Lack of Structural Variation but Extensive Length Polymorphisms and Heteroplasmic Length Variations in the Mitochondrial DNA Control Region of Highly Inbred Crested Ibis,Nipponia nippon

The animal mitochondrial DNA (mtDNA) length polymorphism and heteroplasmy are accepted to be universal. Here we report the lack of structural variation but the presence of length polymorphism as well as heteroplasmy in mtDNA control region of an endangered avian species – the Crested Ibis (Nipponia nippon). 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 of CA₃CA₂CA₃ at the 3′-end of the control region determined the length polymorphisms among and heteroplasmy within individual birds. There were one to three predominant fragments in every bird; nevertheless multiple minor fragments coexist in all birds. These extremely high polymorphisms were suggested to have derived from the ‘replication slippage’ of a perfect microsatellite evolution following the step-wise mutational model. The patterns of heteroplasmy were found to be shifted between generations and among siblings but rather stable between blood and feather samples. This study provides the first evidence of a very extensive mtDNA length polymorphism and heteroplasmy in the highly inbred Crested Ibis which carries an mtDNA genome lack of structural genetic diversity. The analysis of pedigreed samples also sheds light on the transmission of mtDNA length heteroplasmy in birds following the genetic bottleneck theory. Further research focusing on the generation and transmission of particular mtDNA heteroplasmy patterns in single germ line of Crested Ibis is encouraged by this study.     

Gene duplication and fragmentation in the zebra finch major histocompatibility complex

Background

Due to its high polymorphism and importance for disease resistance, the major histocompatibility complex (MHC) has been an important focus of many vertebrate genome projects. Avian MHC organization is of particular interest because the chicken Gallus gallus, the avian species with the best characterized MHC, possesses a highly streamlined minimal essential MHC, which is linked to resistance against specific pathogens. It remains unclear the extent to which this organization describes the situation in other birds and whether it represents a derived or ancestral condition. The sequencing of the zebra finch Taeniopygia guttata genome, in combination with targeted bacterial artificial chromosome (BAC) sequencing, has allowed us to characterize an MHC from a highly divergent and diverse avian lineage, the passerines.

Results

The zebra finch MHC exhibits a complex structure and history involving gene duplication and fragmentation. The zebra finch MHC includes multiple Class I and Class II genes, some of which appear to be pseudogenes, and spans a much more extensive genomic region than the chicken MHC, as evidenced by the presence of MHC genes on each of seven BACs spanning 739 kb. Cytogenetic (FISH) evidence and the genome assembly itself place core MHC genes on as many as four chromosomes with TAP and Class I genes mapping to different chromosomes. MHC Class II regions are further characterized by high endogenous retroviral content. Lastly, we find strong evidence of selection acting on sites within passerine MHC Class I and Class II genes.

Conclusion

The zebra finch MHC differs markedly from that of the chicken, the only other bird species with a complete genome sequence. The apparent lack of synteny between TAP and the expressed MHC Class I locus is in fact reminiscent of a pattern seen in some mammalian lineages and may represent convergent evolution. Our analyses of the zebra finch MHC suggest a complex history involving chromosomal fission, gene duplication and translocation in the history of the MHC in birds, and highlight striking differences in MHC structure and organization among avian lineages.     

陕西宁陕朱鹮再引入种群个体死亡原因分析

2007年5月至2013年5月,通过环志和无线电跟踪及野外观察和记录,对陕西宁陕朱鹮(Nipponia nippon)再引入种群的死亡个体进行了收集和确定。共计死亡朱鹮54只,其中雏鸟26只,亚成体8只,释放朱鹮个体均为成体,死亡总数20只。研究表明,朱鹮死亡的原因包括巢内竞争、亲鸟弃巢、雏鸟先天发育不良;天敌、食物缺乏、飞行撞击、高压电击、恶劣天气等。同时,针对上述原因,提出一些合理的保护措施和管理建议。     

Reduced Dependence of Crested Ibis on Winter-Flooded Rice Fields: Implications for Their Conservation

The Crested Ibis Nipponia nippon was once thought to be extinct in the wild until seven birds were discovered in a remote mountain village in China in 1981. Studies suggested that winter-flooded rice fields play an essential role in nest site selection by the Crested Ibis and hence in their survival. Considerable efforts were therefore made to conserve the winter-flooded rice fields, but these have caused conflicts between the agricultural and conservation communities. The population and geographical range of the wild Crested Ibis has expanded greatly since 1981, but there is no spatial information on the winter-flooded rice fields, nor on the current association of nest sites and winter-flooded rice fields. We mapped winter-flooded rice fields across the entire current range of Crested Ibis using innovative remote sensing and geographical information systems (GIS) techniques. The spatial relationships between the nest site clusters and winter-flooded rice fields were quantified using Ward''s hierarchical clustering method and Ripley''s K-function. We show that both have significantly clumped distribution patterns and that they are positively associated. However, the dependence of Crested Ibis on the winter-flooded rice fields varied significantly among the nest site clusters and has decreased over the years, indicating the absence of winter-flooded rice fields is not constraining their recovery and population expansion. We therefore recommend that efforts should be made to protect the existing winter-flooded rice fields and to restore the functionality of natural and semi-natural wetlands, to encourage both in-situ conservation and the re-introduction of the Crested Ibis. In addition, we recommend that caution should be exercised when interpreting the habitat requirements of species with a narrow distribution, particularly when that interpretation is based only on their current habitat.     

不同野化训练条件下朱鹮的行为差异

2010年7月至201 1年1月,在陕西省洋县朱鹮生态园和华阳镇朱鹮野化训练基地,采用瞬时扫描取样法和行为取样法,对2处野化训练大网笼中朱鹮(Nipponia nippon)(n洋县=30只;n华阳=22只)的行为进行研究,同时调查2处大网笼野化训练条件的不同.结果表明,在觅食行为的时间分配中,秋季洋县群的划动寻觅、探...     

北戴河朱鹮野化种群非繁殖期日间活动时间分配和行为节律

于2019年7月-2020年1月对北戴河朱鹮野化种群非繁殖期的日间活动时间分配和行为节律进行了分析。结果表明,休息（41.8%）、觅食（39.7%）和理羽（13.3%）是北戴河朱鹮非繁殖期的主要行为。在行为节律上,有3个觅食高峰、2个休息高峰和2个理羽高峰。成鸟和幼鸟行为分配相似,但成鸟的警戒和社群等其他行为比例显著较高。与野生种群相比,北戴河野化种群的觅食时间显著较少,主要原因是野生个体需要花费较多时间搜寻食物,且飞行等活动的能耗较高。朱鹮在冬季的休息和理羽行为比例高于夏秋季,觅食行为则相反,这样可以在低温和大风天气降低能耗,是对北戴河气候环境的有效适应。为了提高朱鹮对多种气候环境的适应能力,为今后北戴河朱鹮的再引入奠定基础,我们建议在冬季要确保朱鹮有充足的适宜觅食地,同时在野化网笼中进行必要的环境丰容,如在网笼内增加常绿树种和阔叶树种,部分侧网上安装防风板,或在网笼中设置防风避寒的伞棚等设施,以供朱鹮进行选择。     

Isolation and characterization of three class II MHC genomic clones from the chicken

A genomic library was constructed from sperm DNA from an individual of the inbred chicken line G-B2, MHC haplotype B6. The library was screened with a chicken class II probe (beta 2 exon specific) and three MHC class II beta chain genomic clones were isolated. The restriction maps of the three clones showed that each of the three clones was unique. The position of the beta chain sequence was located in each of the three genomic clones by Southern blot hybridization. Subclones containing the beta chain gene were produced from each of the genomic clones and the orientation of the leader peptide, beta 1, beta 2, transmembrane, and cytoplasmic exons was determined by Southern blot hybridization and nucleotide sequencing. The complete nucleotide sequence of two of the three subclones was determined. Comparison of the nucleotide and predicted amino acid sequences of the two subclones with other class II beta chain sequences showed that the B6 chicken beta chain genes are evolutionarily related to the class II beta chain genes from chickens of other MHC haplotypes, and to class II beta chain genes from other species. Analysis of Southern blots of B6 chicken DNA, as well as the isolation of the three beta chain genes, suggests that chickens of the B6 haplotype possess at least three MHC class II beta chain genes.     

Public attitudes and willingness to pay toward the conservation of Crested Ibis: Insights for management

In order to better design conservation programs to conserve threatened species, social, economic and political factors must be carefully considered. The wild population of the endangered Crested Ibis (Nipponia nippon) is currently only distributed in Yangxian County, Shaanxi province of China, and as the gradual dispersal of its population from the mountains to the plains, the possibility of human-wildlife interaction has increased, which poses certain challenges for conservation and management. Here, we used a questionnaire to investigate public attitudes and willingness to pay (WTP) toward Crested Ibis conservation, and to evaluate relevant influencing factors, with a view to providing recommendations for its management. The results showed that local residents strongly support the conservation of ibis and factors like education level, income, residential area, perception for ibis and prior experiences significantly affected their support for conservation. Results also indicated that the annual WTP was US$18.28 per household, and the potential value of donations is about US$2.45 million per year for Crested Ibis conservation. Regression analysis indicated that income, perception for ibis, prior experience, support for conservation and environmental attitudes had a significant effect on WTP. The comparison between the original research value of the ibis and the predicted value from the Meta-regression analysis function indicated that the benefit transfer values are able to inform policy decisions. Our results can be applied not only to the design of tailored outreach programs and management policies for the Crested Ibis, but also to facilitate conservation and management of other endangered species by encouraging decision makers to use benefit transfer models for the rapid valuation in the absence of species economic information.     

基于人工巢试验分析黄腹角雉巢卵捕食者

2007年至今,我国在陕西宁陕、铜川和千阳,以及河南董寨和浙江德清初步建立了朱鹮(Nipponia nippon)野化放归种群。鸟类的繁殖行为是其生活史策略的重要组成部分,可以反映动物个体的营养状态、所处栖息地的质量,以及对栖息地的适应等。本文报道浙江德清朱鹮野化放归种群的繁殖行为,旨在了解其在我国南方的适应情况,为最终建立中国朱鹮南方种群提供科学支撑。本研究于2018年4和5月及2020年3至5月对浙江德清野化放归朱鹮的3个繁殖巢进行实时监测,记录孵卵期和育雏期繁殖行为,总记录时长为134 d,有效数据时长为2 958 h。利用线性混合模型探究朱鹮衔巢材频率、孵卵期以及育雏期行为的影响因子,采用Spearman相关分析检验朱鹮翻卵频率与孵卵进程的关系,建立Logistic回归模型探究朱鹮亲鸟暖雏时长随雏鸟日龄的变化情况,并利用one-way ANOVA分析德清朱鹮与陕西洋县野生种群和其他野化放归种群的换巢频率差异。结果表明,德清朱鹮平均窝卵数3.7 ± 0.3,孵化成功率90.9%,雏鸟出飞率100.0%;繁殖的不同时期、营巢条件和亲鸟性别均显著影响朱鹮的衔巢材频率;朱鹮的翻卵频率随孵卵进程显著下降;亲鸟的暖雏时长在雏鸟11日龄时下降率最大;育雏的不同时期对朱鹮的暖雏时长、换巢频率以及喂雏频率有极显著的影响;环境温度较高可能是导致德清朱鹮比洋县野生种群提前产卵的原因之一;较高的平均窝卵数与出飞成功率表明,朱鹮对我国南方的栖息地较为适应。鉴于本研究发现利用人工巢筐营巢的亲鸟花费更多时间补充巢材,表明人工巢筐的结构和大小有必要优化,建议在今后设计时加大筐壁仰角并减小深度、扩大外径,使人工巢筐更接近于自然巢的盘状结构,同时,应将巢筐底部设计成多孔透气、利于排水的结构,以适应我国长江中下游地区湿热多雨的气候。     

Phylogeography of the Japanese greater horseshoe bat Rhinolophus nippon (Mammalia: Chiroptera) in Northeast Asia: New insight into the monophyly of the Japanese populations

The Japanese greater horseshoe bat (Rhinolophus nippon) is distributed widely in East Asia. Within the species, R. nippon in Northeast Asia is regarded as the lineage that diverged most recently. However, the monophyly of the Japanese populations is unclear due to insufficient data about phylogenetic relationship of the western Japanese populations. To test the monophyly of the Japanese populations of R. nippon, we sampled R. nippon from western Japan and performed a phylogeographic analysis based on mitochondrial DNA cytochrome b and the D‐loop. The Northeast Asian lineage consisted of three main clades in eastern Japan (clade I), western Japan (clade II), and the continent as well as the Kumamoto population in westernmost Japan (clade III). The results of this study do not support the monophyly of the Japanese population. The findings suggest the “reverse colonization” of R. nippon from the Japanese Archipelago to the Eurasian continent, and provide important insight into the role of the island system in creation and supply of diversity to the continent.     

Multiple divergent haplotypes express completely distinct sets of class I MHC genes in zebrafish

The zebrafish is an important animal model for stem cell biology, cancer, and immunology research. Histocompatibility represents a key intersection of these disciplines; however, histocompatibility in zebrafish remains poorly understood. We examined a set of diverse zebrafish class I major histocompatibility complex (MHC) genes that segregate with specific haplotypes at chromosome 19, and for which donor-recipient matching has been shown to improve engraftment after hematopoietic transplantation. Using flanking gene polymorphisms, we identified six distinct chromosome 19 haplotypes. We describe several novel class I U lineage genes and characterize their sequence properties, expression, and haplotype distribution. Altogether, ten full-length zebrafish class I genes were analyzed, mhc1uba through mhc1uka. Expression data and sequence properties indicate that most are candidate classical genes. Several substitutions in putative peptide anchor residues, often shared with deduced MHC molecules from additional teleost species, suggest flexibility in antigen binding. All ten zebrafish class I genes were uniquely assigned among the six haplotypes, with dominant or codominant expression of one to three genes per haplotype. Interestingly, while the divergent MHC haplotypes display variable gene copy number and content, the different genes appear to have ancient origin, with extremely high levels of sequence diversity. Furthermore, haplotype variability extends beyond the MHC genes to include divergent forms of psmb8. The many disparate haplotypes at this locus therefore represent a remarkable form of genomic region configuration polymorphism. Defining the functional MHC genes within these divergent class I haplotypes in zebrafish will provide an important foundation for future studies in immunology and transplantation.     

Dominant Sequences of Human Major Histocompatibility Complex Conserved Extended Haplotypes from HLA-DQA2 to DAXX

We resequenced and phased 27 kb of DNA within 580 kb of the MHC class II region in 158 population chromosomes, most of which were conserved extended haplotypes (CEHs) of European descent or contained their centromeric fragments. We determined the single nucleotide polymorphism and deletion-insertion polymorphism alleles of the dominant sequences from HLA-DQA2 to DAXX for these CEHs. Nine of 13 CEHs remained sufficiently intact to possess a dominant sequence extending at least to DAXX, 230 kb centromeric to HLA-DPB1. We identified the regions centromeric to HLA-DQB1 within which single instances of eight “common” European MHC haplotypes previously sequenced by the MHC Haplotype Project (MHP) were representative of those dominant CEH sequences. Only two MHP haplotypes had a dominant CEH sequence throughout the centromeric and extended class II region and one MHP haplotype did not represent a known European CEH anywhere in the region. We identified the centromeric recombination transition points of other MHP sequences from CEH representation to non-representation. Several CEH pairs or groups shared sequence identity in small blocks but had significantly different (although still conserved for each separate CEH) sequences in surrounding regions. These patterns partly explain strong calculated linkage disequilibrium over only short (tens to hundreds of kilobases) distances in the context of a finite number of observed megabase-length CEHs comprising half a population''s haplotypes. Our results provide a clearer picture of European CEH class II allelic structure and population haplotype architecture, improved regional CEH markers, and raise questions concerning regional recombination hotspots.     

Isolation of a 97-kb minimal essential MHC B locus from a new reverse-4D BAC library of the golden pheasant

The bacterial artificial chromosome (BAC) system is widely used in isolation of large genomic fragments of interest. Construction of a routine BAC library requires several months for picking clones and arraying BACs into superpools in order to employ 4D-PCR to screen positive BACs, which might be time-consuming and laborious. The major histocompatibility complex (MHC) is a cluster of genes involved in the vertebrate immune system, and the classical avian MHC-B locus is a minimal essential one, occupying a 100-kb genomic region. In this study, we constructed a more effective reverse-4D BAC library for the golden pheasant, which first creates sub-libraries and then only picks clones of positive sub-libraries, and identified several MHC clones within thirty days. The full sequencing of a 97-kb reverse-4D BAC demonstrated that the golden pheasant MHC-B locus contained 20 genes and showed good synteny with that of the chicken. The notable differences between these two species were the numbers of class II B loci and NK genes and the inversions of the TAPBP gene and the TAP1-TAP2 region. Furthermore, the inverse TAP2-TAP1 was unique in the golden pheasant in comparison with that of chicken, turkey, and quail. The newly defined genomic structure of the golden pheasant MHC will give an insight into the evolutionary history of the avian MHC.     

Differential recombination dynamics within the MHC of macaque species

A panel of 15 carefully selected microsatellites (short tandem repeats, STRs) has allowed us to study segregation and haplotype stability in various macaque species. The STRs span the major histocompatibility complex (MHC) region and map in more detail from the centromeric part of the Mhc-A to the DR region. Two large panels of Indian rhesus and Indonesian/Indochinese cynomolgus macaques have been subjected to pedigree analysis, allowing the definition of 161 and 36 different haplotypes and the physical mapping of 10 and 5 recombination sites, respectively. Although most recombination sites within the studied section of the Indian rhesus monkey MHC are situated between the Mhc-A and Mhc-B regions, the resulting recombination rate for this genomic segment is low and similar to that in humans. In contrast, in Indonesian/Indochinese macaques, two recombination sites, which appear to be absent in rhesus macaques, map between the class III and II regions. As a result, the mean recombination frequency of the core MHC, Mhc-A to class II, is higher in Indonesian/Indochinese cynomolgus than in Indian rhesus macaques, but as such is comparable to that in humans. The present communication demonstrates that the dynamics of recombination ‘hot/cold spots’ in the MHC, as well as their frequencies, may differ substantially between highly related macaque species.     

家禽MHC结构研究进展

禽主要组织相容性复合体(Major histocompatibility complex,MHC)的结构与禽病防控、禽免疫学、禽类遗传学研究密切相关。文章对鸡、火鸡、鹌鹑、鸭和鹅的MHC结构方面的研究进展进行了综述,表明其有以下共同特点:都有保守的MHC区域,包括MHC I基因和MHC II基因及一些功能未知基因;基因排列简单而紧凑;MHC I基因内含子的长度都比哺乳动物小;鸡、火鸡、鸭和鹅的MHC I基因组序列都有8个外显子和7个内含子,MHC IIβ基因组序列都有6个外显子和5个内含子;鸡、火鸡和鹌鹑的BG基因结构模式相同;都存在微卫星重复单元。但也存在种属差异:鸡的MHC I基因和MHC II基因是双拷贝,而鸭、鹅和鹌鹑有若干个拷贝;BG基因的拷贝数及其外显子数目不同。对主要家禽MHC结构进行分析比较,将有利于对禽病学及禽免疫遗传学的进究。     

MHC polymorphism in Caribbean African green monkeys

African green monkeys (AGM) are among the most widely used nonhuman primate models used in various fields of medical research. One species of AGM that originated from West Africa, Chlorocebus sabaeus, was introduced three centuries ago in the Caribbean islands. We present here a systematic study of the major histocompatibility complex (MHC) polymorphism of Caribbean AGM which is currently frequently used as an animal model. We studied 54 animals originated from Barbados (N?=?25) or Saint Kitts (N?=?29). The MHC polymorphism was characterized by means of 17 MHC microsatellites spread across MHC and DRB genotyping by DGGE sequencing. We defined nine frequent MHC haplotypes of which two were found in the two insular populations suggesting either past exchanges between the two populations or a common origin of the founders of the two populations. By the analysis of a previously described EST library, we characterized 38 MHC cDNA sequences (17 class I and 21 class II). In conclusion, we characterized for the first time the MHC polymorphism of Barbados and Saint Kitts AGM. We found a restricted polymorphism due to a founding effect, which is responsible for a strong bottleneck. The poorness of MHC polymorphism observed in the Caribbean AGM populations is similar to that observed in the Mauritian cynomolgus macaque population.     

Isolation of chicken major histocompatibility complex class II (B-L) beta chain sequences: comparison with mammalian beta chains and expression in lymphoid organs

By cross-hybridization in low stringency conditions, using a probe derived from an HLA-DQ beta cDNA clone, we have isolated several chicken genomic DNA clones. These clones were mapped to the major histocompatibility complex (MHC) of the chick (B complex) by virtue of their ability to detect restriction enzyme length polymorphisms between congenic lines of chicken. Evidence was obtained for the presence of at least three B-L beta genes in the chicken genome. The B-L beta genes are transcribed specifically in tissues containing cells of the B lymphocyte and myeloid lineages and expressing the B-L antigens. Exons encoding the beta 1, beta 2 and transmembrane domains of a B-L beta chain have been identified with 63, 66 and 62% similarity with the HLA-DQ beta sequence. This first isolation of an MHC class II gene outside of the mammalian class provides insight into the evolution of MHC genes based on the comparison of avian and mammalian class II beta chain amino acid and nucleotide sequences.     

Disruption and pseudoautosomal localization of the major histocompatibility complex in monotremes

Background

The monotremes, represented by the duck-billed platypus and the echidnas, are the most divergent species within mammals, featuring a flamboyant mix of reptilian, mammalian and specialized characteristics. To understand the evolution of the mammalian major histocompatibility complex (MHC), the analysis of the monotreme genome is vital.

Results

We characterized several MHC containing bacterial artificial chromosome clones from platypus (Ornithorhynchus anatinus) and the short-beaked echidna (Tachyglossus aculeatus) and mapped them onto chromosomes. We discovered that the MHC of monotremes is not contiguous and locates within pseudoautosomal regions of two pairs of their sex chromosomes. The analysis revealed an MHC core region with class I and class II genes on platypus and echidna X3/Y3. Echidna X4/Y4 and platypus Y4/X5 showed synteny to the human distal class III region and beyond. We discovered an intron-containing class I pseudogene on platypus Y4/X5 at a genomic location equivalent to the human HLA-B,C region, suggesting ancestral synteny of the monotreme MHC. Analysis of male meioses from platypus and echidna showed that MHC chromosomes occupy different positions in the meiotic chains of either species.

Conclusion

Molecular and cytogenetic analyses reveal new insights into the evolution of the mammalian MHC and the multiple sex chromosome system of monotremes. In addition, our data establish the first homology link between chicken microchromosomes and the smallest chromosomes in the monotreme karyotype. Our results further suggest that segments of the monotreme MHC that now reside on separate chromosomes must once have been syntenic and that the complex sex chromosome system of monotremes is dynamic and still evolving.