A genome-wide survey on basic helix-loop-helix transcription factors in giant panda

The giant panda (Ailuropoda melanoleuca) is a critically endangered mammalian species. Studies on functions of regulatory proteins involved in developmental processes would facilitate understanding of specific behavior in giant panda. The basic helix-loop-helix (bHLH) proteins play essential roles in a wide range of developmental processes in higher organisms. bHLH family members have been identified in over 20 organisms, including fruit fly, zebrafish, mouse and human. Our present study identified 107 bHLH family members being encoded in giant panda genome. Phylogenetic analyses revealed that they belong to 44 bHLH families with 46, 25, 15, 4, 11 and 3 members in group A, B, C, D, E and F, respectively, while the remaining 3 members were assigned into "orphan". Compared to mouse, the giant panda does not encode seven bHLH proteins namely Beta3a, Mesp2, Sclerax, S-Myc, Hes5 (or Hes6), EBF4 and Orphan 1. These results provide useful background information for future studies on structure and function of bHLH proteins in the regulation of giant panda development.     

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.     

Profile of microRNA in Giant Panda Blood: A Resource for Immune-Related and Novel microRNAs

The giant panda (Ailuropoda melanoleuca) is one of the world’s most beloved endangered mammals. Although the draft genome of this species had been assembled, little was known about the composition of its microRNAs (miRNAs) or their functional profiles. Recent studies demonstrated that changes in the expression of miRNAs are associated with immunity. In this study, miRNAs were extracted from the blood of four healthy giant pandas and sequenced by Illumina next generation sequencing technology. As determined by miRNA screening, a total of 276 conserved miRNAs and 51 novel putative miRNAs candidates were detected. After differential expression analysis, we noticed that the expressions of 7 miRNAs were significantly up-regulated in young giant pandas compared with that of adults. Moreover, 2 miRNAs were up-regulated in female giant pandas and 1 in the male individuals. Target gene prediction suggested that the miRNAs of giant panda might be relevant to the expressions of 4,602 downstream genes. Subseuqently, the predicted target genes were conducted to KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis and we found that these genes were mainly involved in host immunity, including the Ras signaling pathway, the PI3K-Akt signaling pathway, and the MAPK signaling pathway. In conclusion, our results provide the first miRNA profiles of giant panda blood, and the predicted functional analyses may open an avenue for further study of giant panda immunity.     

Sequencing,annotation and comparative analysis of nine BACs of the giant panda(Ailuropoda melanoleuca)

A 10-fold BAC library for the 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 the giant panda newly generated by 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 ...     

An improved,chromosome-level genome of the giant panda (Ailuropoda melanoleuca)

BackgroundThe iconic giant panda (Ailuropoda melanoleuca), as both a flagship and umbrella species endemic to China, is a world famous symbol for wildlife conservation. The giant panda has several specific biological traits and holds a relatively small place in evolution. A high-quality genome of the giant panda is key to understanding the biology of this vulnerable species.FindingsWe generated a 2.48-Gb chromosome-level genome (GPv1) of the giant panda named “Jing Jing” with a contig N50 of 28.56 Mb and scaffold N50 of 134.17 Mb, respectively. The total length of chromosomes (n = 21) was 2.39-Gb, accounting for 96.4% of the whole genome. Compared with the previously published four genomes of the giant panda, our genome is characterized by the highest completeness and the correct sequence orientation. A gap-free and 850 kb length of immunoglobulin heavy-chain gene cluster was manually annotated in close proximity to the telomere of chromosome 14. Additionally, we developed an algorithm to predict the centromere position of each chromosome. We also constructed a complete chromatin structure for “Jing Jing”, which includes inter-chromosome interaction pattern, A/B compartment, topologically associated domain (TAD), TAD-clique and promoter-enhancer interaction (PEI).ConclusionsWe presented an improved chromosome-level genome and complete chromatin structure for the giant panda. This is a valuable resource for the future genetic and genomic studies on giant panda.     

Genome of the Giant Panda Roundworm Illuminates Its Host Shift and Parasitic Adaptation

Baylisascaris schroederi, a roundworm (ascaridoid) parasite specific to the bamboo-feeding giant panda (Ailuropoda melanoleuca), represents a leading cause of mortality in wild giant panda populations. Here, we present a 293-megabase chromosome-level genome assembly of B. schroederi to infer its biology, including host adaptations. Comparative genomics revealed an evolutionary trajectory accompanied by host-shift events in ascaridoid parasite lineages after host separations, suggesting their potential for transmission and rapid adaptation to new hosts. Genomic and anatomical lines of evidence, including expansion and positive selection of genes related to the cuticle and basal metabolisms, indicate that B. schroederi undergoes specific adaptations to survive in the sharp-edged bamboo-enriched gut of giant pandas by structurally increasing its cuticle thickness and efficiently utilizing host nutrients through gut parasitism. Additionally, we characterized the secretome of B. schroederi and predicted potential drug and vaccine targets for new control strategies. Overall, this genome resource provides new insights into the host adaptation of B. schroederi to the giant panda as well as the host-shift events in ascaridoid parasite lineages. Our findings on the unique biology of B. schroederi will also aid in the development of prevention and treatment measures to protect giant panda populations from roundworm parasitism.     

基于高分辨率网络的大熊猫姿态估计方法

对圈养大熊猫 (Ailuropoda melanoleuca) 开展长期行为监测能及时了解其所处生理周期和健康状况,有助于繁殖饲养机构迅速采取相应繁育保护措施提高饲养管理水平,但目前无法对大熊猫进行24 h监控并及时地获得相应的行为信息。准确的动物姿态估计是动物行为研究的关键,也是诸多下游应用的基础。了解大熊猫的姿态可以促进大熊猫行为研究并提升保护管理水平。为了提高复杂环境下大熊猫姿态估计的准确率,本文以高分辨率网络 (High resolution net, HRNet) 为基础网络架构提出了一种大熊猫姿态估计方法：针对大熊猫不同部位尺度差异较大的问题,在HRNet-32中引入了空洞空间金字塔池化 (Atrous spatial pyramid pooling, ASPP) 模块,在提升特征感受野的同时捕获多尺度信息;同时对大熊猫身体关键点进行分组,引入基于部位的多分支结构来学习特定于每个部位组的表征。多次对比实验结果表明本文所用模型具有较高的检测精度：在PCK@0.05中所用模型精度达到了81.51%。本文提出的方法可为大熊猫的行为分析和健康评估提供技术支撑。     

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.     

Genome-wide survey and analysis of microsatellites in giant panda (Ailuropoda melanoleuca), with a focus on the applications of a novel microsatellite marker system

Background

The giant panda (Ailuropoda melanoleuca) is a critically endangered species endemic to China. Microsatellites have been preferred as the most popular molecular markers and proven effective in estimating population size, paternity test, genetic diversity for the critically endangered species. The availability of the giant panda complete genome sequences provided the opportunity to carry out genome-wide scans for all types of microsatellites markers, which now opens the way for the analysis and development of microsatellites in giant panda.

Results

By screening the whole genome sequence of giant panda in silico mining, we identified microsatellites in the genome of giant panda and analyzed their frequency and distribution in different genomic regions. Based on our search criteria, a repertoire of 855,058 SSRs was detected, with mono-nucleotides being the most abundant. SSRs were found in all genomic regions and were more abundant in non-coding regions than coding regions. A total of 160 primer pairs were designed to screen for polymorphic microsatellites using the selected tetranucleotide microsatellite sequences. The 51 novel polymorphic tetranucleotide microsatellite loci were discovered based on genotyping blood DNA from 22 captive giant pandas in this study. Finally, a total of 15 markers, which showed good polymorphism, stability, and repetition in faecal samples, were used to establish the novel microsatellite marker system for giant panda. Meanwhile, a genotyping database for Chengdu captive giant pandas (n = 57) were set up using this standardized system. What’s more, a universal individual identification method was established and the genetic diversity were analysed in this study as the applications of this marker system.

Conclusion

The microsatellite abundance and diversity were characterized in giant panda genomes. A total of 154,677 tetranucleotide microsatellites were identified and 15 of them were discovered as the polymorphic and stable loci. The individual identification method and the genetic diversity analysis method in this study provided adequate material for the future study of giant panda.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1268-z) contains supplementary material, which is available to authorized users.     

Exploring the genome of Trypanosoma vivax through GSS and in silico comparative analysis

A survey of the Trypanosoma vivax genome was carried out by the genome sequence survey (GSS) approach resulting in 1,086 genomic sequences. A total of 455 high-quality GSS sequences were generated, consisting of 331 non-redundant sequences distributed in 264 singlets and 67 clusters in a total of 135.5 Kb of the T. vivax genome. The estimation of the overall G+C content, and the prediction of the presence of ORFs and putative genes were carried out using the Glimmer and Jemboss packages. Analysis of the obtained sequences was carried out by BLAST programs against 12 different databases and also using the Conserved Domain Database, InterProScan, and tRNAscan-SE. Along with the existing 23 T. vivax entries in the GenBank, the 32 putative genes predicted and the 331 non-redundant GSS sequences reported herein represent new potential markers for the development of PCRbased assays for specific diagnosis and typing of Trypanosoma vivax.     

Spliceosomal genes in the D. discoideum genome: a comparison with those in H. sapiens, D. melanogaster, A. thaliana and S. cerevisiae

Little is known about pre-mRNA splicing in Dictyostelium discoideum although its genome has been completely sequenced. Our analysis suggests that pre-mRNA splicing plays an important role in D. discoideum gene expression as two thirds of its genes contain at least one intron. Ongoing curation of the genome to date has revealed 40 genes in D. discoideum with clear evidence of alternative splicing, supporting the existence of alternative splicing in this unicellular organism. We identified 160 candidate U2-type spliceosomal proteins and related factors in D. discoideum based on 264 known human genes involved in splicing. Spliceosomal small ribonucleoproteins (snRNPs), PRP19 complex proteins and late-acting proteins are highly conserved in D. discoideum and throughout the metazoa. In non-snRNP and hnRNP families, D. discoideum orthologs are closer to those in A. thaliana, D. melanogaster and H. sapiens than to their counterparts in S. cerevisiae. Several splicing regulators, including SR proteins and CUG-binding proteins, were found in D. discoideum, but not in yeast. Our comprehensive catalog of spliceosomal proteins provides useful information for future studies of splicing in D.βdiscoideum where the efficient genetic and biochemical manipulation will also further our general understanding of pre-mRNA splicing.     

大熊猫脐带间充质干细胞外泌体的分离鉴定及miRNAs富集分析

大熊猫脐带间充质干细胞(Umbilical cord mesenchymal stem cells, UC-MSCs)通过旁分泌所释放的外泌体在大熊猫保健与疾病治疗方面具有一定的应用前景。本研究旨在建立大熊猫UC-MSCs外泌体分离方法,开展生物学特征分析和分子鉴定,并研究UC-MSCs外泌体中miRNAs的种类与功能。采用超速离心法从大熊猫UCMSCs培养上清中成功分离外泌体,通过透射电子显微镜进行形态学观察,纳米颗粒跟踪分析仪检测粒径大小,蛋白免疫印迹法检测特异性分子标记表达。采用Small RNA测序技术对UC-MSCs外泌体中的miRNAs进行鉴定,并对其靶基因进行了预测与功能分析。结果显示,大熊猫UC-MSCs外泌体呈圆形杯托状结构,直径为(79.15±4.81) nm,外泌体标志蛋白CD81与TSG101呈阳性表达而CALNEXIN呈阴性表达。大熊猫UC-MSCs外泌体中的miRNA主要为miR-148-3p (30.28%)与miR-21-5p (21.72%)。本研究首次从大熊猫UC-MSCs培养上清中分离出外泌体,并对其所含的miRNAs进行富集分析及功能预测,为大熊...     

大熊猫肠道正常菌群降解纤维素的机制

大熊猫的主食竹类粗纤维含量很高,而大熊猫自身的消化系统不能降解纤维素。现已从大熊猫的肠道正常菌群中鉴定出涵盖7个菌门的22种菌,相关的研究证明大熊猫的肠道正常菌群能降解纤维素。大熊猫肠道中的假单胞菌产生的漆酶能对竹纤维中的木质素进行氧化,使纤维素得以暴露,梭菌属、淀粉芽胞杆菌等产生的纤维素酶将其降解成大熊猫可利用的糖类。其具体机制有待进一步研究。     

全基因组预测目标基因的新方法及其应用

运用隐马尔可夫模型, 利用Perl编程, 以几种模式生物的蛋白质数据库为基础, 构建了目标基因的全基因组预测的新方法。该方法具有高通量, 准确度高且操作简易等优点, 特别在多结构域蛋白家族预测上更显优势。应用该方法对几种模式生物的全基因组PPR和TPR蛋白家族进行了预测, 其中粳稻日本晴中含有536个PPR蛋白、199个TPR蛋白; 籼稻9311中含有519个PPR蛋白、177个TPR蛋白; 拟南芥中含有735个PPR蛋白、292个TPR蛋白; 红藻中6个PPR蛋白、32个TPR蛋白; 蓝细菌以及古细菌中没有PPR蛋白, 但蓝细菌含有10个TPR蛋白, 古细菌有4个TPR蛋白, 并对所得结果进行了进一步生物信息学分析。     

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。     

<Emphasis Type="Italic">Ab initio</Emphasis> gene identification: Prokaryote genome annotation with GeneScan and GLIMMER

We compare the annotation of three complete genomes using theab initio methods of gene identification GeneScan and GLIMMER. The annotation given in GenBank, the standard against which these are compared, has been made using GeneMark. We find a number of novel genes which are predicted by both methods used here, as well as a number of genes that are predicted by GeneMark, but are not identified by either of the nonconsensus methods that we have used. The three organisms studied here are all prokaryotic species with fairly compact genomes. The Fourier measure forms the basis for an efficient non-consensus method for gene prediction, and the algorithm GeneScan exploits this measure. We have bench-marked this program as well as GLIMMER using 3 complete prokaryotic genomes. An effort has also been made to study the limitations of these techniques for complete genome analysis. GeneScan and GLIMMER are of comparable accuracy insofar as gene-identification is concerned, with sensitivities and specificities typically greater than 0.9. The number of false predictions (both positive and negative) is higher for GeneScan as compared to GLIMMER, but in a significant number of cases, similar results are provided by the two techniques. This suggests that there could be some as-yet unidentified additional genes in these three genomes, and also that some of the putative identifications made hitherto might require re-evaluation. All these cases are discussed in detail.     

Hopes and challenges for giant panda conservation under climate change in the Qinling Mountains of China

One way that climate change will impact animal distributions is by altering habitat suitability and habitat fragmentation. Understanding the impacts of climate change on currently threatened species is of immediate importance because complex conservation planning will be required. Here, we mapped changes to the distribution, suitability, and fragmentation of giant panda habitat under climate change and quantified the direction and elevation of habitat shift and fragmentation patterns. These data were used to develop a series of new conservation strategies for the giant panda. Qinling Mountains, Shaanxi, China. Data from the most recent giant panda census, habitat factors, anthropogenic disturbance, climate variables, and climate predictions for the year 2050 (averaged across four general circulation models) were used to project giant panda habitat in Maxent. Differences in habitat patches were compared between now and 2050. While climate change will cause a 9.1% increase in suitable habitat and 9% reduction in subsuitable habitat by 2050, no significant net variation in the proportion of suitable and subsuitable habitat was found. However, a distinct climate change‐induced habitat shift of 11 km eastward by 2050 is predicted firstly. Climate change will reduce the fragmentation of suitable habitat at high elevations and exacerbate the fragmentation of subsuitable habitat below 1,900 m above sea level. Reduced fragmentation at higher elevations and worsening fragmentation at lower elevations have the potential to cause overcrowding of giant pandas at higher altitudes, further exacerbating habitat shortage in the central Qinling Mountains. The habitat shift to the east due to climate change may provide new areas for giant pandas but poses severe challenges for future conservation.     

Characterizing the spatial distribution of giant pandas (Ailuropoda melanoleuca) in fragmented forest landscapes

Aim To examine the effects of forest fragmentation on the distribution of the entire wild giant panda (Ailuropoda melanoleuca) population, and to propose a modelling approach for monitoring the spatial distribution and habitat of pandas at the landscape scale using Moderate Resolution Imaging Spectro‐radiometer (MODIS) enhanced vegetation index (EVI) time‐series data. Location Five mountain ranges in south‐western China (Qinling, Minshan, Qionglai, Xiangling and Liangshan). Methods Giant panda pseudo‐absence data were generated from data on panda occurrences obtained from the third national giant panda survey. To quantify the fragmentation of forests, 26 fragmentation metrics were derived from 16‐day composite MODIS 250‐m EVI multi‐temporal data and eight of these metrics were selected following factor analysis. The differences between panda presence and panda absence were examined by applying significance testing. A forward stepwise logistic regression was then applied to explore the relationship between panda distribution and forest fragmentation. Results Forest patch size, edge density and patch aggregation were found to have significant roles in determining the distribution of pandas. Patches of dense forest occupied by giant pandas were significantly larger, closer together and more contiguous than patches where giant pandas were not recorded. Forest fragmentation is least in the Qinling Mountains, while the Xiangling and Liangshan regions have most fragmentation. Using the selected landscape metrics, the logistic regression model predicted the distribution of giant pandas with an overall accuracy of 72.5% (κ = 0.45). However, when a knowledge‐based control for elevation and slope was applied to the regression, the overall accuracy of the model improved to 77.6% (κ = 0.55). Main conclusions Giant pandas appear sensitive to patch size and isolation effects associated with fragmentation of dense forest, implying that the design of effective conservation areas for wild giant pandas must include large and dense forest patches that are adjacent to other similar patches. The approach developed here is applicable for analysing the spatial distribution of the giant panda from multi‐temporal MODIS 250‐m EVI data and landscape metrics at the landscape scale.     

繁殖-育幼期雌性大熊猫的空间利用与活动模式特征

繁殖期间的空间利用与活动模式特征是野生动物长期适应环境的具体表现,研究野生动物繁殖期间行为特征与策略是动物生态学研究中最重要的基础问题之一。2010年3月至2012年3月,我们利用内置记录活动水平传感器的GPS项圈记录了1只受孕、产仔并育幼的野生大熊猫（Ailuropoda melanoleuca）的活动位点和活动水平。研究了该大熊猫各繁殖阶段（交配后期、产仔期和育幼期）的空间利用与活动模式特征。结果表明：在交配后期（2010年春季,即4-6月）,雌性大熊猫活动空间范围较大为3.49 km²,其日平均活动距离较大,活动水平也相对较强;在产仔期前后（2010年夏秋季,即7-10月）,雌性大熊猫的活动范围明显缩小为0.42 km²,日平均活动距离和活动水平都明显减低;自2010年冬季（2010年11月至2011年3月）开始雌性大熊猫带仔生活,其家域面积逐渐增大,且趋向稳定 （0.84~1.19 km²）,日平均活动距离和活动水平都开始逐渐增大。雌性大熊猫在求偶交配、受孕、产仔和育幼过程中的空间利用与活动模式特征反映了大熊猫在繁殖期间的行为策略。本研究结果可为繁殖期间大熊猫及栖息地的保护管理政策制定提供科学依据。