SSCP analysis of Mhc class IIB genes in the threespine stickleback

Due to its universality, speed, sensitivity, precision and reproducibility, PCR followed by fluorescence SSCP analysis represents an attractive tool for the characterization of Mhc class II B genotypes and the estimation of DNA sequence variability of Mhc genes in natural stickleback Gasterosteus aculeatus populations.     

Microbial diversity: the importance of exploration and conservation

Microbial diversity is fundamental to maintenance and conservation of global genetic resources. As extreme environments are explored, the richness of microbial diversity is increasingly evident. Measures must be taken to estimate, record, and conserve microbial diversity, not only to sustain human health but also to enrich the human condition globally through wise use and conservation of genetic resources of the microbial world. Received 24 April 1996/ Accepted in revised form 07 January 1997     

Genetic structure and diversity of the Diachasmimorpha longicaudata species complex in Thailand: SSCP analysis of mitochondrial 16S rDNA and COI DNA sequences

Genetic variation among 20 populations of Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae) in Thailand was investigated using single strand conformation polymorphism (SSCP) analysis of mitochondrial DNA sequences. From a total 641 individual parasitoids, seven distinct haplotypes containing a total of 32 polymorphic sites were observed from cytochrome oxidase subunit I (COI) sequences along with five distinct haplotypes containing a total 16 polymorphic sites from 16S rDNA sequences. Values obtained through pairwise F_ST comparisons and analysis of molecular variance (AMOVA) indicated significant genetic differentiation among D. longicaudata populations in Thailand. Congruent relationships showing separation of these populations into three groups were obtained from Neighbor joining and Bayesian phylogenetic tree analyses along with the use of haplotype networks. This SSCP analysis of populations of the D. longicaudata species complex is the first report using molecular population genetic methods to analyze the structure of this parasitoid species in Thailand. This may provide useful information for release of parasitoid strains to maximize their benefit in biological control programs.     

皂荚种质资源SCoT遗传多样性分析及指纹图谱的构建

采用SCoT标记分析了18个皂荚种质的遗传多样性,并采用UPGMA法对18个皂荚种质进行了聚类分析。在此基础上,通过筛选出的多态性条带构建了18个皂荚种质的SCoT指纹图谱。扩增结果表明:从51个SCoT引物中筛选了15个引物进行PCR扩增,共扩增出226条带,其中多态性条带216条,多态性比率为96.61%。各引物多态性信息含量(PIC)、观测等位基因数(Na)、有效等位基因数(Ne)、Nei’s基因多样性指数(H)和Shannon’s信息指数(I)的平均值分别为0.875 9、1.964 9、1.440 1、0.272 6、0.426 1。18个皂荚种质的遗传相似系数在0.491 4~0.938 1之间,表明供试材料之间具有较丰富的遗传多样性。聚类分析结果表明:在遗传相似系数为0.60处可将18个皂荚种质分为3组,其中野皂荚单独为一组,山皂荚和皂荚-T聚为一组,其它皂荚材料聚为一组。利用3个引物扩增的8个多态性位点构建了18份皂荚种质资源的DNA指纹图谱,可以将其区分并精准鉴定。该研究结果为皂荚种质的鉴定和新品种选育提供了一定的理论依据。     

On the risks of using dendrograms to measure functional diversity and multidimensional spaces to measure phylogenetic diversity: a comment on Sobral et al. (2016)

Sobral et al. (Ecology Letters, 19, 2016, 1091) reported that the loss of bird functional and phylogenetic diversity due to species extinctions was not compensated by exotic species introductions. Here, we demonstrate that the reported changes in biodiversity were underestimated because of methodological pitfalls.     

利用SSR标记与毛细管电泳对甘蔗属进行的遗传分析

为探讨甘蔗属内不同种之间的遗传多样性,利用SSR标记与毛细管电泳技术,对来自甘蔗属3个不同种的12个材料19对引物进行检测,共检测到229个DNA多态性条带,19对引物扩增的DNA条带范围集中在100～260bp之间。12个甘蔗材料的Jaccard遗传相似度,最小0.09,最大0.65,平均为0.26。通过遗传相似性系数分析,UPGMA聚类图内12个甘蔗材料可分为两个群,三个割手密种材料分为一个亚群,甘蔗栽培品种与甘蔗热带种合为一个亚群。结果表明:热带种比割手密种具有和甘蔗栽培品种更亲近的遗传关系;SSR分子标记与毛细管技术结合,相比别的分子标记技术或电泳技术,具有更准确、简便、自动化等优点。     

PCR扩增循环数对细菌群落多样性测序分析的影响

运用高通量测序技术分析复杂样品中微生物群落组成及变化趋势,已经成为目前微生物研究领域的热点之一。本研究以复杂土壤样品和应用范围较广的瘤胃食糜样品为对象,选取20、25和30三个扩增循环数分别对样品的16S r RNA基因的V3区进行扩增,然后进行文库构建和测序。最后通过数据分析比较不同的扩增循环数对细菌多样性测定结果的影响。结果表明,扩增循环数越多,捕获到的细菌数量和种类越多;但并非循环数越多,群落中的微生物组成比例最优。整体来看,当扩增循环数为25时,样品中物种的数量和组成是最优的。     

Discriminating glacial races of burbot (Lota lota) by means of PCR‐RF‐SSCP (PRS) analysis of the mitochondrial control region

We used closely related haplotypes of the mitochondrial control region of burbot (Lota lota) (Teleostei; Gadidae) with known sequences to assess the sensitivity of a standardized PRS (PCR‐RF‐SSCP) approach. In total, 537 pairwise comparisons between PRS patterns were evaluated. Only seven mutations did not result in a detectable change that resulted in 11 pairwise comparisons (2%) with indistinguishable haplotypes. The haplotypes that could not be discriminated were always characteristic for a certain assemblage (glacial race). As such, this method provides a reliable alternative to direct sequencing for phylogeographic studies in case intrapopulation diversity is limited.     

Assessing microbial diversity using recent lake sediments and estimations of spatio‐temporal diversity

Aim Recent papers have used large palaeolimnological datasets to reveal the biodiversity patterns of aquatic microorganisms. However, scant attention has been paid to the influence of time on these patterns. Where lake surficial sediment samples are used as integrals of diversity, the time interval of each sample varies according to differences in sediment accumulation rates. This paper aims to test the reliability of using lake surface sediments to measure and to compare microbial diversity when the potential influences of the species–time relationships are taken into account. Location Alpine lakes in Europe. Methods We analysed microorganism (siliceous microalgae) assemblages in three European Alpine lakes using short sediment cores (²¹⁰Pb‐dated) and annual sediment trap samples from 12 UK lakes. The same number of individuals was pooled for each sample 500 times to avoid sampling effort effects and to standardize species diversity estimation. The influence of time on the diversity score was assessed by simulating an increase of time span for surface sediment samples by cumulatively adding in successive sediment core samples (from the most recent to the oldest). We used species richness (S) and the exponential of the bias‐corrected Shannon entropy index (exp(H_b‐c)) to estimate diversity. Results Increasing the time interval represented by a surficial sediment sample did not affect the diversity results. The estimation of diversity was similar for cumulative and non‐cumulative samples. Diversity estimation was only altered in lakes experiencing high community turnover due to strong environmental forcing during the time period spanned by the cumulative sample. Main conclusions The use of surface lake sediments is suitable for estimating the average site diversity of free‐living microorganisms. Diversity is integrated in a single sample and species assemblage composition is derived from microbial communities living in distinct lake microhabitats. Species remains, accumulated in a single sample over several years of environmental variability, represent a diversity integral that captures a spatio‐temporal component equivalent to the γ‐diversity measure.     

Metabolomic analysis using optimized NMR and statistical methods

NMR-based metabolomics requires robust automated methodologies, and the accuracy of NMR-based metabolomics data is greatly influenced by the reproducibility of data acquisition and processing methods. Effective water resonance signal suppression and reproducible spectral phasing and baseline traces across series of related samples are crucial for statistical analysis. We assess robustness, repeatability, sensitivity, selectivity, and practicality of commonly used solvent peak suppression methods in the NMR analysis of biofluids with respect to the automated processing of the NMR spectra and the impact of pulse sequence and data processing methods on the sensitivity of pattern recognition and statistical analysis of the metabolite profiles. We introduce two modifications to the excitation sculpting pulse sequence whereby the excitation solvent suppression pulse cascade is preceded by low-power water resonance presaturation pulses during the relaxation delay. Our analysis indicates that combining water presaturation with excitation sculpting water suppression delivers the most reproducible and information-rich NMR spectra of biofluids.     

基于SSR分子标记的地被菊遗传多样性分析和指纹图谱构建

为了从分子水平上研究地被菊(Chrysanthemum×morifolium)种质资源的遗传多样性并建立地被菊品种的指纹图谱库,筛选出多态性高的引物用于地被菊品种间鉴定、亲缘关系分析和分子标记辅助选种体系的建立,本研究利用多态性好、条带清晰、重复性好的12对引物对91份地被菊材料和14份菊属近缘种材料进行简单重复序列(simple sequence repeat,SSR)分子标记和遗传多样性分析,从12对引物中筛选出9对核心引物对受试材料进行指纹图谱构建。结果显示,在105个样品中,12对引物检测出104个等位位点,范围为2−26,平均每个点位检测出9.25个等位基因数,平均每个点位检测得到的有效等位基因数(number of effective alleles,Ne)为2.7456,范围为1.2760−4.7425;Shannon信息指数(Shannon genetic diversity index,I)变化范围是0.5133−2.2399,均值是1.2090;Nei’s基因多样性指数(Nei’s gene diversity index,H)范围是0.2163−0.7891,均值是0.5780;观测杂合度(observed heterozygosity,Ho)的范围是0.2233−0.8952,均值是0.5575;期望杂合度(expected heterozygosity,He)的范围是0.2174−0.7933,均值是0.5808;多态信息含量(polymorphism information content,PIC)值变化范围是0.2115−0.7740,均值是0.5329;遗传相似性系数(genetic similarity,GS)范围为0.2285−1.0000,均值是0.6083。聚类分析表明,在遗传距离(genetic distance,GD)=0.30时,受试材料可以分为两个类群。Structure群体结构分析将受试材料分为3个种群和1个混合种群。从12对引物中筛选出可完全区分105份受试材料的9对核心引物,构建了91份地被菊材料和14份菊属近缘种材料的指纹图谱。地被菊材料之间具有显著的遗传差异和丰富的遗传多样性,对于地被菊的园林应用和品种选育具有重要意义。地被菊品种和菊属近缘种的指纹图谱库的构建,一定程度上揭示了105份实验材料的亲缘关系,为今后地被菊品种鉴定与筛选体系的研究提供了技术支撑。     

生物群落多样性的测度方法VI：与多样性测度有关的统计问题

物种多样性测度是（群落的）总体参数,它们常常是未知的,需要通过抽样将它们估计出来,因此,必须了解估计量的抽样性质。本文对与一些多样性测度的均值、方差的估计和假设检验以及大、小样本分布等有关的问题作了综述。可以看出大多数多样性测度的抽样性质还不清楚,有些甚至根本就没有研究过。Ｐｉｅｌｏｕ的合并样方法和刀切法是两个比较通用的方法,用它们可以解决其中的一些问题。但相比之下,刀切法更实用。     

Applicability of SSCP analysis for MHC genotyping: fingerprinting of Ovar-DRB1 exon 2 alleles from Finnish and Russian breeds

The applicability of single strand conformation polymorphism (SSCP) analysis for major histocompatibility complex (MHC) genotyping in sheep was studied. A panel of Ovar-DRB1 exon 2 'allele fingerprints' was defined. The panel could accelerate DRB1 genotyping of new breeds when already existing sequences are used as references in SSCP analysis. In this study, seven new exon 2 sequences and 19 different alleles in total were detected from 31 animals of Finnish and Russian sheep breeds. Ovar-DRB1 * 0201 was detected in all the six grey Finnsheep animals included in this study, suggesting reduced MHC diversity within these animals.     

Analysis of bacterial and archaeal diversity in coastal microbial mats using massive parallel 16S rRNA gene tag sequencing

Coastal microbial mats are small-scale and largely closed ecosystems in which a plethora of different functional groups of microorganisms are responsible for the biogeochemical cycling of the elements. Coastal microbial mats play an important role in coastal protection and morphodynamics through stabilization of the sediments and by initiating the development of salt-marshes. Little is known about the bacterial and especially archaeal diversity and how it contributes to the ecological functioning of coastal microbial mats. Here, we analyzed three different types of coastal microbial mats that are located along a tidal gradient and can be characterized as marine (ST2), brackish (ST3) and freshwater (ST3) systems. The mats were sampled during three different seasons and subjected to massive parallel tag sequencing of the V6 region of the 16S rRNA genes of Bacteria and Archaea. Sequence analysis revealed that the mats are among the most diverse marine ecosystems studied so far and consist of several novel taxonomic levels ranging from classes to species. The diversity between the different mat types was far more pronounced than the changes between the different seasons at one location. The archaeal community for these mats have not been studied before and revealed a strong reaction on a short period of draught during summer resulting in a massive increase in halobacterial sequences, whereas the bacterial community was barely affected. We concluded that the community composition and the microbial diversity were intrinsic of the mat type and depend on the location along the tidal gradient indicating a relation with salinity.     

Estimating T-cell repertoire diversity: limitations of classical estimators and a new approach

A highly diverse T-cell receptor (TCR) repertoire is a fundamental property of an effective immune system, and is associated with efficient control of viral infections and other pathogens. However, direct measurement of total TCR diversity is impossible. The diversity is high and the frequency distribution of individual TCRs is heavily skewed; the diversity therefore cannot be captured in a blood sample. Consequently, estimators of the total number of TCR clonotypes that are present in the individual, in addition to those observed, are essential. This is analogous to the ‘unseen species problem’ in ecology. We review the diversity (species richness) estimators that have been applied to T-cell repertoires and the methods used to validate these estimators. We show that existing approaches have significant shortcomings, and frequently underestimate true TCR diversity. We highlight our recently developed estimator, DivE, which can accurately estimate diversity across a range of immunological and biological systems.     

ribosort: a program for automated data preparation and exploratory analysis of microbial community fingerprints

ribosort is a computer package for convenient editing of automated ribosomal intergenic spacer analysis (ARISA) and terminal restriction fragment length polymorphism (TRFLP) data. It is designed to eliminate the labourious task of manually classifying community fingerprints in microbial ecology studies. This program automatically assigns detected fragments and their respective relative abundances to appropriate ribotypes. It permits simultaneous sorting of multiple profiles and facilitates direct workflow from TRFLP and ARISA output through to community analyses. ribosort also provides several options to merge repeat profiles of a sample into a single composite profile. By creating a 'ribotypes by samples' matrix ready for statistical analyses, use of the package saves time and simplifies the preparation of DNA fingerprint data sets for statistical analysis. In addition, ribosort performs exploratory analysis on the data by creating multidimensional scaling plots that compare the similarity of sample profiles using the statistical software r.