Sequencing,annotation and comparative analysis of nine BACs of giant panda (<Emphasis Type="Italic">Ailuropoda melanoleuca</Emphasis>)

A 10-fold BAC library for giant panda was constructed and nine BACs were selected to generate finish sequences. These BACs could be used as a validation resource for the de novo assembly accuracy of the whole genome shotgun sequencing reads of giant panda newly generated by the Illumina GA sequencing technology. Complete sanger sequencing, assembly, annotation and comparative analysis were carried out on the selected BACs of a joint length 878 kb. Homologue search and de novo prediction methods were used to annotate genes and repeats. Twelve protein coding genes were predicted, seven of which could be functionally annotated. The seven genes have an average gene size of about 41 kb, an average coding size of about 1.2 kb and an average exon number of 6 per gene. Besides, seven tRNA genes were found. About 27 percent of the BAC sequence is composed of repeats. A phylogenetic tree was constructed using neighbor-join algorithm across five species, including giant panda, human, dog, cat and mouse, which reconfirms dog as the most related species to giant panda. Our results provide detailed sequence and structure information for new genes and repeats of giant panda, which will be helpful for further studies on the giant panda.     

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.     

Does one size fit all? A multispecies approach to regional landscape corridor planning

Aim

The practical value of the single‐species approach to conserve biodiversity could be minimal or negligible when sympatric species are limited by factors that are not relevant to the proposed umbrella species. In this study, we quantitatively evaluated as follows: (1) habitat suitability and potential movement corridors of a single umbrella species, giant panda (Ailuropoda melanoleuca); (2) habitat suitability of sympatric mammals; and (3) the potential effectiveness of the single‐species corridor planning to preserve suitable habitat and its connectivity of other focal species.

Location

Qinling Mountains, central part of China (15,000 km²).

Methods

We collected species distribution, environmental and anthropogenic data and conducted species occupancy modelling for giant panda and six other sympatric species (i.e., takin Budorcas taxicolor, tufted deer Elaphodus cephalophus, Chinese goral Naemorhedus griseus, Reeve's muntjac Muntiacus reevesi, leopard cat Prionailurus bengalensis and yellow‐throated marten Martes flavigula). We then conducted circuit models to identify potential corridors for each species and evaluated the effectiveness of giant panda corridors to restore the habitat connectivity for these sympatric mammals.

Results

Occupancy modelling revealed that each species had a unique set of environmental variables associated with its distribution in the Qinling Mountains. We found that giant panda and all other focal species had some degree of fragmentation to their suitable habitat that required restoring habitat connectivity. Among the eight potential giant panda corridors, conservation efforts to reduce anthropogenic impacts would significantly improve the effectiveness of six corridors, while the other two corridors would require altering the vegetation. Five proposed giant panda corridors had remarkable overlap with corridors proposed for other species. We suggest two giant panda corridors as a priority due to their potential to maximize the benefits to both giant panda and a broader suite of mammals.

Main conclusions

Corridor planning in this region of China will likely continue using the single‐species policy, but our results highlight that not all potential giant panda corridors have equal effectiveness for other wildlife species. When offered multiple alternative actions, conservation planners can prioritize corridor development based on a multispecies perspective without loss of connectivity for the priority species. This approach has strong implications to the conservation of wildlife communities in China, and elsewhere, where conservation plans developed for a single‐species garner most available funding and institutional support.

     

大熊猫和亚洲黑熊TNNC1 基因的克隆、表达与序列分析

慢肌肌钙蛋白C (Troponin C type 1,TNNC1)具有高度保守性,调控骨骼肌慢肌和心肌的收缩,影响肌蛋白的生成,从而可能导致动物肌肉的生长、进化和功能的差异。本研究以大熊猫和亚洲黑熊骨骼肌为材料,提取总RNA 和基因组DNA,运用RT-PCR 和Touch-down PCR 分别扩增出TNNC1 基因的cDNA 序列和结构基因序列,并且构建了含有TNNC1 cDNA 的重组表达载体,转化进入E. coli BL21 进行超表达研究。结果表明大熊猫TNNC1 基因的cDNA 片段长602 bp,包含一个编码161 个氨基酸的开放阅读框,其结构基因全长2 831 bp,包含6 个外显子和5 个内含子。亚洲黑熊TNNC1 基因的cDNA 片段长486 bp,亦包含一个编码161 个氨基酸的开放阅读框,其结构基因全长2 758 bp,同样包含6 个外显子和5 个内含子。该两个物种的TNNC1 基因与已报道的13种动物的TNNC1 基因具有很高的相似性。拓扑预测表明,大熊猫和亚洲黑熊TNNC1 蛋白有1 个蛋白激酶C 磷酸化位点,5 个酪蛋白激酶Ⅱ磷酸化位点,1 个N-豆蔻酰化位点,3 个EF 手性钙结合域及1 个N - 糖基化位点。将TNNC1 基因在大肠杆菌中表达发现TNNC1 蛋白与氮端多聚组氨酸标签蛋白(His6) 融合成大小为23. 5kD 左右的多肽,这与预期结果相一致。本研究结果为进一步深入探讨大熊猫和亚洲黑熊TNNC1 基因及蛋白的结构、功能和进化关系提供资料。     

Nucleotide Sequences of an Important Functional Gene hnRNPA2/B1 from Ailuropoda melanoleuca and Ursus thibetanus mupinensis and Its Potential Value in Phylogenetic Study

The cDNA fragments of hnRNPA2/B1 were cloned from the giant panda and black bear using RT-PCR method, which were, respectively, 1029bp and 1026bp in length encoding 343 and 341 amino acids. Analysis indicated the cDNA cloned from the giant panda encoded variant B1 while the cDNA cloned from black bear encoded variant A2.

Analyzing the hnRNPA2B1 peptide of the giant panda and black bear, 76 glycine residues and 86 glycine residues were, respectively, found, and moreover, most glycine are concentrated in the latter halves of the hnRNPA2B1 peptides. Functional sites prediction also showed many N-myristoylation sites existed in the glycine-rich domain, which is probably related to the role of telomere maintenance.

From base bias and substitution analysis, we can conclude that the ORF of hnRNPA2/B1 biased G while hated C, and transition of the third site did not achieve the level of saturation.

Orthology analysis indicated that both the nucleotide sequence and the deduced amino acid sequence showed high identity to other 26 hnRNPA2/B1 sequences from mammals and nonmammals reported. These sequences were used to construct phylogenetic trees employing the NJ method with 1000 bootstrap, and the obtained tree demonstrated similar topology with the classical systematics, which suggested the potential value of hnRNPA2/B1 in phylogenetic analysis.

This report will be the first step to the study function of hnRNPA2/B1 in the giant panda and black bear, and will provide a scientific basis to disease surveillance, captive breeding, and conservation of the endangered species.     

大熊猫苦味受体T2R2 基因的克隆与序列分析

Based on bitter taste receptor T2R2 gene sequence of domesticated dog(AB249685), one pair of primers were designed and used to amplify an approximately 1.1 kb DNA fragment from genomic DNA sample of giant panda by using PCR. The PCR products were ligated into the pMD-18T vector, and then transformed into competent cells of E.coli DH5α. The identified positive clone was sequenced. The result showed that the T2R2 gene of giant panda was 1 008 bp in length, and contained complete exon, and 915 bp, encoding 304...     

Assessing vulnerability of giant pandas to climate change in the Qinling Mountains of China

Climate change might pose an additional threat to the already vulnerable giant panda (Ailuropoda melanoleuca). Effective conservation efforts require projections of vulnerability of the giant panda in facing climate change and proactive strategies to reduce emerging climate‐related threats. We used the maximum entropy model to assess the vulnerability of giant panda to climate change in the Qinling Mountains of China. The results of modeling included the following findings: (1) the area of suitable habitat for giant pandas was projected to decrease by 281 km² from climate change by the 2050s; (2) the mean elevation of suitable habitat of giant panda was predicted to shift 30 m higher due to climate change over this period; (3) the network of nature reserves protect 61.73% of current suitable habitat for the species, and 59.23% of future suitable habitat; (4) current suitable habitat mainly located in Chenggu, Taibai, and Yangxian counties (with a total area of 987 km²) was predicted to be vulnerable. Assessing the vulnerability of giant panda provided adaptive strategies for conservation programs and national park construction. We proposed adaptation strategies to ameliorate the predicted impacts of climate change on giant panda, including establishing and adjusting reserves, establishing habitat corridors, improving adaptive capacity to climate change, and strengthening monitoring of giant panda.     

同域分布的珍稀野生动物对放牧的行为响应策略

保护区内放牧活动对野生动物保护存在负面影响,明确不同物种对放牧干扰的行为响应对制定更有针对性的保护管理政策具有重要意义。使用红外相机研究卧龙自然保护区放牧活动对多种珍稀野生动物的影响,分析放牧激励政策实施前后大熊猫(Ailuropoda melanoleuca)及其同域分布的小熊猫(Ailurus fulgens)、川金丝猴(Rhinopithecus roxellana)、水鹿(Rusa unicolor) 4种珍稀野生动物的照片数、空间分布以及活动模式的变化,探讨这4种动物对放牧的行为响应策略。结果表明:(1)一期(2012—2013),2012年10月实施了禁马政策,同年12月实施放牧(牛羊)激励政策)家畜照片数量很少,4种野生动物照片数相对较多。二期(2014—2015)家畜的照片数显著增加(P0.01),小熊猫(P0.05)与川金丝猴(P0.01)的照片数均显著减少,大熊猫、水鹿的照片数也呈减少趋势;到三期(2016—2017),大熊猫、小熊猫及水鹿3种关注野生动物的照片数基本回升到激励政策实施前的水平,无川金丝猴照片记录。(2)一期,4种野生动物在研究区域有较广的分布;二期,大熊猫、小熊猫的空间分布范围均缩小,无川金丝猴空间分布信息,而家畜、水鹿的空间分布范围有所增加;到三期,大熊猫、小熊猫的空间分布基本恢复到放牧激励政策实施前的区域,无川金丝猴的空间分布信息。(3)放牧激励政策实施前后,大熊猫、小熊猫及川金丝猴活动模式无明显变化,但水鹿的活动更加集中于傍晚,以避开人类与家畜的活动高峰。同域分布的不同的野生动物对人类活动(如放牧)的行为响应策略不同,各保护区在制定相关保护政策时应综合考虑人类干扰对多个物种的影响,增加决策的科学性与合理性。     

Giant Panda (Ailuropoda melanoleuca) Buccal Mucosa Tissue as a Source of Multipotent Progenitor Cells

Since the first mammal was cloned, the idea of using this technique to help endangered species has aroused considerable interest. However, several issues limit this possibility, including the relatively low success rate at every stage of the cloning process, and the dearth of usable tissues from these rare animals. iPS cells have been produced from cells from a number of rare mammalian species and this is the method of choice for strategies to improve cloning efficiency and create new gametes by directed differentiation. Nevertheless information about other stem cell/progenitor capabilities of cells from endangered species could prove important for future conservation approaches and adds to the knowledge base about cellular material that can be extremely limited. Multipotent progenitor cells, termed skin-derived precursor (SKP) cells, can be isolated directly from mammalian skin dermis, and human cheek tissue has also been shown to be a good source of SKP-like cells. Recently we showed that structures identical to SKPs termed m-SKPs could be obtained from monolayer/ two dimensional (2D) skin fibroblast cultures. Here we aimed to isolate m-SKPs from cultured cells of three endangered species; giant panda (Ailuropoda melanoleuca); red panda (Ailurus fulgens); and Asiatic lion (Panthera leo persica). m-SKP-like spheres were formed from the giant panda buccal mucosa fibroblasts; whereas dermal fibroblast (DF) cells cultured from abdominal skin of the other two species were unable to generate spheres. Under specific differentiation culture conditions giant panda spheres expressed neural, Schwann, adipogenic and osteogenic cell markers. Furthermore, these buccal mucosa derived spheres were shown to maintain expression of SKP markers: nestin, versican, fibronectin, and P75 and switch on expression of the stem cell marker ABCG2. These results demonstrate that giant panda cheek skin can be a useful source of m-SKP multipotent progenitors. At present lack of sample numbers means that we can only postulate why we were unable to obtain m-SKPs from the lion and red panda cultures. However the giant panda observations point to the value of archiving cells from rare species, and the possibilities for later progenitor cell derivation.     

Mandible of the giant panda (Ailuropoda melanoleuca) compared with other Chinese carnivores: functional adaptation

The aim of this study was to understand the mandible of the giant panda in morphometric terms to explore differences between the giant panda and other carnivores distributed in China, in terms of functional adaptation. Twelve mandibular variables were studied using bivariate (allometry) and multivariate (principal components analysis, PCA, and discriminant functional analysis, DFA) tools. When deviations were produced from allometric baselines consisting of all the species studied, the giant panda displayed a much more developed mandibular structure than the bear, leopard, and tiger. This may be related to its specific dietary preference for bamboo, which has very strong fibers. Results also indicate that the mandibular structure among carnivores mainly reflects the differences in their dietary preferences and functional adaptation. Three groups were found referring to dispersal profiles expressed by the first two axes of PCA and DFA: (1) the two panda species – the herbivorous carnivores; (2) the black bear – the omnivorous carnivore; and (3) the tiger and leopard – the hypercarnivores. Nevertheless, a significant separation between the two panda species was also found with the profiles displayed by the first and third axes of DFA. In addition to no close evolutionary relationship and phylogenetic development, a noticeable separation between the two panda species found in DFA analysis may be associated with their variation in consuming different parts of the bamboo plant: the giant panda feeds on stems and the red panda feeds on leaves.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 92 , 449–456.     

大熊猫等五种食肉动物血清蛋白和LDH同工酶盘电泳比较

大熊猫(Ailuropoda melanoleuca)是世界珍稀动物之一,近年来,兽类科学工作者对它进行了多方面的研究,也包括生化方面的工作(潘文石等,1982;Sarich,1973),但与其他食肉哺乳动物相比研究较少,而此又为探讨其分类地位所需。本文应用聚丙烯酰胺凝胶盘电泳,对大熊猫、小熊猫(AilurSs fulgens)、黑熊(Selenarctos thibetanus)、家猫及犬等5种食肉动物的血清蛋白和LDH同工酶进行比较分析,目的在于了解这5种动物的血清蛋白图象和LDH同工酶酶谱以及它们之间的谱型、相对活力、迁移率的异同,进而讨论大熊猫的分类地位。     

Black and white and read all over: the past,present and future of giant panda genetics

Few species attract much more attention from the public and scientists than the giant panda (Ailuropoda melanoleuca), a popular, enigmatic but highly endangered species. The application of molecular genetics to its biology and conservation has facilitated surprising insights into the biology of giant pandas as well as the effectiveness of conservation efforts during the past decades. Here, we review the history of genetic advances in this species, from phylogeny, demographical history, genetic variation, population structure, noninvasive population census and adaptive evolution to reveal to what extent the current status of the giant panda is a reflection of its evolutionary legacy, as opposed to the influence of anthropogenic factors that have negatively impacted this species. In addition, we summarize the conservation implications of these genetic findings applied for the management of this high‐profile species. Finally, on the basis of these advances and predictable future changes in genetic technology, we discuss future research directions that seem promising for giant panda biology and conservation.     

Automatically detecting the wild giant panda using deep learning with context and species distribution model

The giant panda is a flagship species in ecological conservation. The infrared camera trap is an effective tool for monitoring the giant panda. Images captured by infrared camera traps must be accurately recognized before further statistical analyses can be implemented. Previous research has demonstrated that spatiotemporal and positional contextual information and the species distribution model (SDM) can improve image detection accuracy, especially for difficult-to-see images. Difficult-to-see images include those in which individual animals are only partially observed and it is challenging for the model to detect those individuals. By utilizing the attention mechanism, we developed a unique method based on deep learning that incorporates object detection, contextual information, and the SDM to achieve better detection performance in difficult-to-see images. We obtained 1169 images of the wild giant panda and divided them into a training set and a test set in a 4:1 ratio. Model assessment metrics showed that our proposed model achieved an overall performance of 98.1% in mAP_0.5 and 82.9% in recall on difficult-to-see images. Our research demonstrated that the fine-grained multimodal-fusing method applied to monitoring giant pandas in the wild can better detect the difficult-to-see panda images to enhance the wildlife monitoring system.     

大熊猫及其近缘种rDNA序列变异和系统进化关系

应用ｒＤＮＡ间隔区Ｓｏｕｔｈｅｒｎ转换技术研究大熊猫及其近缘种的分子系统关系。通过比较大熊猫、小熊猫、黑熊、马来熊、浣熊和猞猁的ｒＤＮＡ间隔区限制性内切酶图谱,用最大似然法和简约法构建它们的分子系统树。结果表明大熊猫与熊具有较近的亲缘关系,与小熊猫和浣熊的亲缘关系较远。     

Basic Fibroblast Growth Factor Stimulates the Proliferation of Bone Marrow Mesenchymal Stem Cells in Giant Panda (Ailuropoda melanoleuca)

It has been widely known that the giant panda (Ailuropoda melanoleuca) is one of the most endangered species in the world. An optimized platform for maintaining the proliferation of giant panda mesenchymal stem cells (MSCs) is very necessary for current giant panda protection strategies. Basic fibroblast growth factor (bFGF), a member of the FGF family, is widely considered as a growth factor and differentiation inducer within the stem cell research field. However, the role of bFGF on promoting the proliferation of MSCs derived from giant panda bone marrow (BM) has not been reported. In this study, we aimed to investigate the role of bFGF on the proliferation of BM-MSCs derived from giant panda. MSCs were cultured for cell proliferation analysis at 24, 48 and 72 hrs following the addition of bFGF. With increasing concentrations of bFGF, cell numbers gradually increased. This was further demonstrated by performing 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) cell proliferation assay, 5-Bromo-2-deoxyUridine (BrdU) labeling and cell cycle testing. Furthermore, the percentage of MSCs that were OCT4 positive increased slightly following treatment with 5 ng/ml bFGF. Moreover, we demonstrated that the extracellular signal-regulated kinase (ERK) signaling pathway may play an important role in the proliferation of panda MSCs stimulated by bFGF. In conclusion, this study suggests that giant panda BM-MSCs have a high proliferative capacity with the addition of 5 ng/ml bFGF in vitro.     

A reliable genetic technique for sex determination of giant panda (<Emphasis Type="Italic">Ailuropoda melanoleuca</Emphasis>) from non-invasively collected hair samples

Extractions from non-invasive hair samples usually yield low amounts of highly degraded DNA. Previously developed mammal molecular sexing methods were not designed with such sub-optimal conditions in mind. We developed a simple and reliable PCR-based sexing method aimed at degraded, low yield DNA extractions from the giant panda (Ailuropoda melanoleuca). Comparisons of this new primer set with others showed that the reliability of sex determination from low-yield, degraded DNA extractions was improved if; amplification products were short (<170 bp); and the Y-chromosome amplification product was shorter than the X-chromosome amplification product. The primers developed in this study appear useful for sex determination in other bear species.     

大熊猫生长激素受体(GHR) cDNA 的克隆与序列分析

根据已报道的若干物种GHR 基因cDNA 序列设计引物, 利用RT- PCR 技术首次从大熊猫肝脏组织总RNA中扩增出GHR 基因编码区全长cDNA 序列, 克隆于pGEM®-T 载体后进行测序和序列分析。结果表明,大熊猫GHR 的ORF为1 917 bp , 编码638 个氨基酸的前体蛋白, 由18 个氨基酸的信号肽和620 个氨基酸的成熟肽组成,与人、狗、猪GHR 结构相似, 大熊猫GHR 成熟肽由246 个氨基酸的胞外区、24 个氨基酸的跨膜区和350 个氨基酸的胞内区组成, 并具GHR 的特征性结构。序列相似性比较显示, 大熊猫GHR 与哺乳类GHR 具有69 %～93 %的高序列相似性, 与爬行类和鸟类的序列相似性也达到60 % , 而与鱼类的序列相似性较低, 仅为30 %左右。与其它哺乳动物GHR 相比, 大熊猫GHR 在氨基酸序列上也存在明显的特异性。     

Molecular characterization of major histocompatibility complex class I genes from the giant panda (Ailuropoda melanoleuca)

The major histocompatibility complex class I genes play crucial roles in the adaptive immune system of vertebrates against intracellular pathogens. To date, no class I genes from the giant panda (Ailuropoda melanoleuca) has been reported, even none from species of Ursidae. In this study, we successfully identified three class I genes from a giant panda bacterial artificial chromosome library and designated them as Aime-128, 152, and 1906, respectively. Pairwise sequence alignments revealed that (1) the Aime-1906 always possessed the lowest identities (52–86%) in different regions compared with the Aime-128 and 152 and (2) the Aime-128 also varied from the Aime-152 in the regions of 5′ untranslated region (UTR), 3′ UTR, and exon1, whose similarities were 83%, 87%, and 91%, respectively. Comparison of structure characteristics indicated that the Aime-128 possessed all conserved amino acids important to the function of antigen presentation while the Aime-152 and 1906 presented two and five mutated residues. Analysis of phylogenetic trees demonstrated that the Aime-128, 152, and 1906 were clustered into three different branches with 99% or 100% bootstrap values. As a result, these three kinds of evidence supported that the Aime-1906, 152, and 128 should be derived from different loci. Furthermore, in view of a prestop codon in the exon 7 and patterns of amino acid replacement within alleles, the Aime-1906 gene is predicted to be a nonclassical locus, which is most closely related to dog leukocyte antigen 79 in the phylogenetic tree constructed with various mammalian class I loci. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

气候变化对邛崃山系大熊猫主食竹和栖息地分布的影响

气候变化对生物多样性的影响,特别是珍稀濒危物种的影响是当前的研究热点。全球气候变化对大熊猫的影响一直受到广泛关注。根据野外调查的大熊猫活动痕迹点、竹类分布点和主食竹扩散距离数据,采用Maxent模型,利用植被、地形、气候等因素,在RCP8.5下分析了2050年和2070年邛崃山系大熊猫主食竹分布及栖息地变化趋势。结果显示:(1)未来大熊猫适宜生境及主食竹气候适宜区面积均有所减少,到2070年分别减少37.2%和4.7%;(2)未来主食竹分布范围总体向高海拔扩展,但面积持续减少,到2070年分布面积比当前减少8.3%;(3)大熊猫栖息地未来有向高海拔扩张的趋势,在低海拔地区退缩明显,到2070年较当前减少27.2%;但到2070年大熊猫栖息地面积加上非栖息地有主食竹分布的面积,较现有大熊猫栖息地面积大1.5%;(4)受气候变化影响较严重的区域是邛崃山系南部以及低海拔地区,其余区域所受影响相对较小;(5)未来需要加强对受气候变化影响严重区域的监测与保护,特别是邛崃山系中部的大熊猫集中分布区。