Effects of single nucleotide polymorphism marker density on degree of genetic variance explained and genomic evaluation for carcass traits in Japanese Black beef cattle.

BACKGROUND: Japanese Black cattle are a beef breed whose meat is well known to excel in meat quality, especially in marbling, and whose effective population size is relatively low in Japan. Unlike dairy cattle, the accuracy of genomic evaluation (GE) for carcass traits in beef cattle, including this breed, has been poorly studied. For carcass weight and marbling score in the breed, as well as the extent of whole genome linkage disequilibrium (LD), the effects of equally-spaced single nucleotide polymorphisms (SNPs) density on genomic relationship matrix (G matrix), genetic variance explained and GE were investigated using the genotype data of about 40,000 SNPs and two statistical models. RESULTS: Using all pairs of two adjacent SNPs in the whole SNP set, the means of LD (r2) at ranges 0-0.1, 0.1-0.2, 0.2-0.5 and 0.5-1 Mb were 0.22, 0.13, 0.10 and 0.08, respectively, and 25.7, 13.9, 10.4 and 6.4% of the r2 values exceeded 0.3, respectively. While about 90% of the genetic variance for carcass weight estimated using all available SNPs was explained using 4,000-6,000 SNPs, the corresponding percentage for marbling score was consistently lower. With the conventional linear model incorporating the G matrix, correlation between the genomic estimated breeding values (GEBVs) obtained using 4,000 SNPs and all available SNPs was 0.99 for carcass weight and 0.98 for marbling score, with an underestimation of the former GEBVs, especially for marbling score. CONCLUSIONS: The Japanese Black is likely to be in a breed group with a relatively high extent of whole genome LD. The results indicated that the degree of marbling is controlled by only QTLs with relatively small effects, compared with carcass weight, and that using at least 4,000 equally-spaced SNPs, there is a possibility of ranking animals genetically for these carcass traits in this breed.

Genetic diversity and structure in the Sado captive population of the Japanese crested ibis.

The Japanese crested ibis Nipponia nippon is a critically threatened bird. We assessed genetic diversity and structure in the Sado captive population of the Japanese crested ibis based on 24 and 50 microsatellite markers developed respectively for the same and related species. Of a total of 74 loci, 19 showed polymorphisms in the five founder birds of the population, and therefore were useful for the analysis of genetic diversity and structure. Genetic diversity measures, A, ne, He, Hoand PIC, obtained by genotyping of the 138 descendants were similar to those of other species with population bottlenecks, and thus considerably low. The low level of genetic diversity resulting from such bottlenecks was consistent with the results of lower genetic diversity measures for the Sado captive relative to the Chinese population that is the source population for the Sado group as determined using previously reported data and heterozygosity excess by Hardy-Weinberg equilibrium tests. Further, individual clustering based on the allele-sharing distance and Bayesian model-based clustering revealed that the founder genomes were equally at population in total, and with various admixture patterns at individual levels inherited by the descendants. The clustering results, together with the result of inheritance of all alleles of the microsatellites from the founders to descendants, suggest that planned mating in captive-breeding programs for the population has succeeded in maintaining genetic diversity and minimizing kinship. In addition, the Bayesian model-based clustering assumed two different components of genomes in the Sado captive Japanese crested ibis, supporting a considerably low level of genetic diversity.

Fitting Genomic Prediction Models with Different Marker Effects among Prefectures to Carcass Traits in Japanese Black Cattle.

We fitted statistical models, which assumed single-nucleotide polymorphism (SNP) marker effects differing across the fattened steers marketed into different prefectures, to the records for cold carcass weight (CW) and marbling score (MS) of 1036, 733, and 279 Japanese Black fattened steers marketed into Tottori, Hiroshima, and Hyogo prefectures in Japan, respectively. Genotype data on 33,059 SNPs was used. Five models that assume only common SNP effects to all the steers (model 1), common effects plus SNP effects differing between the steers marketed into Hyogo prefecture and others (model 2), only the SNP effects differing between Hyogo steers and others (model 3), common effects plus SNP effects specific to each prefecture (model 4), and only the effects specific to each prefecture (model 5) were exploited. For both traits, slightly lower values of residual variance than that of model 1 were estimated when fitting all other models. Estimated genetic correlation among the prefectures in models 2 and 4 ranged to 0.53 to 0.71, all <0.8. These results might support that the SNP effects differ among the prefectures to some degree, although we discussed the necessity of careful consideration to interpret the current results.

Estimation of the autosomal contribution to total additive genetic variability of carcass traits in Japanese Black cattle.

We attempted to estimate the additive genetic variance explained by each autosome, using genotype data of 33,657 single nucleotide polymorphism (SNP) markers in 2271 Japanese Black fattened steers. Traits were cold carcass weight, ribeye area, rib thickness, subcutaneous fat thickness, estimated yield percentage, and marbling score. Two mixed linear models were used: One is that (model 1) incorporating a genomic relationship matrix (G matrix) constructed by using all available SNPs, and another (model 2), incorporating two G matrices constructed by using the SNPs on one autosome and using those on the remaining autosomes. Genomic heritabilities estimated using model 1 were moderate to high. The sums of the proportions of the additive genetic variance explained by each autosome to the total genetic variance estimated by using model 2 were >90%. For carcass weight, the proportions explained by Bos taurus autosomes 6, 8, and 14 were higher than those explained by the remaining autosomes. In some cases, the estimated proportion was close to 0. The results obtained from model 2 could provide a novel insight into the genetic architecture, such as heritability per chromosome, of carcass traits in Japanese Black cattle, although further careful investigation would be required.

The spatial and temporal expression of delta-like protein 1 in the rat pituitary gland during development.

An analysis of secreted proteins by the signal sequence trap method using a cDNA library of the rat pituitary anlage at embryonic days (E) 13.5 revealed the abundant expression of delta-like protein 1 (Dlk1) in the pituitary gland. Dlk1, an epidermal growth factor-like repeat protein in preadipocytes, functions in maintaining the preadipose state. Expression of Dlk1 mRNA in the pituitary at E13.5 and in the adult pituitary was confirmed by in situ hybridization. The expression pattern of Dlk1 during pituitary development was also studied by immunohistochemistry. Dlk1 protein first appeared in Rathke's pouch and the infundibulum at E11.5; as development proceeded, expression became restricted to the pars distalis and pars tuberalis (PT). Dlk1 was expressed in most ACTH cells during the embryonic stages, but its expression was limited to only a few ACTH cells in the adult pituitary. It was also expressed in a small population of TSH, GTH, and PRL cells throughout development, whereas it was present in the cytoplasm of most GH cells at all developmental stages. Similarly, Dlk1 was localized in the cytoplasm of PT cells during development. These findings provide new insights into the mechanism of Dlk1 regarding its regulation of pituitary hormone-secreting cells during development.

An intersection network based on combining SNP coassociation and RNA coexpression networks for feed utilization traits in Japanese Black cattle.

Genome-wide association studies (GWAS) of quantitative traits have detected numerous genetic associations, but they encounter difficulties in pinpointing prominent candidate genes and inferring gene networks. The present study used a systems genetics approach integrating GWAS results with external RNA-expression data to detect candidate gene networks in feed utilization and growth traits of Japanese Black cattle, which are matters of concern. A SNP coassociation network was derived from significant correlations between SNPs with effects estimated by GWAS across 7 phenotypic traits. The resulting network genes contained significant numbers of annotations related to the traits. Using bovine transcriptome data from a public database, an RNA coexpression network was inferred based on the similarity of expression patterns across different tissues. An intersection network was then generated by superimposing the SNP and RNA networks and extracting shared interactions. This intersection network contained 4 tissue-specific modules: nervous system, reproductive system, muscular system, and glands. To characterize the structure (topographical properties) of the 3 networks, their scale-free properties were evaluated, which revealed that the intersection network was the most scale-free. In the subnetwork containing the most connected transcription factors (URI1, ROCK2, and ETV6), most genes were widely expressed across tissues, and genes previously shown to be involved in the traits were found. Results indicated that the current approach might be used to construct a gene network that better reflects biological information, providing encouragement for the genetic dissection of economically important quantitative traits.

Structure and polymorphisms of the major histocompatibility complex in the Oriental stork, Ciconia boyciana.

The major histocompatibility complex (MHC) is highly polymorphic and plays a central role in the vertebrate immune system. Despite its functional consistency, the MHC genomic structure differs substantially among organisms. In birds, the MHCs of Galliformes and the Japanese crested ibis (Pelecaniformes) are well-characterized, but information about other avian MHCs remains scarce. The Oriental stork (Ciconia boyciana, order Ciconiiformes) is a large endangered migrant. The current Japanese population of this bird originates from a few founders; thus, understanding the genetic diversity among them is critical for effective population management. We report the structure and polymorphisms in C. boyciana MHC. One contig (approximately 128 kb) was assembled by screening of lambda phage genomic library and its complete sequence was determined, revealing a gene order of COL11A2, two copies of MHC-IIA/IIB pairs, BRD2, DMA/B1/B2, MHC-I, TAP1/2, and two copies each of pseudo MHC-I and TNXB. This structure was highly similar to that of the Japanese crested ibis, but largely different from that of Galliformes, at both the terminal regions. Genotyping of the MHC-II region detected 10 haplotypes among the six founders. These results provide valuable insights for future studies on the evolution of the avian MHCs and for conservation of C. boyciana.

Estimation of variance and genomic prediction using genotypes imputed from low-density marker subsets for carcass traits in Japanese black cattle.

The influence of genotype imputation using low-density single nucleotide polymorphism (SNP) marker subsets on the genomic relationship matrix (G matrix), genetic variance explained, and genomic prediction (GP) was investigated for carcass weight and marbling score in Japanese Black fattened steers, using genotype data of approximately 40,000 SNPs. Genotypes were imputed using equally spaced SNP subsets of different densities. Two different linear models were used. The first (model 1) incorporated one G matrix, while the second (model 2) used two different G matrices constructed using the selected and remaining SNPs. When using model 1, the estimated additive genetic variance was always larger when using all SNPs obtained via genotype imputation than when using only equally spaced SNP subsets. The correlations between the genomic estimated breeding values obtained using genotype imputation with at least 3,000 SNPs and those using all available SNPs without imputation were higher than 0.99 for both traits. While additive genetic variance was likely to be partitioned with model 2, it did not enhance the accuracy of GP compared with model 1. These results indicate that genotype imputation using an equally spaced low-density panel of an appropriate size can be used to produce a cost-effective, valid GP.

Accuracy of imputation of single nucleotide polymorphism marker genotypes from low-density panels in Japanese Black cattle.

Using target and reference fattened steer populations, the performance of genotype imputation using lower-density marker panels in Japanese Black cattle was evaluated. Population imputation was performed using BEAGLE software. Genotype information for approximately 40,000 single nucleotide polymorphism (SNP) markers by Illumina BovineSNP50 BeadChip was available, and imputation accuracy was assessed based on the average concordance rates of the genotypes, varying equally spaced SNP densities, and the number of individuals in the reference population. Two additional statistics were also calculated as indicators of imputation performance. The concordance rates tended to be lower for SNPs with greater minor allele frequencies, or those located near the ends of the chromosomes. Longer autosomes yielded greater imputation accuracies than shorter ones. When SNPs were selected based on linkage disequilibrium information, relative imputation accuracy was slightly improved. When 3000 and 10,000 equally spaced SNPs were used, the imputation accuracies were greater than 90% and approximately 97%, respectively. These results indicate that combining genotyping using a lower-density SNP chip with genotype imputation based on a population of individuals genotyped using a higher-density SNP chip is a cost-effective and valid approach for genomic prediction.

Bifunctional properties of peroxisome proliferator-activated receptor gamma1 in KDR gene regulation mediated via interaction with both Sp1 and Sp3.

Vascular endothelial growth factor receptor 2 (KDR) plays a critical role in mediating a variety of vasculogenic and angiogenic processes, including diabetic retinopathy. We previously demonstrated that the promoter activity of the KDR gene in retinal capillary endothelial cells (RCECs) was regulated in part by the relative concentration of positive/negative transcription factors Sp1/Sp3. We also reported that the peroxisome proliferator-activated receptor (PPAR)gamma ligand could inhibit intraocular angiogenesis. In the present study, the role of PPARgamma1 in KDR gene regulation in RCECs was examined. PPARgamma1 protein physically interacted with both Sp1 and Sp3. Transactivation and electrophoretic mobility shift assays clearly demonstrated novel findings that PPARgamma1 increased KDR promoter activity by enhancing the interaction between Sp1, but not Sp3, and KDR promoter region without its ligand in RCECs. The ligand-binding site but not the DNA binding site of PPARgamma1 enhanced the interaction between Sp1 and KDR promoter region. Conversely, PPARgamma1 ligand 15-deoxy Delta (12,14)-prostaglandin J2 dose-dependently suppressed the binding of KDR promoter region with both Sp1 and Sp3, resulting an inhibition of KDR gene expression. In conclusion, PPARgamma1 has bifunctional properties in the regulation of KDR gene expression mediated via interaction with both Sp1 and Sp3.

A congenic strain (F344.OLETF-Imfm) displays the existence of intramuscular fat accumulation QTL on rat chromosome 1.

A genomic region between D1Wox8 and D1Rat90 on rat chromosome 1 was previously shown to be linked to intramuscular fat accumulation by quantitative trait locus (QTL) analysis using a F2 population derived from the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, which exhibits an increase in the levels of intramuscular fat content in Musculus longissimus, and the F344 rat. There exist two regions showing major and minor lod peaks for linkage to intramuscular fat accumulation, in the chromosomal region. We constructed a congenic strain introgressing the OLETF allele on the minor but not the major lod peak region in the F344 rat strain. The congenic strain had higher levels of intramuscular fat content in Musculus longissimus than the inbred partner F344 rat, thereby proving the existence of a QTL, designated Imfm (for Intramuscular fat-minor), responsible for the intramuscular fat accumulation in the congenic region of the minor lod peak region of about 10 cM. The F344.OLETF-Imfm congenic strain might provide a refined tool for the analysis of the gene causing intramuscular fat accumulation.

Detection of a quantitative trait locus for intramuscular fat accumulation using the OLETF rat.

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model for obese type 2 diabetes. We showed that the OLETF rat exhibits higher levels of intramuscular fat content in Musculus longissimus as compared to the Fischer-344 (F344) rat. Our investigation was designed to identify quantitative trait loci (QTLs) contributing to the increased levels of intramuscular fat content by performing a whole-genome search using 108 F2 intercross obtained by mating the OLETF and the F344 rats. We identified one QTL responsible for intramuscular fat accumulation on rat chromosome 1 with a maximum lod score of 3.4, which accounts for 5% of the total variance. As expected, the OLETF allele corresponds to the increased levels of intramuscular fat content.

Predicting the phenotypic values of physiological traits using SNP genotype and gene expression data in mice.

Predicting phenotypes using genome-wide genetic variation and gene expression data is useful in several fields, such as human biology and medicine, as well as in crop and livestock breeding. However, for phenotype prediction using gene expression data for mammals, studies remain scarce, as the available data on gene expression profiling are currently limited. By integrating a few sources of relevant data that are available in mice, this study investigated the accuracy of phenotype prediction for several physiological traits. Gene expression data from two tissues as well as single nucleotide polymorphisms (SNPs) were used. For the studied traits, the variance of the effects of the expression levels was more likely to differ among the genes than were the effects of SNPs. For the glucose concentration, the total cholesterol amount, and the total tidal volume, the accuracy by cross validation tended to be higher when the gene expression data rather than the SNP genotype data were used, and a statistically significant increase in the accuracy was obtained when the gene expression data from the liver were used alone or jointly with the SNP genotype data. For these traits, there were no additional gains in accuracy from using the gene expression data of both the liver and lung compared to that of individual use. The accuracy of prediction using genes that were selected differently was examined; the use of genes with a higher tissue specificity tended to result in an accuracy that was similar to or greater than that associated with the use of all of the available genes for traits such as the glucose concentration and total cholesterol amount. Although relatively few animals were evaluated, the current results suggest that gene expression levels could be used as explanatory variables. However, further studies are essential to confirm our findings using additional animal samples.

Structure and polymorphism of the major histocompatibility complex class II region in the Japanese Crested Ibis, Nipponia nippon.

The major histocompatibility complex (MHC) is a highly polymorphic genomic region that plays a central role in the immune system. Despite its functional consistency, the genomic structure of the MHC differs substantially among organisms. In birds, the MHC-B structures of Galliformes, including chickens, have been well characterized, but information about other avian MHCs remains sparse. The Japanese Crested Ibis (Nipponia nippon, Pelecaniformes) is an internationally conserved, critically threatened species. The current Japanese population of N. nippon originates from only five founders; thus, understanding the genetic diversity among these founders is critical for effective population management. Because of its high polymorphism and importance for disease resistance and other functions, the MHC has been an important focus in the conservation of endangered species. Here, we report the structure and polymorphism of the Japanese Crested Ibis MHC class II region. Screening of genomic libraries allowed the construction of three contigs representing different haplotypes of MHC class II regions. Characterization of genomic clones revealed that the MHC class II genomic structure of N. nippon was largely different from that of chicken. A pair of MHC-IIA and -IIB genes was arranged head-to-head between the COL11A2 and BRD2 genes. Gene order in N. nippon was more similar to that in humans than to that in chicken. The three haplotypes contained one to three copies of MHC-IIA/IIB gene pairs. Genotyping of the MHC class II region detected only three haplotypes among the five founders, suggesting that the genetic diversity of the current Japanese Crested Ibis population is extremely low. The structure of the MHC class II region presented here provides valuable insight for future studies on the evolution of the avian MHC and for conservation of the Japanese Crested Ibis.

Identification of novel genetic markers and evaluation of genetic structure in a population of Japanese crested ibis.

Japanese population of the Japanese crested ibis Nipponia nippon was founded by five individuals gifted from the People's Republic of China. In order to exactly evaluate genetic structure, we first performed development of novel genetic makers using 89 microsatellite primer pairs of related species for cross-amplification. Of these, only three primer pairs were useful for the genetic markers. Additionally, we sequenced allelic PCR products of these three markers together with 10 markers previously identified. Most markers showed typical microsatellite repeat units, but two markers were not simple microsatellites. Moreover, over half of the markers did not have the same repeat units as those of the original species. These results suggested that development of novel genetic markers in this population by cross-amplification is not efficient, partly because of low genetic diversity. Furthermore, the cluster analysis by STRUCTURE program using 17 markers showed that the five founders were divided into two clusters. However, the genetic relationships among the founders indicated by the clustering seemed to be questionable, because the analysis relied largely on a small number of triallelic markers, in spite of the addition of the three useful markers. Therefore, more efficient methods for identifying large numbers of single nucleotide polymorphisms are desirable.

Genome-wide SNP and STR discovery in the Japanese crested ibis and genetic diversity among founders of the Japanese population.

The Japanese crested ibis is an internationally conserved, critically threatened bird. Captive-breeding programs have been established to conserve this species in Japan. Since the current Japanese population of crested ibis originates only from 5 founders donated by the Chinese government, understanding the genetic diversity between them is critical for an effective population management. To discover genome-wide single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs) while obtaining genotype data of these polymorphic markers in each founder, reduced representation libraries were independently prepared from each of the founder genomes and sequenced on an Illumina HiSeq2000. This yielded 316 million 101-bp reads. Consensus sequences were created by clustering sequence reads, and then sequence reads from each founder were mapped to the consensus sequences, resulting in the detection of 52,512 putative SNPs and 162 putative STRs. The numbers of haplotypes and STR alleles and the investigation of genetic similarities suggested that the total genetic diversity between the founders was lower, although we could not identify a pair with closely related genome sequences. This study provided important insight into protocols for genetic management of the captive breeding population of Japanese crested ibis in Japan and towards the national project for reintroduction of captive-bred individuals into the wild. We proposed a simple, efficient, and cost-effective approach for simultaneous detection of genome-wide polymorphic markers and their genotypes for species currently lacking a reference genome sequence.

QTL/microarray approach using pathway information.

BACKGROUND: A combined quantitative trait loci (QTL) and microarray-based approach is commonly used to find differentially expressed genes which are then identified based on the known function of a gene in the biological process governing the trait of interest. However, a low cutoff value in individual gene analyses may result in many genes with moderate but meaningful changes in expression being missed. RESULTS: We modified a gene set analysis to identify intersection sets with significantly affected expression for which the changes in the individual gene sets are less significant. The gene expression profiles in liver tissues of four strains of mice from publicly available microarray sources were analyzed to detect trait-associated pathways using information on the QTL regions of blood concentrations of high density lipoproteins (HDL) cholesterol and insulin-like growth factor 1 (IGF-1). Several metabolic pathways related to HDL levels, including lipid metabolism, ABC transporters and cytochrome P450 pathways were detected for HDL QTL regions. Most of the pathways identified for the IGF-1 phenotype were signal transduction pathways associated with biological processes for IGF-1's regulation. CONCLUSION: We have developed a method of identifying pathways associated with a quantitative trait using information on QTL. Our approach provides insights into genotype-phenotype relations at the level of biological pathways which may help to elucidate the genetic architecture underlying variation in phenotypic traits.

The W- and Z-linked EE0.6 sequences used for molecular sexing of captive Japanese crested ibis on Sado Island.

The Japanese crested ibis Nipponia nippon is a critically threatened bird. Accurate sexing is necessary to perform effective management of captive breeding toward a national project for a tentative release of the Japanese crested ibis on Sado Island. A PCR-based sexing method targeting a 0.6 kb EcoRI fragment (EE0.6) sequence on W chromosome with AWS03 and USP3 primers has been developed for the Japanese crested ibis. However, the primers were selected from the EE0.6 sequences from bird species other than the Japanese crested ibis. In this study, we determined the W- and Z-linked EE0.6 sequences in the Japanese crested ibis, and clarified Japanese crested ibis sequence mismatch in the binding sites of the primers. Further, we found no polymorphism in the primer binding sites among five founder birds for the Sado captive Japanese crested ibis population. These findings validated the PCR-based sexing method with the AWS03 and USP3 as accurate molecular sexing methods of captive Japanese crested ibis on the Sado Island. Additionally, we designed a primer set for a novel PCR-based sexing, based on the EE0.6 sequences obtained in this study. This novel sexing method may be useful for future ecological research following the release of Japanese crested ibis on Sado Island. This is the first report to show the EE0.6 sequences in Japanese crested ibis.

Non-invasive sampling technique for DNA extraction from captive Japanese Crested Ibis on Sado Island.

The Japanese Crested Ibis Nipponia nippon is a critically threatened bird. The post-hatch eggs of the current captive population of this species on Sado Island have been stored at room temperature for the long-term. In this study, we investigated the suitability of the vascularized chorioallantois membrane from the eggs as a non-invasive DNA source. Using microsatellite loci developed for the Japanese Crested Ibis, we performed three experiments for comparison of genotypes obtained among DNA. First, DNA from five different sites of the identical membrane showed the same genotypes at either of two loci examined. Second, DNA from the membrane of each full-sibling birds and blood of their parents showed the genotypes that were consistent with Mendelian parent-offspring relationships at any of eight loci examined. Third, DNA from the membrane and blood of the same bird showed the matched genotypes at any of eight loci examined. These results indicate that the vascularized chorioallantois membrane from post-hatch eggs stored at room temperature for the long- term can be used as a reliable DNA source of offspring that had hatched from the egg. This study will promote a molecular genetics study on genetic diversity of the current captive Japanese Crested Ibis population on Sado Island.

Identification of positional candidates for bovine placental genes responsible for early embryonic death during cloning-attempted pregnancy.

Our previous study detected 291 and 77 genes showing early embryonic death-associated elevation and reduction of expression, respectively, in the fetal placenta of the cow carrying somatic nuclear transfer-derived cloned embryo. In this study, we mapped the 10 genes showing the elevation and the 10 genes doing the reduction most significantly, using somatic cell hybrid and bovine draft genome sequence. We then compared the mapped positions for these genes with the genomic locations of bovine quantitative trait loci for still-birth and/or abortion. Among the mapped genes, peptidylglycine alpha-amidating monooxygenase (PAM), spectrin, beta, nonerythrocytic 1 (SPTBNI), and an unknown novel gene containing AU277832 expressed sequence tag were intriguing, in that the mapped positions were consistent with the genomic locations of bovine still-birth and/or abortion quantitative trait loci, and thus identified as positional candidates for bovine placental genes responsible for the early embryonic death during the pregnancy attempted by somatic nuclear transfer-derived cloning.