| The crested ibis (Nipponia nippon), as a globally endangered species with state grade I class protection, was ever widely dispersed in East Asia. However, during the19th to20th centuries, it declined dramatically and was near extinction due to the human and natural impact. The seven wild birds rediscovered in Yangxian. Shanxi province, China, became the founders of all the current individuals. Nowadays, it has increased to2000birds mainly distributed in one wild and six captive populations. Nevertheless, owing to the severe bottleneck effect and intensive inbreeding, it is still at risk under changeably environmental pressures.The major histocompatibility complex (MHC) is a multigene family with striking diversity in the vertebrate immune system. It encodes protein receptors that can recognize and present pathogen-derived peptides, triggering the adaptive immune reaction. Thus, it has become a popular adaptive molecular marker. However, previous studies of MHC mainly focused on the level of alleles, and the relative studies of crested ibis are deficient. Accordingly, based on the BAC library with physics map constructed by our laboratory, we established a marker system of all the Nini MHC class Ⅰ and Ⅱ genes among seven populations with307individuals. Also, based on the multilocus MHC haplotype and microsatellite markers, we discussed the evolutionary mechanism of MHC genes, genetic variation, population differentiation, adaptive potentials and conservation strategies. The main results are as follow:(1) The exons2and3genotyping results of the five la loci showed that Nini-UCA and-UEA were monomorphic whereas Nini-UAA,-UBA and-UDA were dimorphic. There was only a single silent nucleotide variation between the two alleles of both UBA and UDA. The expression detection showed that UAA and UEA were strongly and weakly expressed in all tissues respectively, and UBA, UCA and UDA were all expressed in various levels among tissues. The full-length cDNA sequences indicated that UEA was premature in exon5. According to the results described above and sequence analysis, we suggested that UAA was a major gene, UBA, UCA and UDA were minor genes, and UEA was a nonclassical gene.(2) Based on the recombination and phylogenetic analysis, we proposed that UBA and UCA (or UDA) might be the ancestral major and minor genes respectively, and generated UAA and UEA through gene duplication and recombination. The presence of UCA and UDA with nearly identical full-length genomic sequences also revealed a recent gene duplication event.(3) The exon2genotyping results of the four Ⅱα (DAA) and four Ⅱβ (DAB) loci showed that all loci were monomorphic except Nini-DAB1with four alleles. However, the distribution of three DAA genes (DAA2,3,4) and three DAB genes (DAB2,3,4) was various among individual, resulting in the structural diversity. All alleles were expressed. Based on the distribution and organization of the eight class Ⅱ genes and the major class I gene UAA, seven different haplotypes were identified. Genomic sequences of the entire class Ⅱ region with structural diversity among seven haplotypes were further obtained.(4) The seven haplotypes can be divided into four groups, i.e. HT01(haplotype of BAC library), HT02and06, HT03and07, HT04and05, respectively with4,3,2and1repeated "αβ" units containing a set of DAA and DAB locus. Each group has the same class Ⅱ region but different alleles of UAA. The full-length sequences of the three duplicated genes DAA2,3,4were nearly identical and all divergent from DAA1across all haplotypes. The DAB genes were more complicated for the mosaic structures of several recombinant sequences. However, all the divergent regions were also dimorphic (the polymorphic exon2was also clustered into two groups).(5) Based on the recombination and phylogenetic analysis we proposed an evolutionary model for the Nini MHC haplotypes:HT03and07with the two "αβ" units corresponding to the two previously proposed ancestral class Ⅱ lineage genes derived from gene duplication in avain, might be the ancestral Nini haplotypes and may even represent the original structure of avain MHC class Ⅱ region. Through two rounds of unequal crossing over accompanied by homologous recombination between the two ancestral Nini Ⅱβ loci, different haplotypes with structural diversity and mosaic DAB sequences were generated, and were further diversified through recombination between the class Ⅱ region and the class Ⅰ locus UAA.(6) There were two evolutionary fates for the Nini class Ⅰ and Ⅱ duplicated genes: Some gene copies maintained the original sequences, resulting in the genetic redundancy with benefits from buffering effect and extra products. Other gene copies rapidly evolved into divergent functional sequences under positive selection. Besides, the common roles of gene duplication, gene conversion and recombination events in class Ⅰ and Ⅱ genes indicated that the crested ibis may have undergone a common evolutionary pattern, namely, birth-and-death model and concerted evolution in different timescales. (7) Population analysis based on the Nini MHC haplotypes and microsatellite markers showed:depleted genetic diversities were shown by both markers and their allelic richness was significantly correlated. With the strong detected historical positive selection, the crested ibis might harbor high level of MHC diversity in history. However, the selection was unable to counter the drift effect, and the diversities of both markers were depleted after the bottleneck effect. Same genetic differentiation for both markers was detected among Seven Nini populations which can be divided into group1(DQ, YX, LGT and Wild), group2(SD and HN), and group3(BJ). Group1maintained the most comprehensive genetic resource, whereas group2and3were genetically monomorphic. By comparing the population structures indicated by MHC and microsatellite, we found that the selection pressures were slight and nearly uniform across the seven populations, and the founder effect was actually the main cause of population differentiation.(8) MHC haplotype should be taken as an evaluating index while making conservation and management strategies for crested ibis. Differences in haplotypes and current population structure should be taken into consideration during captive breeding, individual exchange, captive population establishment and reintroduction. The correlation between the structurally diversified MHC haplotypes and immunological diseases should be analyzed in the future studies. |
PDF Full Text Request