Microfungi from decaying leaves of two rain forest trees in Puerto Rico

Fungal species richness and abundance were compared in leaf litter of two tree species,Guarea guidonia andManilkara bidentata, in the Luquillo Mountains of Puerto Rico. Four litter samples yielded a total of 3337 isolates, ranging from 591 to 1259 isolates/sample. The number of species/sample ranged from 134 to 228. Many uncommon litter hyphomycetes were recovered as well as coelomycetes, sterile strains, endophytes, and phytopathogens. Species-abundance distributions revealed a typical pattern of a few abundant species and a high proportion of rare species. Similarities in fungal species composition were not correlated with host species or with the site. Replicate samples examined by the moist chamber technique yielded a total of 24 species among the four litter samples. The particle filtration method indicated that leaves ofG. guidonia were more species-rich, while moist chambers indicated leaves ofM. bidentata were more species-rich. The moist chamber technique underestimated the number and species of viable fungi.     

A rapid method for tissue collection and high-throughput isolation of genomic DNA from mature trees

Collection of tissue and subsequent isolation of genomic DNA from mature tree species often proves difficult. DNA extraction from needles, leaves, or buds is recommended in many protocols. Collecting these tissues from mature trees generally requires the use of firearms or climbing if sampling is to be nondestructive. As a result, sample collection is a major expense of many tree-based projects. Tree (and plant) tissues generally contain large amounts of polysaccharides and phenolic compounds that are difficult to separate from DNA. Many methods aim to overcom these problems, with most involving extraction in buffers containing the nonionic detergent cetyltrimethyl-ammonium bromide (CTAB), followed by numerous steps to clean contaminants from the DNA, using organic solvents and differential salt precipitation. These steps are time-consuming, such that isolation of DNA becomes the bottleneck in many molecular studies. This paper presents a new, efficient, cambium collection method for tree species and a DNA extraction protocol based on that of Doyle and Doyle (1987), with follow-up purification using the Wizard nuclei lysis and protein precipitation solutions (Promega). Results show a significant improvement in yield and DNA purity compared with other published methods, with consistently high yields of pure genomic DNA and high sample throughput. The relatively low cost per extraction, no requirement for use of liquid nitrogen, no requirement for freezer storage, and long-term sample stability after collection are important additional benefits.     

Additive biomass equations for native eucalypt forest trees of temperate Australia

Biomass additivity is a desirable characteristic of a system of equations for predicting component as well as total tree biomass since it eliminates the inconsistency between the sum of predicted values for components such as stem, bark, branch and leaf and the prediction for the total tree. Besides logical consistency, a system of additive biomass equations when estimated by taking into account the inherent correlation among the biomass components has greater statistical efficiency than separately estimated equations for individual components. Using mostly small sample data from both published and unpublished sources, a system of non-linear additive biomass equations was developed for 15 native eucalypt forest tree species of temperate Australia. Diameter at breast height was used as the independent variable for all 15 species, while the combined variable of diameter and tree height was used for 14 species with height data. The system of additive equations provided more accurate biomass estimates than the common approach of separately fitting total tree and component biomass equations using log transformed data through least squares regression. Residual error variances were collectively estimated for each species by pooling small sample data across species and using indicator variables to represent the scale factor for each species in a residual variance function. This method overcame a common problem in estimating heteroscedastic error variance in non-linear biomass equations with additive error terms for small samples. From the estimated residual variance functions, approximate confidence bands containing about 95% of the observed data about the mean curve of predicted biomass were derived for all biomass components of each species. This system of additive biomass equations will prove to be useful for biomass estimation of native eucalypt forests of temperate Australia.     

Method for automated extraction and purification of nucleic acids and its implementation in microfluidic system

A method and a microfluidic device for automated extraction and purification of nucleic acids from biological samples have been developed. The method involves disruption of bacterial cells and/or viral particles by combining enzymatic and chemical lysis procedures followed by solid-phase sorbent extraction and purification of nucleic acids. The procedure is carried out in an automated mode in a microfluidic module isolated from the outside environment, which minimizes contact of the researcher with potentially infectious samples and, consequently, decreases the risk of laboratory-acquired infections. The module includes reservoirs with lyophilized components for lysis and washing buffers; a microcolumn with a solid-phase sorbent; reservoirs containing water, ethanol, and water-ethanol buffer solutions for dissolving freeze-dried buffer components, rinsing the microcolumn, and eluting of nucleic acids; and microchannels and valves needed for directing fluids inside the module. The microfluidic module is placed into the control unit that delivers pressure, heats, mixes reagents, and flows solutions within the microfluidic module. The microfluidic system performs extraction and purification of nucleic acids with high efficiency in 40 min, and nucleic acids extracted can be directly used in PCR reaction and microarray assays.     

The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when?

Museums and pathology collections around the world represent an archive of genetic material to study populations and diseases. For preservation purposes, a large portion of these collections has been fixed in formalin-containing solutions, a treatment that results in cross-linking of biomolecules. Cross-linking not only complicates isolation of nucleic acid but also introduces polymerase "blocks" during PCR. A wide variety of methods exists for the recovery of DNA and RNA from archival tissues, and although a number of previous studies have qualitatively compared the relative merits of the different techniques, very few have undertaken wide scale quantitative comparisons. To help address this issue, we have undertaken a study that investigates the quality of nucleic acids recovered from a test panel of fixed specimens that have been manipulated following a number of the published protocols. These include methods of pre-treating the samples prior to extraction, extraction and nucleic acid purification methods themselves, and a post-extraction enzymatic repair technique. We find that although many of the published methods have distinct positive effects on some characteristics of the nucleic acids, the benefits often come at a cost. In addition, a number of the previously published techniques appear to have no effect at all. Our findings recommend that the extraction methodology adopted should be chosen carefully. Here we provide a quick reference table that can be used to determine appropriate protocols for particular aims.     

A Duplex PCR-Based Assay for Measuring the Amount of Bacterial Contamination in a Nucleic Acid Extract from a Culture of Free-Living Protists

Background

Cultures of heterotrophic protists often require co-culturing with bacteria to act as a source of nutrition. Such cultures will contain varying levels of intrinsic bacterial contamination that can interfere with molecular research and cause problems with the collection of sufficient material for sequencing. Measuring the levels of bacterial contamination for the purposes of molecular biology research is non-trivial, and can be complicated by the presence of a diverse bacterial flora, or by differences in the relative nucleic acid yield per bacterial or eukaryotic cell.

Principal Findings

Here we describe a duplex PCR-based assay that can be used to measure the levels of contamination from marine bacteria in a culture of loricate choanoflagellates. By comparison to a standard culture of known target sequence content, the assay can be used to quantify the relative proportions of bacterial and choanoflagellate material in DNA or RNA samples extracted from a culture. We apply the assay to compare methods of purifying choanoflagellate cultures prior to DNA extraction, to determine their effectiveness in reducing bacterial contamination. Together with measurements of the total nucleic acid concentration, the assay can then be used as the basis for determining the absolute amounts of choanoflagellate DNA or RNA present in a sample.

Conclusions

The assay protocol we describe here is a simple and relatively inexpensive method of measuring contamination levels in nucleic acid samples. This provides a new way to establish quantification and purification protocols for molecular biology and genomics in novel heterotrophic protist species. Guidelines are provided to develop a similar protocol for use with any protistan culture. This assay method is recommended where qPCR equipment is unavailable, where qPCR is not viable because of the nature of the bacterial contamination or starting material, or where prior sequence information is insufficient to develop qPCR protocols.     

Evaluation of the Cepheid GeneXpert system for detecting Bacillus anthracis

AIMS: The Cepheid GeneXpert is a four-site, automated sample preparation and real-time PCR detection system. In this study, the capability of the GeneXpert to isolate and detect nucleic acid from Bacillus anthracis Ames spores was assessed. METHODS AND RESULTS: A four-plex, dried-down bead cartridge containing PCR reagents specific for the pXO1 and pXO2 plasmids as well as sample processing and inhibition controls was evaluated. For B. anthracis Ames spores harbouring pXO1 and pXO2, samples containing 68 CFU per ml (148 spores per ml) were positive in all four replicates. A limited cross-reactivity panel, which included closely related Bacillus species, was also tested to determine the specificity of the pXO1 and pXO2 assays. No cross-reactivity occurred. Further, B. anthracis Sterne spore samples were analysed to compare results when processed using the GeneXpert to those run directly on the Cepheid SmartCycler without sample processing. The GeneXpert detection capability was three logs lower than the SmartCycler indicating the benefit of incorporating a nucleic acid extraction procedure. CONCLUSIONS: This study demonstrates that the GeneXpert is a rapid and reliable system for simultaneously detecting the B. anthracis virulence plasmids pXO1 and pXO2. SIGNIFICANCE AND IMPACT OF THE STUDY: The GeneXpert is the only platform currently available that is capable of both nucleic acid purification and real-time PCR detection enclosed within a single system. Further, all sample manipulations are automated, thus reducing errors associated with manual processing.     

应用于预生物合成研究的核酸碱基分析方法

应用于预生物合成研究的核酸碱基分析方法何裕建,戚生初（中国科学院生物物理研究所,北京１０Ｏ１０１）（北京大学技术物理系,北京１０Ｏ８７１）关键词ＨＰＬＣ分析;核酸碱基;离子交换色谱在生命起源研究中,核酸大分子起源是化学进化的一个重要课题,众所周知,核...     

滇中喀斯特森林土壤种子库的种-面积关系

土壤中的种子是植物群落更新与再恢复的种源基础。国内对土壤种子库的取样面积与种子检出之间的关系还很少涉及。在采用萌发方法对采集自滇中喀斯特山地的100个10 cm×10 cm小样方的物种数进行研究后,分析了土壤种子库的种-面积关系。面积与总物种和乔、灌、草本物种数之间的关系与二次方程和对数方程之间的拟合关系都较好。总物种数和草本物种数的基数较大且随面积增长较明显,而乔木和灌木的基数相对较小,增长较慢。总物种数的增加在面积位于0.15~0.2 m²,也即15~20个10 cm×10 cm样方之间时逐渐趋缓,可将这一面积和取样数量作为同类地区种子库取样的参考。     

Rapid method for the detection of genetically engineered microorganisms by polymerase chain reaction from soil and sediments

A rapid and sensitive method for the detection of genetically engineered microorganisms in soil and sediments has been devised by in vitro amplification of the target DNAs by a polymerase chain reaction. A cloned catechol 2,3-dioxygenase gene located on the recombinant plasmid pOH101 was transferred to Pseudomonas putida MMB2442 by triparental crossing and used as a target organism. For the polymerase chain reaction from soil and sediment samples, the template DNA was released from a 100-mg soil sample. Bacterial seeded soil samples were washed with Tris-EDTA buffer (pH 8.0) and treated with a detergent lysis solution at 100°C. After addition of 1% polyvinylpolypyrrolidine solution, the samples were boiled for 5 min. Supernatant containing nucleic acid was purified with a PCR purification kit. The purified DNA was subjected to polymerase chain reaction, using two specific primers designed for the amplification of catechol 2,3-dioxygenase gene sequences. The detection limit was 10² cells per gram of soil. This method is rapid and obviates the need for lengthy DNA purification from soil samples. Received 28 February 1997/ Accepted in revised form 23 November 1997     

Comparative application of the coefficient of variation and range-based statistics for assessing the taxonomic composition of fossil samples

Dental variation has been used commonly to assess taxonomic composition in morphologically homogeneous fossil samples. While the coefficient of variation (CV) has been used traditionally, range-based measures of variation, such as the range as a percentage of the mean (R%) and the maximum/minimum index (I_max/min) have recently become popular alternatives. The current study compares the performance of these statistics when applied to single- and pooled-species dental samples of extant Cercopithecus species. A common methodology for such problems of species discrimination has been to simply compare the maximum value of a variation statistic observed in extant samples with that observed in the fossil sample. However, regardless of what statistic is used, this approach has an unknowable Type I error rate, and usually has low power to detect multiple species. A more appropriate method involves a formal hypothesis test. The null hypothesis is that the level of variation in the fossil sample does not exceed what might be expected in a sample drawn randomly from a reference population, taking into account sampling error and the size of the fossil sample. Previous research using this method with the CV has indicated that it offers considerable power at an acceptable Type I error rate. In the current study, the data of primary interest were posterior dental dimensions for single- and pooled species samples from extant Cercopithecus species. In addition, the study also investigated the relative performance of variation statistics when applied to highly dimorphic canine dimensions, since much recent work has employed sexually dimorphic dental dimensions for assessing single-species hypotheses. The results indicate that the CV consistently out-performed the range-based statistics when using posterior dental dimensions to test a single-species hypothesis. Regardless of which statistic was used, tests on sexually dimorphic dimensions offered minimal power. In consideration of these results and the problem of studywise Type I error rates, we recommend against the use of multiple measures of variation to test for multiple species composition, and advocate the CV as the statistic of choice when using the method of Cope & Lacy (1992). For similar reasons, we argue for careful selection of dental variables for inclusion in such analyses, and in particular recommend against including sexually dimorphic dimensions when testing for multiple species composition.     

Evaluation of six methods for extraction and purification of viral DNA from urine and serum samples

The sensitivity and reliability of PCR for diagnostic and research purposes require efficient unbiased procedures of extraction and purification of nucleic acids. One of the major limitations of PCR-based tests is the inhibition of the amplification process by substances present in clinical samples. This study used specimens spiked with a known amount of plasmid pBKV (ATCC 33-1) to compare six methods for extraction and purification of viral DNA from urine and serum samples based on recovery efficiency in terms of yield of DNA and percentage of plasmid pBKV recovered, purity of extracted DNA, and percentage of inhibition. The most effective extraction methods were the phenol/chloroform technique and the silica gel extraction procedure for urine and serum samples, respectively. Considering DNA purity, the silica gel extraction procedure and the phenol/chloroform method produced the most satisfactory results in urine and serum samples, respectively. The presence of inhibitors was overcome by all DNA extraction techniques in urine samples, as evidenced by semiquantitative PCR amplification. In serum samples, the lysis method and the proteinase K procedure did not completely overcome the presence of inhibitors.     

Isolation and characterization of microsatellite markers for <Emphasis Type="Italic">Cedrela odorata</Emphasis> L. (Meliaceae), a high value neotropical tree

We describe 9 primers for amplification of microsatellite loci for the Neotropical tree Cedrela odorata L. (Meliaceae). Loci were isolated from an enriched library derived from a single DNA sample from a tree in Costa Rica. Levels of polymorphism were determined using samples from a large progeny trial. Across loci, the number of alleles ranged from 14 to 30. Observed heterozygosity levels ranged from 0.61 to 0.88. No linkage disequilibria were detected although some departures from Hardy-Weinberg equilibrium (HWE) were found, probably due to a Wahlund effect.     

Estimating species trees using approximate Bayesian computation

Development of methods for estimating species trees from multilocus data is a current challenge in evolutionary biology. We propose a method for estimating the species tree topology and branch lengths using approximate Bayesian computation (ABC). The method takes as data a sample of observed rooted gene tree topologies, and then iterates through the following sequence of steps: First, a randomly selected species tree is used to compute the distribution of rooted gene tree topologies. This distribution is then compared to the observed gene topology frequencies, and if the fit between the observed and the predicted distributions is close enough, the proposed species tree is retained. Repeating this many times leads to a collection of retained species trees that are then used to form the estimate of the overall species tree. We test the performance of the method, which we call ST-ABC, using both simulated and empirical data. The simulation study examines both symmetric and asymmetric species trees over a range of branch lengths and sample sizes. The results from the simulation study show that the model performs very well, giving accurate estimates for both the topology and the branch lengths across the conditions studied, and that a sample size of 25 loci appears to be adequate for the method. Further, we apply the method to two empirical cases: a 4-taxon data set for primates and a 7-taxon data set for yeast. In both cases, we find that estimates obtained with ST-ABC agree with previous studies. The method provides efficient estimation of the species tree, and does not require sequence data, but rather the observed distribution of rooted gene topologies without branch lengths. Therefore, this method is a useful alternative to other currently available methods for species tree estimation.     

鸟类组织稳定同位素分析样品的预处理方法

稳定同位素分析样品在进行同位素质谱分析之前需要适当的处理,然而如何处理尚未形成统一的标准。本文结合鸟类组织稳定同位素分析样品的测试流程,介绍了鸟类学研究领域稳定同位素技术应用中涉及的分析样品解冻清洗、干燥、分离纯化、研磨和储存等前处理方法,并对不同组织常用的处理方法及分析样品测定中有待解决的问题展开了讨论。旨在抛砖引玉,为利用稳定同位素技术开展鸟类学方面的研究提供科学参考和技术支持。     

Buccal swabs allow efficient and reliable microsatellite genotyping in amphibians

Buccal swabs have recently been used as a minimally invasive sampling method in genetic studies of wild populations, including amphibian species. Yet it is not known to date what is the level of reliability for microsatellite genotypes obtained using such samples. Allelic dropout and false alleles may affect the genotyping derived from buccal samples. Here we quantified the success of microsatellite amplification and the rates of genotyping errors using buccal swabs in two amphibian species, the Alpine newt Triturus alpestris and the Green tree frog Hyla arborea, and we estimated two important parameters for downstream analyses, namely the number of repetitions required to achieve typing reliability and the probability of identity among genotypes. Amplification success was high, and only one locus tested required two to three repetitions to achieve reliable genotypes, showing that buccal swabbing is a very efficient approach allowing good quality DNA retrieval. This sampling method which allows avoiding the controversial toe-clipping will likely prove very useful in the context of amphibian conservation.     

Species-specific RAPD fingerprints for the closely related Picea mariana and P. rubens

Species-specific molecular markers were designed to assist in the identification of closely related black spruce (Picea mariana [B.S.P.] Mill.) and red spruce (P. rubens Sarg.) in northeastern North America. Trees from six provenances of black spruce and three provenances of red spruce were sampled from outside the sympatric zone. They were first classified using a composite index of five qualitative morphological traits. The species-specific genetic markers were developed using random amplified polymorphic DNAs (RAPD) and a combination of bulk sample and individual tree analyses. Each species bulk sample was constructed from DNAs obtained from 12 trees that were from outside the sympatric zone and showed a morphological composite index specific of each species. A total of 161 primers were screened with the bulk samples. From these, 52 primers showing segregating fingerprints were further screened with the individual trees. Most of the markers observed were shared by the two species, and there was less diversity in P. rubens. A small number of markers were found to be monomorphic or nearly monomorphic and specific to either P. mariana or P. rubens. These markers remained species-specific when F₁ progenies derived from independent intraspecific crosses were screened, and they were subsequently found to co-segregate in hybrids derived from independent interspecific crosses here used as controls.     

Composite Sampling Approaches for Bacillus anthracis Surrogate Extracted from Soil

Any release of anthrax spores in the U.S. would require action to decontaminate the site and restore its use and operations as rapidly as possible. The remediation activity would require environmental sampling, both initially to determine the extent of contamination (hazard mapping) and post-decon to determine that the site is free of contamination (clearance sampling). Whether the spore contamination is within a building or outdoors, collecting and analyzing what could be thousands of samples can become the factor that limits the pace of restoring operations. To address this sampling and analysis bottleneck and decrease the time needed to recover from an anthrax contamination event, this study investigates the use of composite sampling. Pooling or compositing of samples is an established technique to reduce the number of analyses required, and its use for anthrax spore sampling has recently been investigated. However, use of composite sampling in an anthrax spore remediation event will require well-documented and accepted methods. In particular, previous composite sampling studies have focused on sampling from hard surfaces; data on soil sampling are required to extend the procedure to outdoor use. Further, we must consider whether combining liquid samples, thus increasing the volume, lowers the sensitivity of detection and produces false negatives. In this study, methods to composite bacterial spore samples from soil are demonstrated. B. subtilis spore suspensions were used as a surrogate for anthrax spores. Two soils (Arizona Test Dust and sterilized potting soil) were contaminated and spore recovery with composites was shown to match individual sample performance. Results show that dilution can be overcome by concentrating bacterial spores using standard filtration methods. This study shows that composite sampling can be a viable method of pooling samples to reduce the number of analysis that must be performed during anthrax spore remediation.     

HPCE quantification of 5-methyl-2′-deoxycytidine in genomic DNA: Methodological optimization for chestnut and other woody species

Quantification of deoxynucleosides using micellar high-performance capillary electrophoresis (HPCE) is an efficient, fast and inexpensive evaluation method of genomic DNA methylation. This approach has been demonstrated to be more sensitive and specific than other methods for the quantification of DNA methylation content. However, effective detection and quantification of 5-methyl-2′-deoxycytidine depend of the sample characteristics. Previous works have revealed that in most woody species, the quality and quantity of RNA-free DNA extracted that is suitable for analysis by means of HPCE varies among species of the same gender, among tissues taken from the same tree, and vary in the same tissue depending on the different seasons of the year. The aim of this work is to establish a quantification method of genomic DNA methylation that lends itself to use in different Castanea sativa Mill. materials, and in other angiosperm and gymnosperm woody species. Using a DNA extraction kit based in silica membrane has increased the resolutive capacity of the method. Under these conditions, it can be analyzed different organs or tissues of angiosperms and gymnosperms, regardless of their state of development. We emphasized the importance of samples free of nucleosides, although, in the contrary case, the method ensures the effective separation of deoxynucleosides and identification of 5-methyl-2′-deoxycytidine.     

Development of a magnetic capture hybridization-PCR assay for Listeria monocytogenes direct detection in milk samples

AIMS: A rapid and sensitive method for Listeria monocytogenes direct detection from milk was developed. It is based on a magnetic capture hybridization procedure for selective DNA purification, followed by PCR identification. A comparison with two similar commercial systems from Dynal (Dynabeads) was carried out. METHODS AND RESULTS: The technique used previously developed nanoparticles modified with a 21-mer oligonucleotide. This sequence, sharing homology with all the L. monocytogenes strains, was selected on hlyA gene and located outside the desired specific PCR site to avoid cross-contaminations. Capture probe properties, in term of spacer length and purification, were determined to obtain the highest hybridization efficiency. Its specificity was tested in hybridization experiments with nontarget bacterial species. Any inhibitory effect of the nanoparticles on PCR was also examined. The amplification performed with the purified DNA could reliably identify a 10 CFU ml(-1) contamination rate. CONCLUSIONS: The optimized purification method showed a high specificity and sensitivity, with a detection level one log more sensitive than PCR carried out with nucleic acids obtained using commercial nanoparticles. SIGNIFICANCE AND IMPACT OF THE STUDY: The method, avoiding pre-enrichment, provides a rapid alternative to conventional microbiological detection methods. Furthermore, it is suitable for automation and can be proposed for the screening of a large number of samples.