Hiroaki Iwaisaki

QTL/microarray approach using pathway information

BackgroundA 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.ResultsWe 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-1s regulation.ConclusionWe 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.

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.

Restricted best linear unbiased prediction using canonical transformation

neous equations if there are many traits and a large number of animals to be evaluated. In this paper, a canonical transformation technique through which new independent traits are introduced is presented. Thus only equations of relatively low order for each transformed trait have to be solved. Furthermore, it is shown that the number of independent transformed traits is reduced by the number of restrictions imposed. The technique is applicable when a multiple-trait animal model is assumed.

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.

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.

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.

Estimation of breeding values from large-sized routine carcass data in Japanese Black cattle using Bayesian analysis

Volumes of official data sets have been increasing rapidly in the genetic evaluation using the Japanese Black routine carcass field data. Therefore, an alternative approach with smaller memory requirement to the current one using the restricted maximum likelihood (REML) and the empirical best linear unbiased prediction (EBLUP) is desired. This study applied a Bayesian analysis using Gibbs sampling (GS) to a large data set of the routine carcass field data and practically verified its validity in the estimation of breeding values. A Bayesian analysis like REML-EBLUP was implemented, and the posterior means were calculated using every 10th sample from 90,000 of samples after 10,000 samples discarded. Moment and rank correlations between breeding values estimated by GS and REML-EBLUP were very close to one, and the linear regression coefficients and the intercepts of the GS on the REML-EBLUP estimates were substantially one and zero, respectively, showing a very good agreement between breeding value estimation by the current GS and the REML-EBLUP. The current GS required only one-sixth of the memory space with REML-EBLUP. It is confirmed that the current GS approach with relatively small memory requirement is valid as a genetic evaluation procedure using large routine carcass data.

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.

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.

Efficient computation of the inverse of gametic relationship matrix for a marked QTL.

Best linear unbiased prediction of genetic merits for a marked quantitative trait locus (QTL) using mixed model methodology includes the inverse of conditional gametic relationship matrix (G-1) for a marked QTL. When accounting for inbreeding, the conditional gametic relationships between two parents of individuals for a marked QTL are necessary to build G-1 directly. Up to now, the tabular method and its adaptations have been used to compute these relationships. In the present paper, an indirect method was implemented at the gametic level to compute these few relationships. Simulation results showed that the indirect method can perform faster with significantly less storage requirements than adaptation of the tabular method. The efficiency of the indirect method was mainly due to the use of the sparseness of G-1. The indirect method can also be applied to construct an approximate G-1 for populations with incomplete marker data, providing approximate probabilities of descent for QTL alleles for individuals with incomplete marker data.