Characterization of major histocompatibility complex DRA and DRB genes of the forest musk deer (Moschus berezovskii)

The forest musk deer(Moschus berezovskii) is one of the most endangered species in China.Over the past decades,extensive hunting and poaching have pushed the forest musk deer to the edge of extinction,and conservation biologists are presently pursuing scientific management plans to rescue this species.The major histocompatibility complex(MHC),a cluster of genes responsible for antigen presentation,is a highly polymorphic genomic region in vertebrates that has become a popular functional marker system for studying adaptive variation.In this study,we developed locus-specific genotyping primers for exon 2 fragments of one DRA gene and one DRB locus of the forest musk deer using a suite of comprehensive methods that included universal primer amplification,genome walking,single-strand conformation polymorphism(SSCP),heteroduplex(HD) profiling,and sequence analysis.Each forest musk deer showed no more than two sequences per locus,confirming the specificity of our primers.Genotyping with these primers allowed us to identify two DRA alleles and six DRB alleles in a captive breeding population of the Sichuan Musk Deer Breeding Institution.For the DRA locus,we found a slightly higher observed heterozygosity(N O =0.154) than expected(N E =0.143).In contrast,the DRB locus showed a significant heterozygote deficiency(N O =0.508;N E =0.761;P<0.05),which was probably due to inbreeding in the captive population.An obvious excess of nonsynonymous substitutions over synonymous was observed at the antigen-binding positions of the DRA and DRB loci,showing the presence of positive selection in the forest musk deer DR genes.Finally,generation of phylogenetic trees for the DRA and DRB sequences of the forest musk deer and other ruminants revealed that the DRA and DRB loci identified in this study had homologous relationships with the known ruminant DRA and DRB genes.Based on this analysis,and to facilitate future studies,we named these novel loci Mobe-DRA and Mobe-DRB3.     

Environmental indicators from comparison of sporopollen in early Pleistocene lacustrine sediments from different climatic zones

Two lacustrine sporopollen records obtained from the Qaidam Basin (in the non-monsoonal region) and the Linxia Basin (in the monsoonal transition zone) indicate that during the early Pleistocene open forest-steppe/steppe vegetation developed in the Qaidam Basin, while in the Linxia Basin an open cypress forest-steppe shifted to a conifer/conifer-steppe and then to open forest-steppe vegetation. Existing sporopollen records from the Chinese Loess Plateau (CLP) (in the monsoonal region) indicate that around the Sanmen Paleolake open forest-steppe shifted to pine/conifer-broadleaved mixed forest. The conifer cover then changed to a subtropical evergreen broadleaved forest distributed around the Nihewan Paleolake. These changes suggest that wetter conditions progressed from western China to the east, and the climate became more humid than that of today indicating a stronger Asian summer monsoon during the early Pleistocene.     

Northern Tibetan Plateau cooling and aridification linked to Cenozoic global cooling: Evidence from <Emphasis Type="Italic">n</Emphasis>-alkane distributions of Paleogene sedimentary sequences in the Xining Basin

The Xining Basin on the northeastern Tibetan Plateau holds the longest continuous Cenozoic stratigraphic record in China. The sequence record contains considerable information on the history of Tibetan uplift and associated climatic change. In particular, high resolution n-alkane biomarker proxy and pollen records have been obtained from the Paleogene sediments of the Xiejia section of the basin. A combination of the n-alkane and palynological records reveals that the paleoclimate in the Xining Basin experienced a long-term cooling trend from 50.2 to 28.2 Ma with a distinctive ecological event spanning 37.5 to 32.7 Ma. Since this ecological event, a vertical zonation of vegetation from lowland arid grasses, to middle-elevation subtropical broad-leaf plants, to high-elevation coniferous trees was established. We interpret that these changes in climate and vegetation were probably responses to a combination of long term global cooling since the Eocene climatic optimum and uplift of the surrounding mountains on the northern Tibetan Plateau in the early Cenozoic.     

Pollen evidence from Baode of the northern Loess Plateau of China and strong East Asian summer monsoons during the Early Pliocene

The red clay eolian sequence on the Chinese Loess Plateau (CLP) is an important archive for paleoclimate change from the late Miocene to Pliocene,and can provide significant information for the controversial problems of East Asian monsoon evolution and its forcing mechanism.In this study,we present a pollen record from Baode,northern CLP.The record shows four stages of paleoecological evolution.From 5.6-4.4 Ma,a forest steppe ecosystem developed under an extremely warm period with high seasonal precipitation.Since 4.4 Ma,a drier episode occurred,which prompted parkland landscapes to develop.During 3.5-3.05 Ma,the environment changed to a rather open steppe ecosystem with a much cooler and drier climate.After 3.05 Ma,the vegetation evolved to forest steppe.Using the percentages of arboreal plants to mirror precipitation,and comparing with other published pollen data from the CLP,we find the existence of S-N directionality of the precipitation change and high percentage of arboreal plants in the entire CLP during the Early Pliocene,which suggest the CLP was characterized by a strong East Asian summer monsoon.The strong summer monsoon corresponds well to the low global ice volume,which may illuminate global climate mechanism for the summer monsoon evolution in the early Pliocene.     

Isolation and characterization of class I MHC genes in the giant panda (Ailuropoda melanoleuca)

Artificial breeding is an important project to protect,recover and reintroduce endangered species.Knowledge of the population’s genetic diversity at functional loci is important for the establishment of effective captive breeding programs.The major histocompatibility complex(MHC) genes are ideal candidate genetic markers to inform planned breeding,due to their high levels of polymorphism and importance in the main immune coding region of the vertebrate genome.In this study,we constructed BAC-based contigs and isolated six functional MHC class Ⅰ genes from the giant panda(Ailuropoda melanoleuca),which we designated Aime-C,Aime-F,Aime-I,Aime-K,Aime-L and Aime-1906.Analyses of the tissue expression patterns and full-length cDNA sequences of these class I genes revealed that Aime-C,-F,-I and-L could be considered classical class Ⅰ loci,due to their extensive expression patterns and normal exonic structures.In contrast,Aime-K and-1906 appeared to be nonclassical genes based on their tissue-specific expression patterns and the presence of an abnormal exon 7 in both genes.We established techniques for genotyping exons 2 and 3 of the classical loci using locus-specific single strand conformation polymorphism(SSCP) and sequence analysis.In the Chengdu captive population,we identified one monomorphic locus(Aime-F) and three polymorphic loci with different numbers of alleles(4/4/4 exon 2 alleles at Aime-C/I/L and 6/5/5 exon 3 alleles at Aime-C/I/L).The distributions of the Aime-C,-I and-L alleles among members of different families were in good agreement with the known pedigree relationships,suggesting that the genotyping results are reliable.Therefore,the MHC-I genotyping techniques established in this study may provide a powerful tool for the future design of scientific breeding or release/reintroduction programs.     

Characterization of MHC class II A genes in Hainan Eld’s deer (Cervus eldi hainanus)

The major histocompatibility complex(MHC) genes play pivotal roles in the immune system of vertebrates against antigens.They are also significant indicators of genetic structure,and are vital to species-level population viability analyses and disease risk assessments.In this study,two DRA and two DQA sequences were isolated from Hainan Eld’s deer(Cervus eldi hainanus) using rapid amplification of cDNA ends(RACE) and single-strand conformation polymorphism-heteroduplex(SSCP-HD) analysis.Nucleotide sequence analysis revealed large differences between the two DQA sequences,especially in their exon 2 regions,but only minimal differences between the variants of the DRA gene.Comparison of the predicted amino acid sequences of the Ceel-MHC class Ⅱ A variants with those from six other species revealed that these molecules share high homology among ruminants.A phylogenetic tree of four class Ⅱ A sequences from Hainan Eld’s deer and the other species placed the newly identified DQA and DRA genes on two distinct branches(100%-supportively),and further divided the two DQA sequences into 98%-supportive DQA1 and 99%-supportive DQA2 clusters,respectively.Therefore,this study identified monomorphic Ceel-DQA1 and Ceel-DQA2 genes,and one dimorphic Ceel-DRA gene from Hainan Eld’s deer.