Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups.

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Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi

Within uncharacterized groups, DNA barcodes, short DNA sequences that are present in a wide range of species, can be used to assign organisms into species. We propose an automatic procedure that sorts the sequences into hypothetical species based on the barcode gap, which can be observed whenever the divergence among organisms belonging to the same species is smaller than divergence among organisms from different species. We use a range of prior intraspecific divergence to infer from the data a model-based one-sided confidence limit for intraspecific divergence. The method, called Automatic Barcode Gap Discovery (ABGD), then detects the barcode gap as the first significant gap beyond this limit and uses it to partition the data. Inference of the limit and gap detection are then recursively applied to previously obtained groups to get finer partitions until there is no further partitioning. Using six published data sets of metazoans, we show that ABGD is computationally efficient and performs well for standard prior maximum intraspecific divergences (a few per cent of divergence for the five data sets), except for one data set where less than three sequences per species were sampled. We further explore the theoretical limitations of ABGD through simulation of explicit speciation and population genetics scenarios. Our results emphasize in particular the sensitivity of the method to the presence of recent speciation events, via (unrealistically) high rates of speciation or large numbers of species. In conclusion, ABGD is fast, simple method to split a sequence alignment data set into candidate species that should be complemented with other evidence in an integrative taxonomic approach.

ABGD, Automatic Barcode Gap Discovery for primary species delimitation

DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF–atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK–psbI spacer, and trnH–psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL+matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants.

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A DNA barcode for land plants.

. The Model of Emissions of Gases and Aerosols from Nature version 2.1 (MEGAN2.1) is a modeling framework for estimating fluxes of biogenic compounds between terrestrial ecosystems and the atmosphere using simple mechanistic algorithms to account for the major known processes controlling biogenic emissions. It is available as an offline code and has also been coupled into land surface and atmospheric chemistry models. MEGAN2.1 is an update from the previous versions including MEGAN2.0, which was described for isoprene emissions by Guenther et al. (2006) and MEGAN2.02, which was described for monoterpene and sesquiterpene emissions by Sakulyanontvittaya et al. (2008). Isoprene comprises about half of the total global biogenic volatile organic compound (BVOC) emission of 1 Pg (1000 Tg or 1015 g) estimated using MEGAN2.1. Methanol, ethanol, acetaldehyde, acetone, α-pinene, β-pinene, t-β-ocimene, limonene, ethene, and propene together contribute another 30% of the MEGAN2.1 estimated emission. An additional 20 compounds (mostly terpenoids) are associated with the MEGAN2.1 estimates of another 17% of the total emission with the remaining 3% distributed among >100 compounds. Emissions of 41 monoterpenes and 32 sesquiterpenes together comprise about 15% and 3%, respectively, of the estimated total global BVOC emission. Tropical trees cover about 18% of the global land surface and are estimated to be responsible for ~80% of terpenoid emissions and ~50% of other VOC emissions. Other trees cover about the same area but are estimated to contribute only about 10% of total emissions. The magnitude of the emissions estimated with MEGAN2.1 are within the range of estimates reported using other approaches and much of the differences between reported values can be attributed to land cover and meteorological driving variables. The offline version of MEGAN2.1 source code and driving variables is available from http://bai.acd.ucar.edu/MEGAN/ and the version integrated into the Community Land Model version 4 (CLM4) can be downloaded from http://www.cesm.ucar.edu/ .

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The model of emissions of gases and aerosols from nature version 2.1 (MEGAN2.1): An extended and updated framework for modeling biogenic emissions

Few genetic markers, if any, have found such widespread use as microsatellites, or simple/short tandem repeats. Features such as hypervariability and ubiquitous occurrence explain their usefulness, but these features also pose several questions. For example, why are microsatellites so abundant, why are they so polymorphic and by what mechanism do they mutate? Most importantly, what governs the intricate balance between the frequent genesis and expansion of simple repetitive arrays, and the fact that microsatellite repeats rarely reach appreciable lengths? In other words, how do microsatellites evolve?

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Microsatellites: simple sequences with complex evolution

A slot-blot method is described that provides rapid and reliable identification of somatic hybrids. Biotin-labelled probes of defined length were prepared from genomic DNA, and calibrated in conjunction with target DNA to allow for differing amounts of biotin incorporation and hybridisation efficiency. The presence of probe DNA was visualised by chemiluminescence detection. Probes of 1 to 5 kbp in length gave optimal discrimination among target DNAs from different potato species. The advantages of this method over existing ones are described.

A slot-blot hybridisation method for screening somatic hybrids

Transgenes encoding for insecticidal crystal (Cry) proteins from the soil-dwelling bacterium Bacillus Thuringiensis have been widely introduced into Genetically Modified (GM) crops to confer protection against insect pests. Concern that these transgenes may also harm beneficial or otherwise valued insects (so-called Non Target Organisms, NTOs) represents a major element of the Environmental Risk Assessments (ERAs) used by all countries prior to commercial release. Compiling a comprehensive list of potentially susceptible NTOs is therefore a necessary part of an ERA for any Cry toxin-containing GM crop. In partly-characterised and biodiverse countries, NTO identification is slowed by the need for taxonomic expertise and time to enable morphological identifications. This limitation represents a potentially serious barrier to timely adoption of GM technology in some developing countries. We consider Bt Cry1A cowpea (Vigna unguiculata) in Nigeria as an exemplar to demonstrate how COI barcoding can provide a simple and cost-effective means of addressing this problem. Over a period of eight weeks, we collected 163 insects from cowpea flowers across the agroecological and geographic range of the crop in Nigeria. These individuals included 32 Operational Taxonomic Units (OTUs) spanning four Orders and that could mostly be assigned to genus or species level. They included 12 Lepidopterans and two Coleopterans (both potentially sensitive to different groups of Cry proteins). Thus, barcode-assisted diagnoses were highly harmonised across groups (typically to genus or species level) and so were insensitive to expertise or knowledge gaps. Decisively, the entire study was completed within four months at a cost of less than 10,000 US$. The broader implications of the findings for food security and the capacity for safe adoption of GM technology are briefly explored.

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DNA barcoding simplifies environmental risk assessment of genetically modified crops in biodiverse regions.

The preferred form of oil palm for commercial production is termed Tenera. Tenera is heterozygous for a gene at a locus K that controls endocarp thickness: it produces fruit with intermediate endocarp thickness between the two homozygous forms of oil palm, Dura and Pisifera, giving easier processing and a higher yield of oil. The invention provides a method of obtaining a population of Tenera offspring from crossing two Tenera parents, which comprises testing immature progeny from the cross with distinct right and left differentiating molecular markers, and selecting for further cultivation those individuals whose marker inheritance patterns indicate they are likely to be Tenera.

Oil palm and processes for producing it

Adaptation of protoplast regeneration systems for use on untested or recalcitrant potato genotypes can be a time-consuming exercise. Callus growth and xylogenesis were evaluated as early correlates of shooting potential to shorten this process. Callus growth was of limited value for predicting organogenesis but a linear relationship was observed between xylogenesis and shooting frequency. Increases in xylem content above a minimum threshold corresponded with increases in shooting frequency. The predictive value of the relationship was tested using a simple protocol modification (the culture of calli on a filter paper base). Calli on filter paper produced more xylem elements and shoots than those plated directly on medium. The potential of xylem content as a predictive test of shooting frequency is discussed.

Predictive correlates of shoot regeneration from potato protoplast culture.

https://www.cell.com/trends/biotechnology/pdf/S0167-7799(04)00199-4.pdf

Mike J. Wilkinson

Papers

A slot-blot hybridisation method for screening somatic hybrids

DNA barcoding simplifies environmental risk assessment of genetically modified crops in biodiverse regions.

Oil palm and processes for producing it

Predictive correlates of shoot regeneration from potato protoplast culture.

Abandoning ‘responsive’ GM risk assessment