1-positive signals were localized at the apical membrane in the kidneys of unaffected fetuses, whereas they were absent from the apical membrane in the kidneys of affected fetuses. These results suggested that p.Pro372Leu affects the membrane localization of SLC12A1, and in turn, may impair its transporter activity. Surveillance of the risk-allele frequency revealed that the carriers were restricted to the local subpopulation of Japanese Black cattle. Moreover, we identified a founder individual that carried the mutation (g.62382825G > A). CONCLUSIONS: In this study, we mapped the shared haplotypes of affected fetuses using autozygosity mapping and identified a de novo mutation in the SLC12A1 gene that was associated with hydrallantois in Japanese Black cattle. In kidneys of hydrallantois-affected fetuses, the mutation in SLC12A1 impaired the apical membrane localization of SLC12A1 and reabsorption of Na(+)-K(+)-2Cl(-) in the thick ascending limb of the loop of Henle, leading to a defect in the concentration of urine via the countercurrent mechanism. Consequently, the affected fetuses exhibited polyuria that accumulated in the allantoic cavity. Surveillance of the risk-allele frequency indicated that carriers were not widespread throughout the Japanese Black cattle population. Moreover, we identified the founder individual, and thus could effectively manage the disorder in the population.

Identity of neocortical layer 4 neurons is specified through correct positioning into the cortex.

Many cell-intrinsic mechanisms have been shown to regulate neuronal subtype specification in the mammalian neocortex. However, how much cell environment is crucial for subtype determination still remained unclear. Here, we show that knockdown of Protocadherin20 (Pcdh20), which is expressed in post-migratory neurons of layer 4 (L4) lineage, caused the cells to localize in L2/3. The ectopically positioned "future L4 neurons" lost their L4 characteristics but acquired L2/3 characteristics. Knockdown of a cytoskeletal protein in the future L4 neurons, which caused random disruption of positioning, also showed that those accidentally located in L4 acquired the L4 characteristics. Moreover, restoration of positioning of the Pcdh20-knockdown neurons into L4 rescued the specification failure. We further suggest that the thalamocortical axons provide a positional cue to specify L4 identity. These results suggest that the L4 identity is not completely determined at the time of birth but ensured by the surrounding environment after appropriate positioning.

Genome-wide identification of copy number variation using high-density single-nucleotide polymorphism array in Japanese Black cattle.

BACKGROUND: Copy number variation (CNV) is an important source of genetic variability associated with phenotypic variation and disease susceptibility. Comprehensive genome-wide CNV maps provide valuable information for genetic and functional studies. To identify CNV in Japanese Black cattle, we performed a genome-wide autosomal screen using genomic data from 1,481 animals analyzed with the Illumina Bovine High-Density (HD) BeadChip Array (735,293 single-nucleotide polymorphisms (SNPs) with an average marker interval of 3.4 kb on the autosomes). RESULTS: We identified a total of 861 CNV regions (CNVRs) across all autosomes, which covered 43.65 Mb of the UMD3.1 genome assembly and corresponded to 1.74% of the 29 bovine autosomes. Overall, 35% of the CNVRs were present at a frequency of > 1% in 1,481 animals. The estimated lengths of CNVRs ranged from 1.1 kb to 1.4 Mb, with an average of 50.7 kb. The average number of CNVR events per animal was 35. Comparisons with previously reported cattle CNV showed that 72% of the CNVR calls detected in this study were within or overlapped with known CNVRs. Experimentally, three CNVRs were validated using quantitative PCR, and one CNVR was validated using PCR with flanking primers for the deleted region. Out of the 861 CNVRs, 390 contained 717 Ensembl-annotated genes significantly enriched for stimulus response, cellular defense response, and immune response in the Gene Ontology (GO) database. To associate genes contained in CNVRs with phenotypes, we converted 560 bovine Ensembl gene IDs to their 438 orthologous associated mouse gene IDs, and 195 of these mouse orthologous genes were categorized into 1,627 phenotypes in the Mouse Genome Informatics (MGI) database. CONCLUSIONS: We identified 861 CNVRs in 1,481 Japanese Black cattle using the Illumina BovineHD BeadChip Array. The genes contained in CNVRs were characterized using GO analysis and the mouse orthologous genes were characterized using the MGI database. The comprehensive genome-wide CNVRs map will facilitate identification of genetic variation and disease-susceptibility alleles in Japanese Black cattle.

A genome-wide association study reveals a quantitative trait locus for days open on chromosome 2 in Japanese Black cattle.

Days open (DO), which is the interval from calving to conception, is an important trait related to reproductive performance in cattle. To identify quantitative trait loci for DO in Japanese Black cattle, we conducted a genome-wide association study with 33,303 single nucleotide polymorphisms (SNPs) using 459 animals with extreme DO values selected from a larger group of 15,488 animals. We identified a SNP on bovine chromosome 2 (BTA2) that was associated with DO. After imputation using phased haplotype data inferred from 586 812 SNPs of 1041 Japanese Black cattle, six SNPs associated with DO were located in an 8.5-kb region of high linkage disequilibrium on BTA2. These SNPs were located on the telomeric side at a distance of 177 kb from the parathyroid hormone 2 receptor (PTH2R) gene. The association was replicated in a sample of 1778 animals. In the replicated population, the frequency of the reduced-DO allele (Q) was 0.63, and it accounted for 1.72% of the total genetic variance. The effect of a Q-to-q allele substitution on DO was a decrease of 3.74 days. The results suggest that the Q allele could serve as a marker in Japanese Black cattle to select animals with superior DO performance.

Impact of QTL minor allele frequency on genomic evaluation using real genotype data and simulated phenotypes in Japanese Black cattle.

BACKGROUND: Genetic variance that is not captured by single nucleotide polymorphisms (SNPs) is due to imperfect linkage disequilibrium (LD) between SNPs and quantitative trait loci (QTLs), and the extent of LD between SNPs and QTLs depends on different minor allele frequencies (MAF) between them. To evaluate the impact of MAF of QTLs on genomic evaluation, we performed a simulation study using real cattle genotype data. METHODS: In total, 1368 Japanese Black cattle and 592,034 SNPs (Illumina BovineHD BeadChip) were used. We simulated phenotypes using real genotypes under different scenarios, varying the MAF categories, QTL heritability, number of QTLs, and distribution of QTL effect. After generating true breeding values and phenotypes, QTL heritability was estimated and the prediction accuracy of genomic estimated breeding value (GEBV) was assessed under different SNP densities, prediction models, and population size by a reference-test validation design. RESULTS: The extent of LD between SNPs and QTLs in this population was higher in the QTLs with high MAF than in those with low MAF. The effect of MAF of QTLs depended on the genetic architecture, evaluation strategy, and population size in genomic evaluation. In genetic architecture, genomic evaluation was affected by the MAF of QTLs combined with the QTL heritability and the distribution of QTL effect. The number of QTL was not affected on genomic evaluation if the number of QTL was more than 50. In the evaluation strategy, we showed that different SNP densities and prediction models affect the heritability estimation and genomic prediction and that this depends on the MAF of QTLs. In addition, accurate QTL heritability and GEBV were obtained using denser SNP information and the prediction model accounted for the SNPs with low and high MAFs. In population size, a large sample size is needed to increase the accuracy of GEBV. CONCLUSION: The MAF of QTL had an impact on heritability estimation and prediction accuracy. Most genetic variance can be captured using denser SNPs and the prediction model accounted for MAF, but a large sample size is needed to increase the accuracy of GEBV under all QTL MAF categories.