Zinc‐finger intron 7: a new locus for sex identification of giant panda (Ailuropoda melanoleuca)

We developed a single‐reaction test for identifying the sex of giant panda (Ailuropoda melanoleuca) targeted to co‐amplify homologous fragments with size polymorphism that located at zinc‐finger (ZF) intron 7 by using one pair of primers. This assay produced one sex‐specific fragment in females (XX genotypes) whereas two fragments were produced in males (XY genotypes). Indels (insertion/deletion) in intron 7 of Y‐linked allele provide a significant discrimination between ZFX and ZFY, thus the amplification products can be simply distinguished by agarose gel electrophoresis, exhibiting sex‐specific banding patterns (female, 354 bp; male, 354 bp, 135 bp). The new primer set was successfully tested on known‐sex giant pandas by using template DNA extracted from both blood and fecal samples. Cross‐species test was also performed, revealing that this assay could be applied to other Ursidae species. Zoo Biol 29:526–531, 2010. © 2009 Wiley‐Liss, Inc.     

Genotyping faeces of red pandas (<Emphasis Type="Italic">Ailurus fulgens</Emphasis>): implications for population estimation

The red panda (Ailurus fulgens) is an endangered species distributed in the Himalaya and Hengduan Mountains and extremely difficult to monitor because it is elusive, wary and nocturnal. However, recent advances in noninvasive genetics are allowing conservationists to indirectly estimate population size of this animal. Here, we present a pilot study of individual identification of wild red pandas using DNA extracted from faeces. A chain of optimal steps in noninvasive studies were used to maximize genotyping success and minimize error rate across sampling, selection of microsatellite loci, DNA extraction and amplification and data checking. As a result, 18 individual red pandas were identified successfully from 33 faecal samples collected in the field using nine red panda-specific microsatellite loci with a low probability of identity of 1.249 × 10⁻³ for full siblings. Multiple methods of tracking genotyping error showed that the faecal genetic profiles possessed very few genotyping errors, with an overall error rate of 1.12 × 10⁻⁵. Our findings demonstrate the feasibility and reliability of using faeces as an effective source of DNA for estimating and monitoring wild red panda populations.     

A reliable, non-invasive PCR method for giant panda (Ailuropoda melanoleuca) sex identification

We developed an inexpensive, fast and reliable PCR method for sex identification of giant panda (Ailuropoda melanoleuca) by using one pair of primers to co-amplify homologous fragments with size polymorphism that located at amelogenin (AMEL) exon 5. In giant panda, a 63 bp deletion in exon 5 of Y-linked allele provides a significant discrimination between AMELX and AMELY, thus the amplification products can be distinguished simply by agarose gel electrophoresis, exhibiting sex-specific banding patterns (male: 237 bp, 174 bp; female: 237 bp). Both blood and feces samples from known-sex giant pandas were successfully amplified. Cross species test also revealed that this method could be applied to other Ursidae species. These authors contributed equally to this work.     

卧龙圈养大熊猫遗传多样性现状及预测,

以中国最大的大熊猫圈养种群—四川卧龙中国大熊猫保护中心的圈养种群为对象,以８个大熊猫微卫星位点为分子标记, 探讨了大熊猫圈养种群的遗传多样性, 并与邛崃野生种群及其他７个濒危物种进行比较。微卫星数据表明, 圈养种群的遗传多样性水平(A＝5.5, He ＝0.620, Ho＝0.574) 低于邛崃野生种群(A＝9.8,He＝0.779,Ho＝0.581),但高于其他7 个濒危物种的种群(He＝0.13~0.46)。在此数据的基础上对未来100个世代内圈养种群遗传多样性的变化情况做出了预测。结果表明假设种群数量比现在扩大一倍, 经历100个世代后也只会使平均等位基因数少减少0.4。因此继续增加野生个体对保持遗传多样性的意义已经不大, 建议该圈养种群的保护策略应将重点放到制定更有效的繁殖计划以避免近交上。     

Determination of Baylisascaris schroederi Infection in Wild Giant Pandas by an Accurate and Sensitive PCR/CE-SSCP Method

It has been recognized that other than habitat loss, degradation and fragmentation, the infection of the roundworm Baylisascaris schroederi (B. schroederi) is one of the major causes of death in wild giant pandas. However, the prevalence and intensity of the parasite infection has been inconsistently reported through a method that uses sedimentation-floatation followed by a microscope examination. This method fails to accurately determine infection because there are many bamboo residues and/or few B. schroederi eggs in the examined fecal samples. In the present study, we adopted a method that uses PCR and capillary electrophoresis combined with a single-strand conformation polymorphism analysis (PCR/CE-SSCP) to detect B. schroederi infection in wild giant pandas at a nature reserve, and compared it to the traditional microscope approach. The PCR specifically amplified a single band of 279-bp from both fecal samples and positive controls, which was confirmed by sequence analysis to correspond to the mitochondrial COII gene of B. schroederi. Moreover, it was demonstrated that the amount of genomic DNA was linearly correlated with the peak area of the CE-SSCP analysis. Thus, our adopted method can reliably detect the infectious prevalence and intensity of B. schroederi in wild giant pandas. The prevalence of B. schroederi was found to be 54% in the 91 fecal samples examined, and 48% in the fecal samples of 31 identified individual giant pandas. Infectious intensities of the 91 fecal samples were detected to range from 2.8 to 959.2 units/gram, and from 4.8 to 959.2 units/gram in the fecal samples of the 31 identified giant pandas. For comparison, by using the traditional microscope method, the prevalence of B. schroederi was found to be only 33% in the 91 fecal samples, 32% in the fecal samples of the 31 identified giant pandas, and no reliable infectious intensity was observed.     

圈养小熊猫的昼夜活动节律

Daily activity rhytlms of three radio-collared captive red pandas were monitored individually at 15 minute intervals for 3 euntinuous days each month at Yele Nature Reserve, Xiangling Mountains, June- October 1995. Our data indicated that mean rate of activity [0.51 ) of captive red pandas was lower than that of wild red pandas. Three captive red pandas showed similar daily activity patterns, being least active during night and more active during daytime. Mean rate of activity during daylight (0.58)was higher than during nighttime (0.41). Daily mean durations of activity and rest were 12.21 hours and 11.79 hours, respectively. Active times of captive red pandas accounted for 50.6% of each 24 hours, of which 66.5% were recorded during daylight and 33.5% during night. Two active peaks appeared at07: 30-09: 15 and 17: 30- 19: 00. We recorded a mean of 2.17, 2.13 and 0.88 long, mid-length, and short resting bouts daily, which had mean durations of 3.47, 1.65 and 0.87 hour, respectively.Among these long rests, 46.1% occurred during daytime and 53.8% during nighttime.     

圈养小熊猫遗传多样性与种群遗传结构分析

小熊猫是亚洲特有的珍稀濒危动物,目前受到栖息地减少、片断化和人类活动干扰等威胁。中国圈养小熊猫已经有60 多年历史,约55 个机构曾经饲养过小熊猫,现今圈养数量有400 多只,评估小熊猫圈养种群的遗传多样性和遗传结构对科学维持圈养种群和保存遗传种质资源意义重大。本研究利用19 个微卫星座位,对中国境内11 个小熊猫圈养种群的116 只个体进行了遗传多样性评估及遗传结构分析。结果显示11 个种群都具有较高的遗传多样性,平均基因丰富度3.505 ± 1.033 (北京)至4.026 ± 1.219 (冕宁),期望杂合度0.631 ± 0.225(黄山)至0.782 ±0.171 (温岭)。其中福州和无锡种群极显著偏离Hardy-Weinberg 平衡。整个圈养群体内各个种群遗传分化系数为0.055,呈显著分化,表明11 个种群遗传分化水平较高。Bayesian 遗传聚类分析将11 个种群聚为三个遗传簇,与野生种群的遗传聚类结果一致。结论:小熊猫圈养种群与野生种群相比,同样具有较高的遗传多样性。因此,圈养小熊猫遗传管理的重点不再是引进野生个体充实圈养种群,应制订科学的繁殖计划,避免近交,从而维持圈养种群的遗传多样性。     

Sex identification of pigs using polymerase chain reaction amplification of the amelogenin gene

The amelogenin (AMEL) gene exists on both sex chromosomes of various mammalian species and the length and sequence of the noncoding regions differ between the two chromosome-specific alleles. Because both forms can be amplified using a single primer set, the use of AMEL in polymerase chain reaction (PCR)-based methods has facilitated sex identification in various mammalian species, including cattle, sheep and humans. In this study, we designed PCR primers to yield different-sized products from the AMEL genes on the X (AMELX) and Y (AMELY) chromosomes of pigs. PCR amplification of genomic DNA samples collected from various breeds of pigs (European breeds: Landrace, Large White, Duroc and Berkshire; Chinese breeds: Meishan and Jinhua and their crossbreeds) yielded the expected products. For all breeds, DNA from male pigs produced two bands (520 and 350 bp; AMELX and AMELY, respectively), whereas samples from female pigs generated only the 520 bp product. We then tested the use of PCR of AMEL for sex identification of in vitro-produced (IVP) porcine embryos sampled at 2 or 5 to 6 days after fertilization; germinal vesicle (GV)-stage oocytes and electroactivated embryos were used as controls. More than 88% of the GV-stage oocytes and electroactivated embryos yielded a single 520 bp single band and about 50% of the IVP embryos tested produced both bands. Our findings show that PCR analysis of the AMEL gene is reliable for sex identification of pigs and porcine embryos.     

Microsatellite variability reveals the necessity for genetic input from wild giant pandas (Ailuropoda melanoleuca) into the captive population

Recent success in breeding giant pandas in captivity has encouraged panda conservationists to believe that the ex situ population is ready to serve as a source for supporting the wild population. In this study, we used 11 microsatellite DNA markers to assess the amount and distribution of genetic variability present in the two largest captive populations (Chengdu Research Base of Giant Panda Breeding, Sichuan Province and the China Research and Conservation Center for the Giant Panda at Wolong, Sichuan Province). The data were compared with those samples from wild pandas living in two key giant panda nature reserves (Baoxing Nature Reserve and Wanglang Nature Reserve). The results show that the captive populations have retained lower levels of allelic diversity and heterozygosity compared to isolated wild populations. However, low inbreeding coefficients indicate that captive populations are under careful genetic management. Excessive heterozygosity suggests that the two captive populations have experienced a genetic bottleneck, presumably caused by founder effects. Moreover, evidence of increased genetic divergence demonstrates restricted breeding options within facilities. Based on these results, we conclude that the genetic diversity in the captive populations is not optimal. Introduction of genetic materials from wild pandas and improved exchange of genetic materials among institutions will be necessary for the captive pandas to be representative of the wild populations.     

圈养小熊猫几种病毒的PCR检测

本文采用巢式PCR/ RT-PCR 方法,对我国10 个动物园中无临床症状的圈养小熊猫的71 个肛拭子和61 个唾液拭子样品,进行犬瘟热病毒(CDV)、犬细小病毒(CPV)、犬冠状病毒(CCV)、犬腺病毒(CAV)和犬疱疹病毒(CHV)的检测,以评估我国圈养小熊猫是否面临这几种病毒的威胁。对阳性PCR 结果进行测序分析,并与GenBank 上的序列进行比较。结果,在肛拭子样品中检测到3 个CPV 和6 个CCV 阳性结果,经测序后,与GenBank 上序列的同源性分别达99% 和100% 。而在唾液拭子样品中没有检测到任何阳性结果,且CDV、CAV和CCV 的检测结果均为阴性。从阳性CPV 的肛拭子样品中分离到一株细小病毒毒株,表明圈养小熊猫已受到细小病毒和犬冠状病毒的感染,今后应加强这两种病毒的预防工作。本文所采用的PCR 方法检测病毒性疾病,能检测到微量的病毒模板,可对小熊猫病毒性感染进行早期诊断。     

Assessing the use of the mitochondrial cox1 marker for use in DNA barcoding of red algae (Rhodophyta)

The red algae, a remarkably diverse group of organisms, are difficult to identify using morphology alone. Following the proposal to use the mitochondrial cytochrome c oxidase subunit I (cox1) for DNA barcoding animals, we assessed the use of this gene in the identification of red algae using 48 samples plus 31 sequences obtained from GenBank. The data set spanned six orders of red algae: the Bangiales, Ceramiales, Corallinales, Gigartinales, Gracilariales and Rhodymeniales. The results indicated that species could be discriminated. Intraspecific variation was between 0 and 4 bp over 539 bp analyzed except in Mastocarpus stellatus (0-14 bp) and Gracilaria gracilis (0-11 bp). Cryptic diversity was found in Bangia fuscopurpurea, Corallina officinalis, G. gracilis, M. stellatus, Porphyra leucosticta and P. umbilicalis. Interspecific variation across all taxa was between 28 and 148 bp, except for G. gracilis and M. stellatus. A comparison of cox1 with the plastid Rubisco spacer for Porphyra species revealed that it was a more sensitive marker in revealing incipient speciation and cryptic diversity. The cox1 gene has the potential to be used for DNA barcoding of red algae, although a good taxonomic foundation coupled with extensive sampling of taxa is essential for the development of an effective identification system.     

Development, multiplexing, and application of ARMS tests for common mutations in the CFTR gene.

The amplification refractory mutation system (ARMS) is a simple, rapid and reliable method for the detection of any mutation involving single base changes or small deletions. We have applied ARMS methodology to the detection of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Single ARMS tests have been developed for 11 CFTR mutations found in the northwest of England. ARMS reactions for the most common mutations have been multiplexed to give a test which will detect the presence of the delta F508, G551D, G542X, and 621 + 1G----T mutations in a DNA sample. The multiplex test has been validated by the analysis of over 500 previously genotyped samples and has been found to be completely accurate. The rapid detection of the most common mutations has enabled early molecular confirmation of suspected cystic fibrosis in neonates, rapid typing of cystic fibrosis patients and their relatives, and testing of sperm and egg donors.     

Habitat correlates of the red panda in the temperate forests of Bhutan

Anthropogenic activities and associated global climate change are threatening the biodiversity in the Himalayas against a backdrop of poor knowledge of the region's threatened species. The red panda (Ailurus fulgens) is a threatened mammal confined to the eastern Himalayas, and because of Bhutan's central location in the distributional range of red pandas, its forests are integral to the long-term viability of wild populations. Detailed habitat requirements of the red panda are largely speculative, and there is virtually no ecological information available on this species in Bhutan. Between 2007 and 2009, we established 615 presence/absence plots in a systematic sampling of resident habitat types within Jigme Dorji and Thrumshingla National Parks, Bhutan, to investigate broad and fine-scale red panda habitat associations. Additional locality records of red pandas were obtained from interviewing 664 park residents. Red pandas were generally confined to cool broadleaf and conifer forests from 2,110-4,389 m above sea level (asl), with the majority of records between 2,400-3,700 m asl on south and east-facing slopes. At a finer scale, multivariate analysis revealed that red pandas were strongly associated with old growth Bhutan Fir (Abies densa) forest dominated by a dense cover of Yushania and Arundanaria bamboo with a high density of fallen logs and tree stumps at ground level; a high density of trees, dead snags, and rhododendron shrubs in the mid-storey; and locations that were close to water. Because Bhutan's temperate forests that encompass prime red panda habitat are also integral to human subsistence and socio-economic development, there exists an inadvertent conflict between the needs of people and red pandas. As such, careful sustainable management of Bhutan's temperate forests is necessary if a balance is to be met between the socioeconomic needs of people and the conservation goals for red pandas.     

大熊猫血液生理指标的测定

大熊猫(Aluropoda melanoleuca)是举世瞩目的珍稀濒危动物,被誉为中国的国宝,越来越受到国内外各界人士的喜爱和重视。关于大熊猫的血液生理生化指标资料已有较多报道。Chen(1987)对6只大熊猫,王强等(1998)对18只大熊猫,     

Differences of the porcine amelogenin X and Y chromosome genes (AMELX and AMELY) and their application for sex determination in pigs

Molecular sexing in wild and domestic animals has becoming an important issue in several fields including reproduction. X and Y chromosome-specific sequence differences of the amelogenin genes (AMELX and AMELY) have been described in different mammalian species and used for sex determination. We studied the possibility to use sequence variability between the porcine AMELX and AMELY genes for sex determination in pigs. Sequence analysis of about 400 bp of intron 3 of the porcine amelogenin genes showed the presence of a 9-10 bp deletion in AMELY gene compared to AMELX sequences. Moreover, one single nucleotide polymorphism (SNP) was detected for the AMELY sequence. Four other SNPs and 1 bp insertion differentiated three AMELX haplotypes indicating an unexpected quite high nucleotide diversity for a chromosome X region. Two sex determination assays targeting the 9-10 bp difference between AMELX and AMELY were developed. Assessment of the accuracy of the amelogenin assays to correctly sex individuals was tested on 329 pigs belonging to different breeds/lines. All analysed animals were correctly sexed with the new designed amelogenin tests. No amplification was obtained in human, cattle, goat, sheep, and horse genomic DNA. These assays can be used for sex diagnosis of small amounts of genomic DNA (20 pg) obtained from different sources including embryo biopsies, hair, meat, and other biological specimens. Thus, apart from the application in the reproduction field, these tests can be useful in several other sectors including forensics, archaeozoology, meat production, and processing as well as for quality control in sample identification.     

大熊猫与同域动物在海拔分布上的生态位分化

大熊猫（Ailuropoda melanoleuca）是中国的特有物种,其成功放归可能受放归地气候、栖息地、同域动物等多种因素的影响。本研究在野外调查的基础上,基于生态位理论等方法,探讨了栗子坪国家级自然保护区大熊猫与其大中型同域动物的空间关系。调查结果表明,该保护区内大熊猫同域动物共20种,分别属于2纲5目,其中东洋型分布物种占优势（30%）。大熊猫同域动物分布海拔显著低于大熊猫的分布海拔（P＜0.05）,大熊猫分别与灵长目等5个目的动物在海拔分布上均存在显著的生态位分化（P＜0.001）。灵长目动物在空间分布上与大熊猫分布相异,而其他目的动物与大熊猫空间分布类似。偶蹄目动物痕迹数量占比最高（54.14 %）,而食肉目的小熊猫与大熊猫样线共同遇见率达到了43.75%。本研究结果表明,该地大熊猫同域动物较为丰富,在海拔分布上与大熊猫存在生态位分化。该研究可为圈养大熊猫的放归提供参考依据。     

自动感应照相系统在大熊猫以及同域分布的野生动物研究中的应用

鉴于野生大熊猫种群的濒危现状,已经不允许对其生境进行破坏性或干扰其行为活动较多的调查活动。例如,野生大熊猫个体数量稀少、其栖息地地形复杂或植被茂密,野外直接观察和调查野生大熊猫极为困难。自动感应照相系统是一种非损伤性野生动物调查工具,在很大程度上弥补了传统调查方法的不足,为野生动物的调查和研究提供了新的有效途径。本研究利用自行研究和开发的自动感应照相系统,获得了野生大熊猫及与其同域分布的其它物种的重要生态信息,显示了自动感应照相系统在物种鉴定、区系调查、个体识别、种群监测、性别确定和行为生态学研究等多方面的应用价值[动物学报51(3)：495—500,2005]。     

Direct haplotype determination by double ARMS: specificity, sensitivity and genetic applications.

We have developed a novel double Amplification Refractory Mutation System (double ARMS) using a highly polymorphic region 5' to the human delta-globin gene as a model system. The double ARMS approach involves using two allele-specific ARMS primers simultaneously during DNA amplification by the polymerase chain reaction (PCR). The resulting system is highly sensitive and more specific than single ARMS. In addition, this approach enables the elucidation of the relationship of polymorphic sites on the same chromosome and thus allows the direct determination of haplotypes. We have also demonstrated that this system can be used in conjunction with inverse PCR, the resulting double ARMS inverse PCR (DARMSI-PCR) may allow haplotype determination on polymorphic sites which are separated further apart than the length limit imposed by PCR. The double ARMS approach has numerous other applications in molecular biology including HLA typing, virology, forensic pathology and the investigation of the phenomenon of chimerism following bone marrow transplantation.     

Sex identification assay useful in great apes is not diagnostic in a range of other primate species.

The ability to identify the sex of individuals from noninvasive samples can be a powerful tool for field studies. Amelogenin, a nuclear gene proximate to the pseudoautosomal region of mammalian sex chromosomes, has a 6 base-pair (bp) size difference between human X and Y chromosomes that can be PCR-amplified and sized to distinguish male from female DNA. We examined whether this test can be used to identify sex from different DNA sources across a number of nonhuman primate taxa. Using human amelogenin primers, we were able to amplify diagnostic products from the four great ape species tested, but products from five other primate species were not sexually dimorphic.