Yoshinori Kaneko

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.

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.

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 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 Peoples 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.

Genetic Analyses in the Japanese Captive Population of Japanese Crested Ibis (Nipponia nippon) using Pedigree Information

The Japanese captive population of Japanese crested ibis (Nipponia nippon) was established using 5 founders derived from the Chinese captive population. Its size has increased rapidly, and the maintenance phase is about to start. Thus, this study was designed to perform genetic analyses in this population with pedigree information, considering the adoption of mean kinship strategy as the breeding strategy suited to the maintenance phase. Because the relationships among the 5 founders were unknown, different assumptions were set up ranging from 0 to 0.25 of kinship coefficients between the 5 founders. Assuming that the 5 founders were non-inbred in all the assumptions, the results showed that the gene diversity and the mean inbreeding coefficient would fluctuate largely from ∼65% to ∼82% and from ∼0.07 to ∼0.29, respectively. Moreover, the genetic importance of individuals based on mean kinship shifted largely. This study suggested that the Japanese captive population had low gene diversity and high mean inbreeding coefficient even under the assumption that the 5 founders were unrelated and non-inbred. In addition, the study also suggested that it became more effective to analyze the genetic status and to introduce mean kinship strategy into this population with more credible molecular evaluation of the relationships among founders.

A proteomic analysis of Nipponia nippon (ID#162)

We investigated the proteome of a female Crested Ibis (Nipponia nippon, ID#162) that died on March 10, 2010 at the Sado Japanese Crested Ibis Conservation Center. Protein preparations from the brain, trachea, liver, heart, lung, proventriculus, muscular stomach, small intestine, duodenum, ovary and neck muscle were subjected to in-solution shotgun mass spectrometry (MS)/MS analyses using an LTQ Orbitrap XL mass spectrometer. A search of the National Center for Biotechnology Information Gallus gallus databases revealed 4253 GI (GenInfo Identifier) numbers with the sum of the same 11 tissues examined in the Crested Ibis. To interpret the obtained proteomics data, it was verified in detail with the data obtained from the brain of the Crested Ibis. It has been reported that drebrin A is specifically expressed in adult chicken brain. In the shotgun proteomic analyses of the Crested Ibis, we identified drebrin A as a brain-specific protein. Furthermore, Western blotting analysis of the protein preparations from 10 tissues of the Crested Ibis and 150-day-old hens using anti-drebrin antibodies showed intensive expression of approximately 110 kDa polypeptides of drebrin in both brains. We believe firmly that the present data will contribute to initial and fundamental steps toward understanding the Crested Ibis proteome.