Study On The Molecular Genetic Diversity Of The Crested Ibis(Nipponia Nippon)

The Crested Ibis Nipponia nippon was once thought to have become extinction in the wild in the late1970s. However, the re-discovery of the remnant population consisting of two pairs of adults and three nestlings provided the last opportunity to restore this species in wild and in captivity. In this study, sex identification, mtDNA heteroplasmy and population diversity of Crested Ibis were carried out based on261individuals. The aim of the study is to provide basic scientific information of genetic background to the conservation program of this endangered species. In this study, we found that:1. The primer set2550F/2718R was firstly used to identify sex of Crested Ibis, all the female birds get two amplicons while male only get one. The sequencing result showed the primer set amplified partial sequences spaning the16th intron of two homologous CHD1genes located in the Z and W chromosome. An improved primer set of2467F/2530R was re-designed to be specific to Crested Ibis following their conserved sequences derived from the2550F/2718R primers. The PCR products of the new primers were conveniently visualized with two bands of552base pairs bp and358bp for females, but a single band of552bp for males in routine1.8%agarose gel.2. The complete control region was directly sequenced while the distribution pattern and inheritance of the length variations were examined using both direct sequencing and genotyping of the PCR fragments from captive birds with pedigrees, wild birds and a historical specimen. Our results demonstrated that there was no structural variation in the control region, however, different numbers of short tandem repeats with an identical motif of11bp repeat at the3’-end of the control region determined the length polymorphisms among and heteroplasmy within individual birds. The analysis of pedigreed samples also sheds light on the transmission of mtDNA length heteroplasmy in birds following the genetic bottleneck theory.3. Magnetic beads enrichment method was used to isolate new microsatellite loci of Crested Ibis. In total,118positive clones were chose to sequenced,32primer sets were designed accorded to the sequences and only12primers amplified single consistent products, among them,3microsatellite loci showed polymorphism. Twenty four exist microsatellite loci of Crested Ibis and10loci of scarlet ibis were also used to screen and11loci showed polymorphism.4. The14polymorphism loci were ued to study the population diversity of261individuals from9populations of Crested Ibis. The results demonstrated there were31alleles from the14loci, the captive and wild populations hold low genetic differentiation, the Fst was0.032(P<0.001). The inbreeding coefficient of all populations was0.113(P<0.001), approved high inbreeding of Crested Ibis.5. The population structure of the33pedigreed individuals showed they hold distinct genetic background among the three families, implied the breeding project of the captive population should consider the genetic information.. Siblings in the same nest of the captive population showed no variation of genetic structure from each other while siblings form58%and57%nests of the wild populations from2011and2012showed different form each other.6. The two copies of the MHC class Ⅱ B gene of Crested Ibis was confirmed in this study for the first time. Genotyping of the exon2of these two genes of70captive and145wild Crested lbis showed there were2and4alleles in the two copies respectively, and the populations shared all the alleles. Our data also demonstrated the frequency of rare alleles in the two genes is reduced in the wild populations compared to the captive population.