Genetic assessment of eight zoo populations of golden snub-nosed monkey (Rhinopithecus roxellana) implication to the conservation management of captive populations.

Captive breeding programs play an important role in preserving the genetic diversity of endangered species. It is of utmost importance to conduct genetic assessment for captive populations in order to develop scientific breeding plans and conservation management strategies. Here, we genotyped 10 microsatellite loci and sequenced 368 bp of mitochondrial DNA control region for the golden snub-nosed monkey (Rhinopithecus roxellana) from eight captive populations in China, and compared the genetic indices of captive populations with a wild population. Meanwhile, we performed paternity tests to verify the genealogical records and established genetic lineages. A total of 157 individuals were identified from 161 fecal samples, including 135 captive individuals (approximately 25% of captive individuals in China). Microsatellite analysis showed that the nine populations had moderate levels of genetic diversity, with polymorphism information content (PIC) ranging from 0.43 to 0.542; the genetic diversity of captive populations (average PIC: 0.503) was slightly higher than that of the wild population (PIC: 0.438). The Structure analysis indicated that individuals of the eight captive populations contained two different genetic components. We conducted either single-blind or double-blind paternity testing on 40 offspring of captive individuals and found that five offspring from two zoos (Nanjing Hongshan Forest Zoo and Shanghai Wild Animal Park) showed discrepant kinships from their pedigree records, probably due to the inaccuracies in pedigree records. By constructing genetic pedigrees, inbred offspring were found in Beijing Zoo, Shanghai Zoo, Hangzhou Zoo, and Chengdu Zoo. Analysis based on mitochondrial DNA showed a high level of genetic diversity in the eight captive populations (mean nucleotide diversity: 0.047). However, no nucleotide diversity was found in the wild population. This study conducted a genetic survey for captive golden snub-nosed monkeys and will significantly benefit the genetic conservation management for captive populations in the future.

Comparing the gut microbiota of Sichuan golden monkeys across multiple captive and wild settings: roles of anthropogenic activities and host factors.

BACKGROUND: Captivity and artificial food provision are common conservation strategies for the endangered golden snub-nosed monkey (Rhinopithecus roxellana). Anthropogenic activities have been reported to impact the fitness of R. roxellana by altering their gut microbiota, a crucial indicator of animal health. Nevertheless, the degree of divergence in gut microbiota between different anthropogenically-disturbed (AD) R. roxellana and their counterparts in the wild has yet to be elucidated. Here, we conducted a comparative analysis of the gut microbiota across nine populations of R. roxellana spanning China, which included seven captive populations, one wild population, and another wild population subject to artificial food provision. RESULTS: Both captivity and food provision significantly altered the gut microbiota. AD populations exhibited common variations, such as increased Bacteroidetes and decreased Firmicutes (e.g., Ruminococcus), Actinobacteria (e.g., Parvibacter), Verrucomicrobia (e.g., Akkermansia), and Tenericutes. Additionally, a reduced Firmicutes/Bacteroidetes ratiosuggested diminished capacity for complex carbohydrate degradation in captive individuals. The results of microbial functional prediction suggested that AD populations displayed heightened microbial genes linked to vitamin and amino acid metabolism, alongside decreased genes associated antibiotics biosynthesis (e.g., penicillin, cephalosporin, macrolides, and clavulanic acid) and secondary metabolite degradation (e.g., naphthalene and atrazine). These microbial alterations implied potential disparities in the health status between AD and wild individuals. AD populations exhibited varying degrees of microbial changes compared to the wild group, implying that the extent of these variations might serve as a metric for assessing the health status of AD populations. Furthermore, utilizing the individual information of captive individuals, we identified associations between variations in the gut microbiota of R. roxellana and host age, as well as pedigree. Older individuals exhibited higher microbial diversity, while a closer genetic relatedness reflected a more similar gut microbiota. CONCLUSIONS: Our aim was to assess how anthropogenic activities and host factors influence the gut microbiota of R. roxellana. Anthropogenic activities led to consistent changes in gut microbial diversity and function, while host age and genetic relatedness contributed to interindividual variations in the gut microbiota. These findings may contribute to the establishment of health assessment standards and the optimization of breeding conditions for captive R. roxellana populations.

Identification and expression analysis of lncRNA in seven organs of Rhinopithecus roxellana.

Long non-coding RNA (lncRNA) represents a new direction to identify expression profiles and regulatory mechanisms in various organisms. Here, we report the first dataset of lncRNAs of the golden snub-nosed monkey (GSM), including 12,557 putative lncRNAs identified from seven organs. Compared with mRNA, GSM lncRNA had fewer exons and isoforms, and longer length. LncRNA showed more obvious tissue-specific expression than mRNA. However, for the top ten most abundant genes in each organ, mRNAs expression was more tissue-specific than lncRNAs. By identification of specifically expressed lncRNAs and mRNAs in each organ, it indicates that the expression of SEG-lncRNA (specifically expressed lncRNA) and SEG-mRNA (specifically expressed mRNA) had high correlation. In particular, combined our lncRNA and mRNA data, we identified 92 heart SEG-lncRNAs targeted ten mRNA genes in the oxidative phosphorylation pathway and upregulated the expression of these target genes such as ND4, ATP6, and ATP8. These may contribute to GSM adaption to its high-elevation environment. We also identified 171 liver SEG-lncRNAs, which targeted 27 genes associated with the metabolism of xenobiotics and leaded to high expression of these target genes in liver. These lncRNAs may play important roles in GSM adaptation to a folivory diet.

Identification and expression profile of microRNA in seven tissues of the Golden snub-nosed monkey (Rhinopithecus roxellanae).

MicroRNAs (miRNAs) are key in the post-transcriptional regulation of gene expression and thus characterization of miRNAs and investigation of the relative abundance and specificity of tissue expression are essential for understanding gene expression in the golden snub-nosed monkey (GSM, Rhinopithecus roxellanae). Here, we report the first dataset of GSM miRNAs where we identified 460 miRNAs in seven tissues, with 246 conserved known mature miRNAs and 214 novel mature miRNAs. We determined miRNA abundance and expression in the seven tissues using a Tissue Specificity Index score and found that most novel GSM miRNAs showed a highly tissue-specific expression pattern. In particular, 67 novel miRNAs and the miR-34 family were expressed in abundance only in the lung. Five known miRNAs were highly abundant in digestive organs such as the pancreas and liver, and four novel miRNAs were highly expressed in the heart and muscle. Annotation of target genes of GSM miRNAs indicated that target genes were enriched in many important pathways, such as the HIF-1 signaling pathway and xenobiotic biodegradation-related pathways. Collectively, these results emphasize that miRNAs play important roles in GSM diet and high-elevation adaptation regulation. In summary, this study provides essential information on GSM miRNAs and will benefit further investigations of the function and mechanism of miRNAs in controlling gene expression in the GSM.

Differences in the gut microbiota between Cercopithecinae and Colobinae in captivity.

The gut microbiome of captive primates can provide a window into their health and disease status. The diversity and composition of gut microbiota are influenced by not only host phylogeny, but also host diet. Old World monkeys (Cercopithecidae) are divided into two subfamilies: Cercopithecinae and Colobinae. The diet and physiological digestive features differ between these two subfamilies. Accordingly, highthroughput sequencing was used to examine gut microbiota differences between these two subfamilies, using data from 29 Cercopithecinae individuals and 19 Colobinae individuals raised in captivity. Through a comparative analysis of operational taxonomic units (OTUs), significant differences in the diversity and composition of gut microbiota were observed between Cercopithecinae and Colobinae. In particular, the gut microbiota of captive Old World monkeys clustered strongly by the two subfamilies. The Colobinae microbial diversity was higher than that of Cercopithecinae. Additionally, Firmicutes, Lactobacillaceae, Veillonellaceae, and Prevotella abundance were higher in Cercopithecinae, while Bacteroidetes, Ruminococcaceae, Christensenellaceae, Bacteroidaceae, and Acidaminococcaceae abundance were higher in Colobinae. PICRUSt analysis revealed that the predicted metagenomes of metabolic pathways associated with proteins, carbohydrates, and amino acids were significantly higher in Colobinae. In the context of host phylogeny, these differences between Cercopithecinae and Colobinae could reflect adaptations associated with their respective diets. This well-organized dataset is a valuable resource for future related research on primates and gut microbiota. Moreover, this study may provide useful insight into animal management practices and primate conservation.

The sequence and de novo assembly of hog deer genome.

Hog deer (Axis porcinus) is a small deer species in family Cervidae and has been undergoing a serious and global decline during the past decades. Chengdu Zoo currently holds a captive population of hog deer with sufficient genetic diversity in China. We sequenced and de novo assembled its genome sequence in the present study. A total of six different insert-size libraries were sequenced and generated 395 Gb of clean data in total. With aid of the linked reads of 10X Genomics, genome sequence was assembled to 2.72 Gb in length (contig N50, 66.04 Kb; scaffold N50, 20.55 Mb), in which 94.5% of expected genes were detected. We comprehensively annotated 22,473 protein-coding genes, 37,019 tRNAs, and 1,058 Mb repeated sequences. The newly generated reference genome is expected to significantly contribute to comparative analysis of genome biology and evolution within family Cervidae.

Can-SINE dynamics in the giant panda and three other Caniformia genomes.

BACKGROUND: Although repeat sequences constitute about 37% of carnivore genomes, the characteristics and distribution of repeat sequences among carnivore genomes have not been fully investigated. Based on the updated Repbase library, we re-annotated transposable elements (TEs) in four Caniformia genomes (giant panda, polar bear, domestic dog, and domestic ferret) and performed a systematic, genome-wide comparison focusing on the Carnivora-specific SINE family, Can-SINEs. RESULTS: We found the majority of young recently integrated transposable elements are LINEs and SINEs in carnivore genomes. In particular, SINEC1_AMe, SINEC1B_AMe and SINEC_C1 are the top three most abundant Can-SINE subfamilies in the panda and polar bear genomes. Transposition in transposition analysis indicates that SINEC1_AMe and SINEC1B_AMe are the most active subfamilies in the panda and the polar bear genomes. SINEC2A1_CF and SINEC1A_CF subfamilies show a higher retrotransposition activity in the dog genome, and MVB2 subfamily is the most active Can-SINE in the ferret genome. As the giant panda is an endangered icon species, we then focused on the identification of panda specific Can-SINEs. With the panda-associated two-way genome alignments, we identified 250 putative panda-specific (PPS) elements (139 SINEC1_AMes and 111 SINEC1B_AMes) that inserted in the panda genome but were absent at the orthologous regions of the other three genomes. Further investigation of these PPS elements allowed us to identify a new Can-SINE subfamily, the SINEC1_AMe2, which was distinguishable from the current SINEC1_AMe consensus by four non-CpG sites. SINEC1_AMe2 has a high copy number (> 100,000) in the panda and polar bear genomes and the vast majority (> 96%) of the SINEC1_AMe2 elements have divergence rates less than 10% in both genomes. CONCLUSIONS: Our results suggest that Can-SINEs show lineage-specific retransposition activity in the four genomes and have an important impact on the genomic landscape of different Caniformia lineages. Combining these observations with results from the COSEG, Network, and target site duplication analysis, we suggest that SINEC1_AMe2 is a young mobile element subfamily and currently active in both the panda and polar bear genomes.

Ancient hybridization and admixture in macaques (genus Macaca) inferred from whole genome sequences.

The evolutionary history of the stump-tailed macaque (Macaca arctoides) and its genetic relationship to other macaques is a subject of continuing controversy. Here, we have reported the first genome sequences of two stump-tailed macaques and one Assamese macaque (M. assamensis). Additionally, we have investigated the genetic diversity between macaque species and analyzed ancient hybridization events. Genome-wide analyses demonstrated that the stump-tailed macaque is more closely related to sinica species than to fascicularis/mulatta species. This topology contradicts the mitochondrial sequence-based phylogeny that places the stump-tailed macaque into the fascicularis/mulatta group. However, our results further show that stump-tailed macaques have genetic backgrounds distinct from sinica species, and present evidence of gene flows with rhesus macaques. We suggest that an ancient introgression occurred after stump-tailed macaques diverged from sinica species. The distinct gene flow between proto-arctoides and proto-mulatta resulted in the transfer of rhesus macaque-type mitochondria into proto-arctoides. The rhesus macaque-type mitochondria remained in populations because of genetic drift during the bottleneck. The PSMC results and morphological and geographic evidence are consistent with the mitochondria capture pattern in the stump-tailed macaque. The molecular clock estimates suggest that the mitochondrial transference into stump-tailed macaques occurred 0.4-1.4 million years ago. Furthermore, we detected extensive admixtures between different macaque species, indicating that gene flow has played an important role in the evolutionary history of the genus Macaca.

Cellulose-dependent expression and antibacterial characteristics of surfactin from Bacillus subtilis HH2 isolated from the giant panda.

Surfactin secreted by Bacillus subtilis can confer strong, diverse antipathogenic effects, thereby benefitting the host. Carbon source is an important factor for surfactin production. However, the mechanism that bacteria utilize cellulose, the most abundant substance in the intestines of herbivores, to produce surfactin remains unclear. Here, we used B. subtilis HH2, isolated from the feces of a giant panda, as a model to determine changes in surfactin expression in the presence of different concentrations of cellulose by quantitative polymerase chain reaction and high-performance liquid chromatography. We further investigated the antimicrobial effects of surfactin against three common intestinal pathogens (Escherichia coli, Staphylococcus aureus, and Salmonella enterica) and its resistance to high temperature (60-121°C), pH (1-12), trypsin (100-300 µg/mL, pH 8), and pepsin (100-300 µg/mL, pH 2). The results showed that the surfactin expressed lowest in bacteria cultured in the presence of 1% glucose medium as the carbon source, whereas increased in an appropriate cellulose concentration (0.67% glucose and 0.33% cellulose). The surfactin could inhibit E. coli and Staphylococcus aureus, but did not affect efficiently for Salmonella enterica. The antibacterial ability of surfactin did not differ according to temperature (60-100°C), pH (2-11), trypsin (100-300 µg/mL), and pepsin (100-300 µg/mL; P > 0.05), but decreased significantly at extreme environments (121°C, pH 1 or 12; P < 0.05) compared with that in the control group (37°C, pH = 7, without any protease). In conclusion, our findings indicated that B. subtilis HH2 could increase surfactin expression in an appropriate cellulose environment and thus provide benefits to improve the intestinal health of herbivores.

Discovery of genome-wideSNPs by RAD-seqand the genetic diversity of captive hog deer (Axis porcinus).

The hog deer (Axis porcinus) is a small deer whose natural habitat is the wet or moist tall grasslands in South and Southeast Asia. Wild populations have dramatically decreased in recent decades. While wild hog deer were recently acknowledged to be extinct in China, a few captive populations have been maintained. In the present study, we successfully employed the restriction-site-associated DNA sequencing (RAD-seq) technique to generate a genome-wide profile of single-nucleotide polymorphisms (SNPs) in the captive population of hog deer from Chengdu Zoo, China (N = 11). Up to 4.7 million clean reads per sample were sequenced, and 11,155 SNPs and 8,247 haplotypes were simultaneously observed within more than 80% of sequenced individuals. In this population, the mean frequency of major alleles at each polymorphism site was 0.7903±0.0014, and the average nucleotide diversity (π) and inbreeding coefficient (FIS) were 0.3031±0.0015 and -0.0302±0.0062, respectively. Additionally, the Euclidean distance-based multidimensional scaling method revealed that the pairwise genetic relatedness was evenly distributed. However, the results of homologous searching by short reads did not provide any meaningful explanation of the phylogenetic relationship of hog deer, which should be further investigated. In conclusion, our results revealed current state of genetic diversity in this captive population of hog deer.Furthermore, these genome-wide SNPs would be useful for guiding the mating schedule to avoid sharp increase of inbreeding coefficient.

Correction: First Report of the Human-Pathogenic Enterocytozoon bieneusi from Red-Bellied Tree Squirrels (Callosciurus erythraeus) in Sichuan, China.

[This corrects the article DOI: 10.1371/journal.pone.0163605.].

First Report of the Human-Pathogenic Enterocytozoon bieneusi from Red-Bellied Tree Squirrels (Callosciurus erythraeus) in Sichuan, China.

Enterocytozoon bieneusi is a common opportunistic pathogen causing diarrhea and enteric disease in a variety of animal hosts. Although it has been reported in many animals, there is no published information available on the occurrence of E. bieneusi in red-bellied tree squirrels. To understand the occurrence, genetic diversity, and zoonotic potential of E. bieneusi in red-bellied tree squirrels, 144 fecal specimens from Sichuan province, China, were examined by PCR amplification and sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene of E. bieneusi. The overall infection rate of E. bieneusi 16.7% (24/144) was observed in red-bellied tree squirrels. Altogether five genotypes of E. bieneusi were identified: three known genotypes D (n = 18), EbpC (n = 3), SC02 (n = 1) and two novel genotypes CE01, CE02 (one each). Multilocus sequence typing (MLST) analysis employing three microsatellite (MS1, MS3, MS7) and one minisatellite (MS4) revealed 16, 14, 7 and 14 positive specimens were successfully sequenced, and identified eight, three, three and two genotypes at four loci, respectively. In phylogenetic analysis, the three known genotypes D, EbpC, and SC02 were clustered into group 1 with zoonotic potential, and the two novel genotypes CE01 and CE02 were clustered into group 6. The present study firstly reported the occurrence of E. bieneusi in red-bellied tree squirrels in China, and the E. bieneusi genotypes D and EbpC were found in humans previously. These results indicate that red-bellied tree squirrels may play a potential role in the transmission of E. bieneusi to humans.

Mitochondrial Genome and Nuclear Markers Provide New Insight into the Evolutionary History of Macaques.

The evolutionary history of macaques, genus Macaca, has been under debate due to the short times of divergence. In this study, maternal, paternal, and biparental genetic systems were applied to infer phylogenetic relationships among macaques and to trace ancient hybridization events in their evolutionary history. Using a PCR display method, 17 newly phylogenetically informative Alu insertions were identified from M. assamensis. We combined presence/absence analysis of 84 Alu elements with mitochondrial genomes as well as nuclear sequences (five autosomal genes, two Y chromosomal genes, and one X chromosomal fragment) to reconstruct a robust macaque phylogeny. Topologies generated from different inherited markers were similar supporting six well defined species groups and a close relationship of M. assamensis and M. thibetana, but differed in the placing of M. arctoides. Both Alu elements and nuclear genes supported that M. arctoides was close to the sinica group, whereas the mitochondrial data clustered it into the fascicularis/mulatta lineage. Our results reveal that a sex-biased hybridization most likely occurred in the evolutionary history of M. arctoides, and suggest an introgressive pattern of male-mediated gene flow from the ancestors of M. arctoides to the M. mulatta population followed by nuclear swamping. According to the estimation of divergence dates, the hybridization occurred around 0.88~1.77 mya (nuclear data) or 1.38~2.56 mya (mitochondrial data). In general, our study indicates that a combination of various molecular markers could help explain complicated evolutionary relationships. Our results have provided new insights into the evolutionary history of macaques and emphasize that hybridization might play an important role in macaque evolution.

Cloning and characterization of giant panda (Ailuropoda melanoleuca) IL-18 binding protein.

The giant panda (Ailuropoda melanoleuca) is an endangered species. Interleukin-18 (IL-18) plays an important role in the innate and adaptive immune responses by inducing IFN-γ. IL-18 has been implicated in the pathogenesis of various diseases. IL-18 binding protein (IL-18BP) is an intrinsic inhibitor of IL-18 that possesses higher affinity to IL-18. In this study, we cloned and characterized IL-18BP in giant panda (AmIL-18BP) from the spleen. The amino acid sequence of giant panda IL-18BP ORF shared about 65% identities with other species. To evaluate the effects of AmIL-18BP on the immune responses, we expressed the recombinant AmIL-18BP in Escherichia coli BL21 (DE3).The fusing protein PET-AmIL-18BP was purified by nickel affinity column chromatography. The biological function of purified PET-AmIL-18BP was determined on mice splenocyte by qRT-PCR. The results showed that AmIL-18BP was functional and could significantly reduce IFN-γ production in murine splenocytes. These results will facilitate the study of protecting giant panda on etiology and immunology.

The complete mitochondrial genome of Assamese Macaques (Macaca assamensis).

The complete mitochondrial genome of Assamese macaques (Macaca assamensis) was sequenced in this study. The genome is 16,542 bp long, consisting of 13 protein-coding genes (PCGs), 2 ribosomal RNA genes, 22 transfer RNA genes and 2 non-coding areas. Eight PCGs (COI, COII, ATP6, COIII, ND4L, ND4, ND6, CYTB) initiate with the start codon ATG and another two genes (ND1, ATP8) use GTG, while ND2, ND3 and ND5 start with ATT, ATC and ATA, respectively. Five genes (COII, ATP8, ATP6, ND4L and ND5) use the complete stop codon TAA, whereas four genes have incomplete stop codons, TA- (COIII) and T- - (ND3, ND4, CYTB), while others use standard canonical TAA as their termination codons. The largest non-coding control region with the length of 1091 bp is located between the tRNA-Pro and tRNA-Phe genes.

Complete mitochondrial genome sequence of the Asian golden cat, Catopuma temminckii.

In this study, the mitochondrial genome of Asian golden cat (Catopuma temminckii) is sequenced. The mitochondrial genome was 16,985 bp long, including 13 protein-coding genes, 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, 1 control region and 1 origin of light-strand replication. The overall base composition of the mitochondrial genome was 32.76% A, 27.49 % T, 25.75 % C, and 13.99 % G. The complete mitochondrial genome of Catopuma temminckii could contribute to understanding taxonomic status and phylogenetic relationship of genus Catopuma.

The complete mitochondrial genome of Stump-tailed Macaques (Macaca arctoides).

Stump-tailed macaque (Macaca arctoides) has been enlisted as the Near Threatened species in the IUCN Red List. In this study, the complete mitochondial genome of M. arctoides was determined. The mitogenome was 16,559 bp in length with an A + T content of 56.8%, containing 2 ribosomal RNA genes (rRNA), 22 transfer RNA genes (tRNA), 13 protein-coding genes (PCGs) and a control region (D-loop). Ten protein-coding genes (ND1, COX1, COX2, ND4, COX3, ND4L, ATP8, ATP6, CYTB, ND6) started with ATG codons, while ND2, ND3 and ND5 initiated with ATT, ATC and ATA, respectively. Eight PCGs ended with complete termination codons except for COX3, ND3, ND4, and CYTB terminated with incompleted codon T.

Adjuvant effects of recombinant giant panda (Ailuropoda melanoleuca) IL-18 on the canine distemper disease vaccine in mice.

Canine distemper virus (CDV) is a morbillivirus known to cause morbidity and mortality in a broad range of animals. Giant pandas (Ailuropoda melanoleuca), especially captive ones, are susceptible to natural infection with CDV. Interleukin-18 (IL-18) is a powerful adjuvant molecule that can enhance the development of antigen-specific immunity and vaccine efficacy. In this study, a giant panda IL-18 gene eukaryotic expression plasmid (pcAmIL-18) was constructed. Female BALB/c mice were muscularly inoculated with the plasmids pcAmIL-18, pcDNA3.1 and PBS, respectively. They were subsequently injected with an attenuated CDV vaccine for dogs, and the induced humoral and cellular responses were evaluated. The results showed that pcAmIL-18 remarkably improved the level of specific antibody, IFN-γ and IL-2 in mice sera, the T lymphocyte proliferation index and the percentage of CD4(+) and CD8(+) cells. These data indicated that pcAmIL-18 is a potential adjuvant that promotes specific immunity.

Giant panda BAC library construction and assembly of a 650-kb contig spanning major histocompatibility complex class II region.

BACKGROUND: Giant panda is rare and endangered species endemic to China. The low rates of reproductive success and infectious disease resistance have severely hampered the development of captive and wild populations of the giant panda. The major histocompatibility complex (MHC) plays important roles in immune response and reproductive system such as mate choice and mother-fetus bio-compatibility. It is thus essential to understand genetic details of the giant panda MHC. Construction of a bacterial artificial chromosome (BAC) library will provide a new tool for panda genome physical mapping and thus facilitate understanding of panda MHC genes. RESULTS: A giant panda BAC library consisting of 205,800 clones has been constructed. The average insert size was calculated to be 97 kb based on the examination of 174 randomly selected clones, indicating that the giant panda library contained 6.8-fold genome equivalents. Screening of the library with 16 giant panda PCR primer pairs revealed 6.4 positive clones per locus, in good agreement with an expected 6.8-fold genomic coverage of the library. Based on this BAC library, we constructed a contig map of the giant panda MHC class II region from BTNL2 to DAXX spanning about 650 kb by a three-step method: (1) PCR-based screening of the BAC library with primers from homologous MHC class II gene loci, end sequences and BAC clone shotgun sequences, (2) DNA sequencing validation of positive clones, and (3) restriction digest fingerprinting verification of inter-clone overlapping. CONCLUSION: The identifications of genes and genomic regions of interest are greatly favored by the availability of this giant panda BAC library. The giant panda BAC library thus provides a useful platform for physical mapping, genome sequencing or complex analysis of targeted genomic regions. The 650 kb sequence-ready BAC contig map of the giant panda MHC class II region from BTNL2 to DAXX, verified by the three-step method, offers a powerful tool for further studies on the giant panda MHC class II genes.