Microbial community structure of Arctic multiyear sea ice and surface seawater by 454 sequencing of the 16S RNA gene

Dramatic decreases in the extent of Arctic multiyear ice (MYI) suggest this environment may disappear as early as 2100, replaced by ecologically different first-year ice. To better understand the implications of this loss on microbial biodiversity, we undertook a detailed census of the microbial community in MYI at two sites near the geographic North Pole using parallel tag sequencing of the 16S rRNA gene. Although the composition of the MYI microbial community has been characterized by previous studies, microbial community structure has not been. Although richness was lower in MYI than in underlying surface water, we found diversity to be comparable using the Simpson and Shannon''s indices (for Simpson t=0.65, P=0.56; for Shannon t=0.25, P=0.84 for a Student''s t-test of mean values). Cyanobacteria, comprising 6.8% of reads obtained from MYI, were observed for the first time in Arctic sea ice. In addition, several low-abundance clades not previously reported in sea ice were present, including the phylum TM7 and the classes Spartobacteria and Opitutae. Members of Coraliomargarita, a recently described genus of the class Opitutae, were present in sufficient numbers to suggest niche occupation within MYI.     

The Chloroplast Genome Sequence of Mungbean (Vigna radiata) Determined by High-throughput Pyrosequencing: Structural Organization and Phylogenetic Relationships

Mungbean is an economically important crop which is grown principally for its protein-rich dry seeds. However, genomic research of mungbean has lagged behind other species in the Fabaceae family. Here, we reported the complete chloroplast (cp) genome sequence of mungbean obtained by the 454 pyrosequencing technology. The mungbean cp genome is 151 271 bp in length which includes a pair of inverted repeats (IRs) of 26 474 bp separated by a small single-copy region of 17 427 bp and a large single-copy region of 80 896 bp. The genome contains 108 unique genes and 19 of these genes are duplicated in the IR. Of these, 75 are predicted protein-coding genes, 4 ribosomal RNA genes and 29 tRNA genes. Relative to other plant cp genomes, we observed two distinct rearrangements: a 50-kb inversion between accD/rps16 and rbcL/trnK-UUU, and a 78-kb rearrangement between trnH/rpl14 and rps19/rps8. We detected sequence length polymorphism in the cp homopolymeric regions at the intra- and inter-specific levels in the Vigna species. Phylogenetic analysis demonstrated a close relationship between Vigna and Phaseolus in the phaseolinae subtribe and provided a strong support for a monophyletic group of the eurosid I.     

No support for occurrence of free-living Cladonia mycobionts in dead wood

Lichenised fungi are traditionally assumed to form obligate symbioses with algae or cyanobacteria and to be confined to the surface of their growing substratum. However, in a recent 454 pyrosequencing study of fungal communities in Picea abies logs, lichen-forming fungi were detected at a depth of more than 6 cm in dead wood, implying the existence of free-living lichen mycobionts. To determine whether this was the case, we investigated whether Cladonia spp., the most frequently encountered mycobionts, occurred in wood without their photobionts. We detected green algae in all samples with records of Cladonia spp. Hence, we found no evidence for free-living Cladonia mycobionts in wood. We suggest that the detected Cladonia DNA in these logs originates from vegetative propagules or thallus fragments dispersed into the logs by animals or water. However, the occurrence of free-living stages of other lichen-forming fungal taxa in dead wood cannot be excluded.     

小檗碱对大鼠肠道菌群结构的体外影响

目的研究小檗碱在体外对高脂饮食诱导的肥胖、胰岛素抵抗大鼠(HFD)肠道菌群和正常饮食对照大鼠(NCD)肠道菌群结构的体外影响。方法采用体外厌氧培养、PCR-DGGE和454焦磷酸测序技术研究小檗碱对肠道菌群结构和多样性的影响。结果 DGGE指纹图谱和454焦磷酸测序结果都表明,小檗碱可以改变肠道菌群的结构,高剂量的小檗碱可以减少肠道微生物的多样性。应用偏最小二乘法判别模型分析(PLS-DA)挑选与小檗碱相关的细菌类群(OTU),在HFD组中挑选了55个关键OTUs,其中53个被明显抑制或消除,剩余2个分别属于Proteus和Escherichia/Shigella属的OTUs则被小檗碱富集。在NCD组中挑选的51个关键OTUs中,32个被小檗碱抑制,17个被小檗碱富集。被富集的除了属于Klebsielal属的OTU外,还包括可以产生短链脂肪酸的Lactobacillus、Blautia属的OTUs。结论小檗碱可以直接调节肠道菌群的结构,对不同结构的肠道菌群其作用也不相同,不同浓度的小檗碱对肠道菌群的影响有较大差异。高浓度的小檗碱可以抑制大部分细菌的生长(其中有很多为肠道条件致病菌),减少肠道微生物的多样性,富集Enterobacteriaceae科的细菌(Proteus、Escherichia/Shigella、Klebsielal)。相较于HFD组,小檗碱可以显著富集NCD组大鼠肠道菌群中的短链脂肪酸产生菌。     

Molecular diversity and distribution of marine fungi across 130 European environmental samples

Environmental DNA and culture-based analyses have suggested that fungi are present in low diversity and in low abundance in many marine environments, especially in the upper water column. Here, we use a dual approach involving high-throughput diversity tag sequencing from both DNA and RNA templates and fluorescent cell counts to evaluate the diversity and relative abundance of fungi across marine samples taken from six European near-shore sites. We removed very rare fungal operational taxonomic units (OTUs) selecting only OTUs recovered from multiple samples for a detailed analysis. This approach identified a set of 71 fungal ‘OTU clusters'' that account for 66% of all the sequences assigned to the Fungi. Phylogenetic analyses demonstrated that this diversity includes a significant number of chytrid-like lineages that had not been previously described, indicating that the marine environment encompasses a number of zoosporic fungi that are new to taxonomic inventories. Using the sequence datasets, we identified cases where fungal OTUs were sampled across multiple geographical sites and between different sampling depths. This was especially clear in one relatively abundant and diverse phylogroup tentatively named Novel Chytrid-Like-Clade 1 (NCLC1). For comparison, a subset of the water column samples was also investigated using fluorescent microscopy to examine the abundance of eukaryotes with chitin cell walls. Comparisons of relative abundance of RNA-derived fungal tag sequences and chitin cell-wall counts demonstrate that fungi constitute a low fraction of the eukaryotic community in these water column samples. Taken together, these results demonstrate the phylogenetic position and environmental distribution of 71 lineages, improving our understanding of the diversity and abundance of fungi in marine environments.     

Strong differences in Quercus robur-associated ectomycorrhizal fungal communities along a forest-city soil sealing gradient

Ectomycorrhizal fungi (EM) that associate with tree roots in a symbiotic relationship may be crucial in mediating tree health in urban environments, but research on the effects of urbanization on EM communities is very limited so far. Here, we compared EM communities of adult pedunculate oaks (Quercus robur) between urban and forest environments, and assessed the effect of soil sealing around the trees on their EM community composition and EM diversity. We sampled 32 oak individuals across 4 sampling classes (Street, Lane, Park and Forest), and we characterized their EM communities using 454 amplicon pyrosequencing. The EM communities were not nested but they were significantly different between all sampling classes, with a very strong community differentiation between forest and urban trees. There were indications that EM richness declined with increasing sealed soil surface, with a significant effect of sampling class on estimated EM richness and diversity. We also identified indicator EM of the different sampling classes. The most important soil factor affecting EM community composition was pH, followed by plant available phosphorus, total nitrogen content and organic matter. Our results may have important implications when developing EM inocula for managing tree health in urban environments.     

Particle-size distributions and seasonal diversity of allergenic and pathogenic fungi in outdoor air

Fungi are ubiquitous in outdoor air, and their concentration, aerodynamic diameters and taxonomic composition have potentially important implications for human health. Although exposure to fungal allergens is considered a strong risk factor for asthma prevalence and severity, limitations in tracking fungal diversity in air have thus far prevented a clear understanding of their human pathogenic properties. This study used a cascade impactor for sampling, and quantitative real-time PCR plus 454 pyrosequencing for analysis to investigate seasonal, size-resolved fungal communities in outdoor air in an urban setting in the northeastern United States. From the 20 libraries produced with an average of ∼800 internal transcribed spacer (ITS) sequences (total 15 326 reads), 12 864 and 11 280 sequences were determined to the genus and species levels, respectively, and 558 different genera and 1172 different species were identified, including allergens and infectious pathogens. These analyses revealed strong relationships between fungal aerodynamic diameters and features of taxonomic compositions. The relative abundance of airborne allergenic fungi ranged from 2.8% to 10.7% of total airborne fungal taxa, peaked in the fall, and increased with increasing aerodynamic diameter. Fungi that can cause invasive fungal infections peaked in the spring, comprised 0.1–1.6% of fungal taxa and typically increased in relative abundance with decreasing aerodynamic diameter. Atmospheric fungal ecology is a strong function of aerodynamic diameter, whereby through physical processes, the size influences the diversity of airborne fungi that deposit in human airways and the efficiencies with which specific groups of fungi partition from outdoor air to indoor environments.     

利用双重PCR技术研究长爪沙鼠的微卫星结构

目的为了建立快速检测长爪沙鼠群体遗传多样性的方法及获得Z：ZCLA长爪沙鼠封闭群现用微卫星位点的结构。方法利用17个微卫星位点（9个来自长爪沙鼠,8个来自大小鼠）进行了PCR反应体系及反应条件的优化,组合了6组双重PCR及两个复合式点样,用上述8个组合对普通级Z：ZCLA长爪沙鼠封闭群43、444、5三个世代核心群各100只种鼠进行遗传检测。结果三个世代的300只种鼠的检测结果表明,9个长爪沙鼠位点均为微卫星,其中7个位点为完全型的微卫星,1个为复合型,1个为不完全型,多态性主要表现在核心序列的重复;来自大小鼠的8个微卫星位点,有7个在Z：ZCLA长爪沙鼠核心群中得到有效扩增,只有3个位点在三个世代中均有出现,对测序结果分析后发现,其核心序列均为小卫星。结论来自长爪沙鼠的位点,无论结构还是遗传方式均符合微卫星遗传标记的特点,可用作检测长爪沙鼠的群体遗传多样性。     

Application of two microsatellite sequences in wheat storage proteins as molecular markers

In eukaryotes, tandem arrays of simple-sequence repeat sequences can find applications as highly variable and multi-allelic PCR-based genetic markers. In hexaploid bread wheat, a large-genome inbreeding species with low levels of RFLP, di- and trinucleotide tandem repeats were found in 22 published gene sequences, two of which were converted to PCR-based markers. These were shown to be genome-specific and displayed high levels of variation. These characteristics make them especially suitable for intervarietal breeding applications.     

EST–SSR markers for asparagus genetic diversity evaluation and cultivar identification

Simple sequence repeat (SSR) markers generated from expressed sequence tag (EST) sequences represent useful tools for genotyping and their development is relatively easy because of the public availability of EST databases. We report design and application of EST–SSRs to assess the level of genetic diversity among thirty-five asparagus cultivars and to fingerprint DePaoli, a new variety released by University of California, Riverside. DNA was isolated from bulks of pooled cladophylls coming from five plants of each variety to reduce the number of DNA extractions and PCR reactions. Allele frequencies were estimated from the intensity of the bands in two bulks and two individual plant samples for each variety. Although asparagus varieties derive from a limited germplasm pool, eight EST–SSR loci differentiated all of the analyzed cultivars. Moreover, UPGMA (unweighted pair group method with arithmetic mean) and neighbor-joining trees, as well as principal components analysis separated the cultivars into clusters corresponding to the geographical areas where they originated.     

Cyanobacteria inhabiting biological soil crusts of a polar desert: Sør Rondane Mountains,Antarctica

Molecular and morphological methods were applied to study cyanobacterial community composition in biological soil crusts (BSCs) from four areas (two nunataks and two ridges) in the Sør Rondane Mountains, Antarctica. The sampling sites serve as control areas for open top chambers (OTCs) that were put in place in 2010 at the time of sample collection and will be compared with BSC samples taken from the OTCs in the future. Cyanobacterial cell biovolume was estimated using epifluorescence microscopy, which revealed the dominance of filamentous cyanobacteria in all studied sites except the Utsteinen ridge, where unicellular cyanobacteria were the most abundant. Cyanobacterial diversity was studied by a combination of molecular fingerprinting methods based on the 16S rRNA gene (denaturing gradient gel electrophoresis (DGGE) and 454 pyrosequencing) using cyanobacteria-specific primers. The number of DGGE sequences obtained per site was variable and, therefore, a high-throughput method was subsequently employed to improve the diversity coverage. Consistent with previous surveys in Antarctica, both methods showed that filamentous cyanobacteria, such as Leptolyngbya sp., Phormidium sp. and Microcoleus sp., were dominant in the studied sites.In addition, the studied localities differed in substrate type, climatic conditions and soil parameters, which probably resulted in differences in cyanobacterial community composition. Furthermore, the BSC growing on gneiss pebbles had lower cyanobacterial abundances than BSCs associated with granitic substrates.