Biomedical subjects

Sheng-Guo Fang

Publications and source records attributed to Sheng-Guo Fang.

15 recordsLinked to original sources

PubMed2006

Genetic diversity of Chinese water deer (Hydropotes inermis inermis): implications for conservation.

The Chinese water deer (Hydropotes inermis inermis) is endemic to China. Historically, the species was widely distributed, but now, habitat loss and poaching have reduced its range and number drastically. In order to provide useful information for its conservation, we have investigated the genetic diversity and population structure of the Chinese water deer by analyzing the 403 bp fragment of the mitochondrial DNA (mtDNA) control region (D-loop). Eighteen different haplotypes were detected in 40 samples. Overall, Chinese water deer have a relatively high-genetic diversity compared to other rare cervid species, with a haplotype diversity of 0.923+/-0.025 and nucleotide diversity of 1.318 +/- 0.146%. No obvious phylogenetic structure among haplotypes was found for samples of different origin. An analysis of molecular variance (AMOVA) showed significant differentiation between the Zhoushan and the mainland population (F(ST)= 0.088, P < 0.001; Phi( ST ) = 0.075, P = 0.043), which suggests that exchanges of individuals between Zhoushan and the mainland should be avoided. We also recommend that a breeding center be set up for the mainland population.

Animals↗

PubMed2006

Major histocompatibility complex variation in the endangered crested ibis Nipponia nippon and implications for reintroduction.

The major histocompatibility complex (MHC), with its extraordinary levels of genetic variation, is thought to be an essential aspect of the ability of an organism to recognize different parasites and pathogens. It has also been proposed to regulate reproductive processes in many aspects. Here we examine the genetic variation of the second exon of the MHC class II B genes of the crested ibis, an endangered species known to descend from just two breeding pairs rediscovered in 1981. Only five alleles are identified by single-strand conformation polymorphism (SSCP) analysis of 36 samples taken from both wild and captive populations, and a comparatively low level of divergence between MHC alleles is observed. We suggest that representative sampling of individuals with most of the different MHC allele genotypes to constitute a founder population, together with the monitoring of the pathogen status of candidate sites before release, is of great importance for raising the success rate of reintroduction for the crested ibis.

Amino Acid Sequence↗

PubMed2006

Major histocompatibility complex class II variation in the giant panda (Ailuropoda melanoleuca).

Habitat destruction and human activity have greatly impacted the natural history of the giant panda (Ailuropoda melanoleuca). Although the genetic diversity of neutral markers has been examined in this endangered species, no previous work has examined adaptive molecular polymorphisms in the giant panda. Here, the major histocompatibility complex (MHC) class II DRB locus was investigated in the giant panda, using single-strand conformation polymorphism (SSCP) and sequence analysis. Comparisons of DNA samples extracted from faecal and blood samples from the same individual revealed that the two materials yielded similar quantities and qualities of DNA, as well as identical SSCP patterns and allelic sequences, demonstrating the reliability of DNA isolation from panda faeces. Analysis of faecal samples from 60 giant pandas revealed relatively low number of alleles: seven alleles. However, the alleles were quite divergent, varying from each other by a range of 7-47 nucleotide substitutions (4-25 amino acid substitutions). Construction of a neighbour-joining tree and comparisons among DRB alleles from other species revealed that both similar and highly divergent alleles survived in the bottlenecked panda populations. Despite species-specific primers used and excellent faecal DNA isolated, a lower level of heterozygosity than expected was still observed due to inbreeding. There were three types of evidence supporting the presence of balancing selection in the giant panda: (i) an obvious excess of nonsynonymous substitutions over synonymous at the antigen-binding positions; (ii) trans-species evolution of two alleles between the giant panda and other felids; and (iii) a more even distribution of alleles than expected from neutrality.

Alleles↗

PubMed2006

Population structure and gene flow among wild populations of the black muntjac (Muntiacus crinifrons) based on mitochondrial DNA control region sequences.

The black muntjac (Muntiacus crinifrons) is a rare species endemic to China and its current distribution is confined to partial mountain ranges in eastern China. To assess the population structure and gene flow among the extant populations, we sequenced a portion of the mitochondrial control region (424 bp) for 47 samples collected from its current three large populations (Huangshan, Tiammushan and Suichang). A total of 18 unique haplotypes were defined based on 22 polymorphic sites. Of these haplotypes, 15 ones were population-specific and only one haplotype was shared among the three populations. Significant genetic differentiation was detected between Suichang and Huangshan populations (ø(ST)=0.1677, P<0.001) or between Suichang and Tianmushan populations (ø(ST)=0.2002, P<0.001), indicating that the Suichang population may be spatially structured from other two populations along matriline. The Mantel test revealed that this significant differentiation was not driven by geographic distance (P=0.14), implying that genetic divergence of Suichang population might result from recent human disturbances. Phylogenetic analyses suggested the mitochondrial control region haplotypes were split into two well divergent clades (Clade I and Clade II). Interestingly, the two distinct haplotype clades were found to coexist in Suichang area. The nested clade analysis revealed a significant phylogeographic structure among the black muntjac populations (total cladogram: chi2=18.68; P<0.001), which was inferred to result from past fragmentation followed by range expansion. The population expansion was supported by the analysis of mismatch distribution and the tests of neutrality. Therefore, we suggest that the coexistence of distinct haplotypes in Suichang population was induced by historical population expansion after fragmentation and that the current genetic differentiation should be attributed to the reduction of female-mediated gene flow due to recent habitat fragmentation and subsequent loss.

Animals↗

PubMed2005

Application of mitochondrial DNA sequence analysis in the forensic identification of Chinese sika deer subspecies.

As a direct and indirect consequence of human activities, only two subspecies, Cervus nippon sinchuanicus and Cervus nippon kopschi, currently subsist in the wild of China. However, a large population of Cervus nippon hortulorum and Cervus nippon nippon is raised in order to gain deer parts for Chinese traditional medicine. According to Chinese Wild Animal Conservation Law, hunting, capturing and trading of the wild sika deer are strictly banned, however, raising and trading of the domestic individual are permitted. Thus, it is very necessary to identify the subspecies of sika deer in China in forensic tests. In our study, we used mitochondrial DNA control region sequence analysis and phylogenetic analysis to identify the subspecies of sika deer. Mitochondrial DNA control region sequences analysis revealed that two haplotypes came from the unknown samples. One is the same as the haplotype that came from the samples of wild population of C. n. kopschi. Phylogenetic analysis indicated that the two haplotypes of unknown samples clustered with the haplotypes of C. n. kopschi, and had significant difference from the haplotypes of the other subspecies. These results together revealed that the unknown samples came from two individuals that belong to the wild population of C. n. kopschi living in the Qinglingfeng State Natural Reserve of Zhejiang province. Therefore, the results provide forensic evidence of illegal wild animal hunting.

Animals↗

PubMed2005

Mitochondrial DNA genetic diversity of black muntjac (Muntiacus crinifrons), an endangered species endemic to China.

Genetic diversities based on the mtDNA control region were measured for both a wild population (n = 26) and a captive population (n = 18) of the black muntjac. In total, nine haplotypes were obtained from 44 samples. The wild population exhibited a low nucleotide diversity (pi = 0.00562), which suggests that the black muntjac had a small effective population size historically. In contrast to its low nucleotide diversity, haplotype diversity (h = 0.862) of the wild population was relatively high. Haplotype distribution among local samples shows a distinct difference. As anticipated because of the paucity of available founders, nucleotide diversity (pi = 0.00214) of the captive population was very low. Additionally, a high degree of haplotype identity and an obvious haplotype frequency bias was revealed in the captive population, which implies that the current breeding program should be readjusted to balance distributions of haplotypes, and some new founders should be introduced to the captive population to alleviate potential inbreeding depression.

Animals↗

PubMed2005

A novel nonsense mutation and polymorphisms in the mouse hairless gene.

A novel autosomal recessive mutation arose spontaneously in a breeding colony of Chinese Kunming mice. The characteristics of these mutant mice include progressive irreversible hair loss soon after birth, rhinocerotic appearance, and shorter life span. Histological evaluation of skin revealed the homogeneous enlargement of utriculi, and the formation of several rows of large cysts. Sequencing the complete cDNA of the hairless gene identified two polymorphisms and a homozygous transition for a G-->A at nucleotide position 3110 (exon 12) leading to the substitution of tryptophan by a nonsense codon, designated W911X. This allele was named rhinocerotic and short-lived, with the symbol hr(rhsl). Addition of hairless gene mutation into the expanding hairless mutation database allows further development of genotype/phenotype correlations towards understanding inherited atrichia.

Animals↗

PubMed2005

A genetic diversity comparison between captive individuals and wild individuals of Elliot's Pheasant (Syrmaticus ellioti) using mitochondrial DNA.

Maintaining genetic diversity is a major issue in conservation biology. In this study, we demonstrate the differences of genetic diversity levels between wild and captive individuals of Elliot's Pheasant Syrmaticus ellioti. Wild individuals showed a higher genetic diversity level than that of the captive individuals. Nucleotide diversity and haplotype diversity of wild individuals were 0.00628 and 0.993, while those of captive individuals were 0.00150 and 0.584 respectively. Only 3 haplotypes of mtDNA control region sequence were identified among 36 captive individuals, while 16 unique haplotypes were identified among the 17 wild individuals in this study. One captive haplotype was shared by a wild individual from Anhui Province. It is concluded that a low number of founders was the likely reason for the lower level genetic diversity of the captive group. Careful genetic management is suggested for captive populations, particularly of such an endangered species, to maintain genetic variability levels.

Animals↗

PubMed2005

[Molecular cloning of full-long cDNA sequences encoding hairless gene in the Kunming mouse].

The mouse hairless gene is a crucial nuclear receptor gene associated with the structure of hair and skin. It encodes a putative zinc finger transcription factor, and is a transcriptional corepressor for thyroid hormone receptors. Hairless gene plays a critical role in maintaining the delicate balance between cell proliferation, differentiation, and apoptosis in the hair follicle as well as in the interfollicular epidermis, and is concerned with the control of hair growth cycling. This study was designed to clone and analyze the cDNA encoding Hr from Kunming mouse. The RT-PCR method was developed to clone the Hr cDNA. A full-length cDNA and CDS sequences of Kunming mouse were 4104bp and 3546bp, respectively, which has been accepted by GenBank (Accession Number: AY547391). Accession number of protein encoded by AY547391 sequence in GenBank is AAT45233 , composed of 1181 amino acid residues. The Hr cDNA of Kunming mouse was cloned and sequenced for the first time. The identities of CDS of Hr gene were 99.9%, 94.4%, 83.1%, 78.1%, 81.9% and 82.1% by homologous comparison among Kunming mouse and other six species, and that were 99.9%, 92.2%, 81.7%, 70.8%, 79.9% and 80.1% respectively in amino acid sequences. The results suggested a high degree of conservation and thus functional significance of the Hr gene among different mammalian taxons. The results derived from information searching by Blast program revealed that there were 4 SNP sites and one deletion mutation in the sequences of hairless gene mRNAs between Kunming mice and that collected in GenBank. Three SNP sites did not alter the related amino acids encoded. Two SNP proved to be polymorphism mutant sites with amino acids changes. The results provided new data for SNPs in Hr gene.

Amino Acid Sequence↗

PubMed2004

Which genetic marker for which conservation genetics issue?

Conservation genetics focuses on the effects of contemporary genetic structuring on long-term survival of a species. It helps wildlife managers protect biodiversity by identifying a series of conservation units, which include species, evolutionarily significant units (ESUs), management units (MUs), action units (AUs), and family nets (FNs). Although mitochondrial DNA (mtDNA) evolves 5-10 times faster than single-copy nuclear DNA (scnDNA), it records few traces of contemporary events. Thus, mtDNA can be used to resolve taxonomic uncertainties and ESUs. Variable number of tandem repeats (VNTRs) evolve 100-1000 times faster than scnDNA and provide a powerful tool for analyzing recent and contemporary events. VNTR analysis techniques include polymerase chain reaction (PCR)-based microsatellite assays and oligonucleotide probing. Size homoplasy problems in PCR-based microsatellite assays can strongly affect the inference of recent population history. The high homozygosity in endangered species is reflected in a relatively low number and level of variability in microsatellite loci. This combined with "allelic dropout" and "misprinting" errors contributes to the generation of highly biased genetic data following analyses of natural populations. Thus, in conservation genetics, microsatellites are of limited use for identifying ESUs, MUs, and AUs. In contrast to PCR-based microsatellite analysis, oligonucleotide probing avoids errors resulting from PCR amplification. It is particularly suitable for inferring recent population history and contemporary gene flow between fragmented subpopulations. Oligonucleotide fingerprinting generates individual-specific DNA banding patterns and thus provides a highly precise tool for monitoring demography of natural populations. Hence, DNA fingerprinting is powerful for distinguishing ESUs, MUs, AUs, and FNs. The use of oligonucleotide fingerprinting and fecal DNA is opening new areas for conservation genetics.

Animals↗

PubMed2004

Mitochondrial DNA heteroplasmy in calves cloned by using adult somatic cell.

Adult somatic cell cloned calves were produced by somatic cell nuclear transfer prepared by fusion of cultured ear fibroblast from a Holstein cow into enucleated oocytes of Luxi Yellow cow. In order to determinate the source of mitochondrial DNA of cloned calves, we designed the breed-specific PCR primers by aligning the known D-loop sequences of Bos taurus and analyzed the displacement loop sequences of five live cloned calves by breed-specific primers PCR. The results demonstrated that mtDNA originated from Holstein breed and that from Luxi breed co-exist in all five live calves.

Animals↗

PubMed2003

A simple DNA extraction and rapid specific identification technique for single cells and early embryos of two breeds of Bos taurus.

In the process of nuclear transfer (NT), different cytoplasm from a donor cell and a recipient oocyte are mixed. However, it is unclear what effect the donor cytoplasm has upon the dedifferentiation of donor nuclei in enucleated ooplasm and upon subsequent production of live cloned offspring. Mitochondria are component parts of cytoplasm so the detection of mitochondrial DNA is helpful to reveal changes of donor cytoplasm in the NT reconstructed embryos. In this study, the experiments were designed to develop efficient DNA extraction techniques and specific primer pairs for mitochondrial DNA of Holstein and Chinese Yellow breeds in order to identify the changes of donor cytoplasm in early stage embryos. Firstly, by adding Triton X-100 and Taq DNA polymerase reaction buffer to the DNA extraction mixture, DNA was rapidly isolated from single diploid cells, single oocytes, early stage embryos and from single hairs. Secondly, two specific primer pairs for the two breeds were designed to detect the cytoplasmic DNA in a different amount of single cells and in early stage embryos. The results show that two specific fragments were successfully amplified from single somatic cells, single oocytes, parthenogenetic embryos and from NT reconstructed embryos. As a result, the techniques provide a powerful tool for studying the developmental mechanism in NT reconstructed embryos.

Animals↗

PubMed2003

Application of species-specific polymerase chain reaction in the forensic identification of tiger species.

Globally, tigers are considered to be endangered, and are listed on Appendix I of CITES. A simple test, using a species-specific primer pair, was developed to identify tiger meat, faeces and dried skin, and provide forensic evidence of illegal wildlife trade. The specific fragment of mitochondrial cytochrome b gene was also successfully amplified from raw DNA products extracted from single tiger hairs. This PCR-based approach opens a new avenue to forensic identification of less-than-optimal samples.

Animals↗

PubMed2003

Genetic differentiation and subspecies development of the giant panda as revealed by DNA fingerprinting.

Over the last 100 years giant pandas (Ailuropoda melanoleuca) have been separated into six completely isolated mountain ranges. DNA fingerprinting revealed different differentiation patterns in giant pandas, including early-stage, late-stage, recent divergence and recent genetic depression. A separation around 10 000 years ago resulted in highly significant differences in DNA fingerprints and morphological characteristics between Qinling and Sichuan populations. Supported by morphological differences, the genetic data were used to classify the Qinling population as a new subspecies, A. m. qinlingensis, while the Sichuan populations were classified into the original subspecies, A. m. melanoleuca. Thus, the Qinling population deserves management as a separate unit. In the Sichuan populations, two management units were defined, including Qionglai-Minshan and Daxiangling-Xiaoxiangling-Liangshan. Our data suggest urgent measures are needed to establish green corridors between subpopulations in each mountain range to increase gene flow and genetic variation to ensure long-term survival.

Animals↗

PubMed2002

Formalin removal from archival tissue by critical point drying.

The extraction of high-quality nucleic acid may be problematic in formalin-fixed tissues because of cross-linking between proteins and DNA. Old fixed tissue specimens do produce fragmented DNA (<1.2 kb), which is only used for PCR amplification. Here we show that high molecular weight DNA (>194 kb) can be successfully extracted from fixed tissue samples (16-70 years old) by gradual dehydration and critical point drying. The reliability of extracted DNA was measured by its ability to serve as a template for the amplification of mtDNA fragments (403 and 1198 bp) and an nDNA fragment (1844 bp). In addition, fingerprinting analysis was performed using DNA from fixed human tissue to ensure the ability of extracted DNA to hybridize with the DNA probe. DNA derived by this method can be subject to amplification, complete digestion by restriction endonuclease, and hybridization.

Animals↗