Design and Experimental Application of a Novel Non-Degenerate Universal Primer Set that Amplifies Prokaryotic 16S rRNA Genes with a Low Possibility to Amplify Eukaryotic rRNA Genes

The deep sequencing of 16S rRNA genes amplified by universal primers has revolutionized our understanding of microbial communities by allowing the characterization of the diversity of the uncultured majority. However, some universal primers also amplify eukaryotic rRNA genes, leading to a decrease in the efficiency of sequencing of prokaryotic 16S rRNA genes with possible mischaracterization of the diversity in the microbial community. In this study, we compared 16S rRNA gene sequences from genome-sequenced strains and identified candidates for non-degenerate universal primers that could be used for the amplification of prokaryotic 16S rRNA genes. The 50 identified candidates were investigated to calculate their coverage for prokaryotic and eukaryotic rRNA genes, including those from uncultured taxa and eukaryotic organelles, and a novel universal primer set, 342F-806R, covering many prokaryotic, but not eukaryotic, rRNA genes was identified. This primer set was validated by the amplification of 16S rRNA genes from a soil metagenomic sample and subsequent pyrosequencing using the Roche 454 platform. The same sample was also used for pyrosequencing of the amplicons by employing a commonly used primer set, 338F-533R, and for shotgun metagenomic sequencing using the Illumina platform. Our comparison of the taxonomic compositions inferred by the three sequencing experiments indicated that the non-degenerate 342F-806R primer set can characterize the taxonomic composition of the microbial community without substantial bias, and is highly expected to be applicable to the analysis of a wide variety of microbial communities.     

基于转录组数据的齿缘刺猎蝽微卫星分子标记开发

【目的】齿缘刺猎蝽Sclomina erinacea是我国猎蝽科天敌昆虫常见种类,其不同地理种群存在明显形态差异。本研究旨在利用已经获得的齿缘刺猎蝽转录组数据筛选微卫星位点,为齿缘刺猎蝽种群遗传多样性和遗传分化研究开发可靠的微卫星分子标记。【方法】基于高通量测序技术平台Illumina HiSeq^TM 2000获得齿缘刺猎蝽转录组数据(42 215条unigenes),利用MISA软件进行搜索发掘SSR标记;利用Primer Premier 3软件设计SSR引物,从中随机选取54对SSR引物,利用PCR技术在中国齿缘刺猎蝽9个地理种群上进行验证。【结果】利用MISA软件搜索到微卫星位点2 395个,它们分布在2 107条unigenes上,其主要重复类型是三核苷酸重复(占总SSR的44.43%),其次是二核苷酸重复(占总SSR的40.08%),再次是四核苷酸重复(占总SSR的12.94%)。利用Primer Premier 3 软件成功设计出2 000余对SSR引物。随机选取的54对引物对9个齿缘刺猎蝽不同地理种群进行的SSR位点PCR扩增结果表明,共有16对引物较好地扩增出目的片段。【结论】研究表明利用齿缘刺猎蝽转录组数据可以大量发掘微卫星分子标记。本研究为进一步开展齿缘刺猎蝽的种群遗传学研究奠定了基础。     

人免疫球蛋白重链可变区基因引物设计方法的改良

针对抗体胚系基因数据库的数据不断更新和完善,为获得人全部免疫球蛋白(Ig)重链可变区基因,改进引物设计方法,自主设计针对可变区基因高度保守的框架区1(FR1)和框架区4(FR4)的引物,提取未经免疫的健康人外周血单个核细胞,通过RT-PCR扩增重链可变区基因.其DNA序列与GenBank数据库和IMGT/V-QUEST软件比对,序列分析符合人免疫球蛋白重链基本框架结构,为胚系基因重排产生的序列.多个克隆的测序结果对比分析显示了良好的多样性.获得的重链序列为研制基因工程抗体及构建噬菌体抗体库奠定了物质基础,也为扩增其他物种Ig可变区基因的引物提供新的设计思路.     

Loss of growth homeostasis by genetic decoupling of cell division from biomass growth: implication for size control mechanisms

Growing cells adjust their division time with biomass accumulation to maintain growth homeostasis. Size control mechanisms, such as the size checkpoint, provide an inherent coupling of growth and division by gating certain cell cycle transitions based on cell size. We describe genetic manipulations that decouple cell division from cell size, leading to the loss of growth homeostasis, with cells becoming progressively smaller or progressively larger until arresting. This was achieved by modulating glucose influx independently of external glucose. Division rate followed glucose influx, while volume growth was largely defined by external glucose. Therefore, the coordination of size and division observed in wild‐type cells reflects tuning of two parallel processes, which is only refined by an inherent feedback‐dependent coupling. We present a class of size control models explaining the observed breakdowns of growth homeostasis.     

Genome‐enabled development of DNA markers for ecology,evolution and conservation

Molecular markers have become a fundamental piece of modern biology’s toolkit. In the last decade, new genomic resources from model organisms and advances in DNA sequencing technology have altered the way that these tools are developed, alleviating the marker limitation that researchers previously faced and opening new areas of research for studies of non‐model organisms. This availability of markers is directly responsible for advances in several areas of research, including fine‐scaled estimation of population structure and demography, the inference of species phylogenies, and the examination of detailed selective pressures in non‐model organisms. This review summarizes methods for the development of large numbers of DNA markers in non‐model organisms, the challenges encountered when utilizing different methods, and new research applications resulting from these advances.     

水稻种子的细菌含量和群落结构及其影响因素分析

【目的】种子是植物微生物群代际传递的重要途径,但种子携带的微生物群落尚缺乏系统的研究。本研究以水稻种子为模型,定量分析种子的细菌含量、测定种子的细菌群落结构、探究地域与品种对细菌含量及群落结构的影响和鉴定水稻种子的核心菌群。【方法】选取18个水稻品种,每个品种分别来自中国海南和天津2个地域,共36组样本。每组样本包含5或10个DNA样本,每个DNA样本由3粒种子提取的总DNA构成。使用细菌特异性16S rDNA介导的荧光定量PCR技术测定种子的细菌含量,并分析影响因素;使用16S rDNA扩增子测序技术测定种子的细菌群落结构,并用生物信息学方法分析了影响因素和核心菌群。【结果】本研究测定了1 080粒水稻种子的细菌含量,发现经过表面除菌的水稻种子内部存在共栖细菌,平均每克种子的细菌含量为1.53×10⁶。水稻品种对种子的细菌含量有显著影响,而地域无影响。测定180个扩增子文库的细菌群落结构,发现水稻种子的菌群与水稻植株有相似之处,均以变形菌门为主要的细菌门类;地域对水稻种子的细菌群落结构有重要影响,不同地域的水稻种子在主坐标分析(principal co-ordinate analysis, PCoA)中有明显分离;而粳稻和籼稻之间无显著差异。还发现水稻种子存在核心菌群,且相对丰度高达总菌群的85.56%。【结论】本研究系统地揭示了水稻种子的细菌含量、群落结构及其影响因素,为利用种传微生物促进水稻健康提供了数据和方法支持。     

基于高通量测序技术研究水貂远端肠道细菌群落组成(英文)

【目的】研究水貂远端肠道微生物群落组成及其多样性。【方法】通过高通量测序技术研究水貂远端肠内容物细菌的组成和多样性。【结果】从10只健康水貂远端肠道内容物样品中得到146 287高质量序列代表17个菌门、167个细菌属。水貂肠道细菌以厚壁菌门(59.99%)、拟杆菌门(16.2%)、梭杆菌门(11.5%)、放线菌门(5.9%)和变形菌门(5.3%)为主,其中厚壁菌门最为丰富。厚壁菌门中的梭菌目是最丰富的目,而梭菌目中的链球菌占有50%以上的OTUs,是最大的细菌属。【结论】水貂肠道内存在复杂的微生物区系,这对进一步研究水貂对营养物质吸收利用提供了理论基础。     

微生物类群在叶际微生物群落构建过程中的主导作用

【背景】 叶际微生物群落构建研究对于厘清其多样性的形成和维持机制具有重要意义,但研究进展缓慢。【目的】 探究园林植物叶际微生物群落的构建过程及决定叶际微生物群落构建过程的主要因素。【方法】 在大理苍山国家自然保护区下沿,选择50 m半径范围内的3种园林植物,采集同一朝向的3种不同大小叶片,基于高通量测序技术和群落构建理论模型,分别分析了整体微生物、不同植物种类、不同叶片大小和不同微生物类群的叶际微生物群落构建过程。【结果】 整体叶际微生物群落、不同植物种类和不同大小叶片上的叶际微生物群落构建过程相似,即都由确定性过程的同质选择主导,但细菌和真菌的群落构建存在差异,即叶际真菌与细菌的群落分别由随机性过程的漂变和确定性过程的同质选择主导。通过广义线性模型分析显示,植物种类对叶际微生物的群落构建过程有显著的影响,尤其是微生物类群和微生物类群与植物种类变量组合对叶际微生物群落构建过程有极显著的影响,而叶片大小对叶际微生物群落构建过程有影响但不显著。【结论】 微生物类群是叶际微生物群落构建过程中的主导因素。     

Social influences on ranging patterns among chimpanzees (Pan troglodytes verus) in the Tai National Park, Cote d'Ivoire

Group size is expected to be an important factor to predicthome-range (HR) size in social animals. In chimpanzees adultmales play an important role in defending the HR against neighbors,and therefore it has been suggested that HR size depends onthe number of adult males. In this long-term study on wild WestAfrican chimpanzees, we analyzed the relative importance ofcommunity size and composition on ranging patterns over a 10-yearperiod, using multivariate statistics. Because community sizedecreased from 51 individuals with 6 adult males in 1992 to22 individuals with only 1 adult male in 2001, we expected adecrease in HR size, which should be better predicted by thenumber of males than by community size. We further investigatedthe effect of fruit availability on monthly HRs over a 4-yearperiod. As predicted, HR size decreased during the first 7 yearsof our study but increased during the last 3 years. Overall,the number of adult males was the best predictor of HR size,whereas fruit availability did not correlate with HR size. HRuse remained stable over the entire study period, with a constantproportion of about 35% of the HR used as core area. High HRand core-area overlap values between different years indicatedstrong site-fidelity. Although the number of males within thecommunity explained the decrease in HR size, the recent increasein size remains unexplained. This finding suggests that otherfactors such as relative fighting power of males affect HR size.     

中亚热带地区氮沉降对杉木幼林土壤细菌群落多样性及组成的影响

大气氮沉降量持续增加已经成为当前关注的热点.土壤细菌群落作为土壤环境中大量存在的微生物,在养分循环过程中发挥着不可忽视的作用.在福建三明森林生态系统与全球变化研究站陈大观测点,我们在野外模拟大气氮沉降试验,通过16S rDNA扩增子测序,研究中亚热带地区杉木幼林土壤细菌群落多样性和组成对氮沉降的响应.结果表明:短期施氮对研究区的土壤细菌群落多样性和组成并未产生显著影响,但高氮处理显著改变敏感菌群相对丰度,如富营养型类群丰度增加、贫营养型类群丰度降低.土壤细菌群落的营养策略发生变化,这可能是受到了养分有效性的驱动.因此,了解土壤细菌群落和养分分配格局对氮沉降的响应,有助于提高我们对未来环境的预测能力.     

Inter-sexual differences in the immune response of Eurasian kestrel nestlings under food shortage

When resources are limited, parents should decide the optimal number, size, and sex of progeny, and offspring should decide the optimal allocation of resources to different costly functions, such as growth and immunity. We manipulated clutch sizes of Eurasian kestrels by one egg to estimate possible cumulative effects of incubation and chick rearing costs on parental body condition, feeding effort, and offspring viability. No obvious effects of clutch size manipulations on feeding effort were found while feeding effort was adjusted to the original clutch size. Enlarged and control nests suffered from higher nestling mortality than reduced nests, and chicks of the enlarged group were in poorer body condition than chicks of the reduced group. Controlling for body mass, male chicks exhibited lower cell-mediated immunity assessed by a cutaneous hypersensitivity response than females, but only in treatments suffering from food restrictions, as indicated by chick starvation. These novel results reveal inter-sexual differences in physiological strategies in early life, suggesting sex-related differences in susceptibility to disease and consequently in survival prospects of offspring.     

High-throughput microsatellite isolation through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries

Microsatellites (or SSRs: simple sequence repeats) are among the most frequently used DNA markers in many areas of research. The use of microsatellite markers is limited by the difficulties involved in their de novo isolation from species for which no genomic resources are available. We describe here a high-throughput method for isolating microsatellite markers based on coupling multiplex microsatellite enrichment and next-generation sequencing on 454 GS-FLX Titanium platforms. The procedure was calibrated on a model species (Apis mellifera) and validated on 13 other species from various taxonomic groups (animals, plants and fungi), including taxa for which severe difficulties were previously encountered using traditional methods. We obtained from 11,497 to 34,483 sequences depending on the species and the number of detected microsatellite loci ranged from 199 to 5791. We thus demonstrated that this procedure can be readily and successfully applied to a large variety of taxonomic groups, at much lower cost than would have been possible with traditional protocols. This method is expected to speed up the acquisition of high-quality genetic markers for nonmodel organisms.