If you are not sure what ‘culture’ means, you are not alone. In 1952, anthropologists Kroeber and Kluckhohn identified 164 definitions of culture and there has been growth rather than rationalisation ...in the ensuing 70 years. In everyday English, culture is the knowledge and behaviour that characterises a particular group of people. Under this umbrella definition, culture was for many decades the exclusive province of the humanities and social sciences, where anthropologists, historians, linguists, sociologists and other scholars studied and compared the language, arts, cuisine, and social habits of particular human groups. Of course, that important work continues, but since the 1980s culture has also been a major focus of enquiry in the natural sciences.
In this Primer, Cecilia Heyes explains how and why cultural evolutionists define culture. Social learning is ubiquitous in animals, it often produces group-typical behaviour, and more rarely yields improvement over generations. In humans, complex cognitive processes may be fruits rather than seeds of cultural selection.
Abstract
Hosts and pathogens impose coevolutionary pressure on each other as pathogens strive to establish themselves and hosts seek to suppress infection. RNA interference (RNAi) is a mechanism by ...which cells repress viruses and transposable elements, thereby serving as a form of immune defense. Previous studies have shown that antiviral RNAi genes evolve extraordinarily quickly in the fruit fly Drosophila melanogaster, suggesting that they may adaptively coevolve with viruses and transposable elements. An article by Palmer and colleagues extends this observation to nematodes and multiple insects. Their article can be combined with this Primer to demonstrate the use of comparative genomics and molecular evolutionary analyses in the measurement of natural selection.
Related article in GENETICS: Palmer, W. H., J. D. Hadfield, and D. J. Obbard, 2018 RNA-Interference pathways display high rates of adaptive protein evolution in multiple invertebrates. Genetics 208: 1585–1599.
MicroRNAs (miRNAs) are 18-25 nucleotides (nt) of highly conserved, noncoding RNAs involved in gene regulation. Because of miRNAs' short length, the design of miRNA primers for PCR amplification ...remains a significant challenge. Adding to the challenge are miRNAs similar in sequence and miRNA family members that often only differ in sequences by 1 nt. Here, we describe a novel empirical-based method, miPrimer, which greatly reduces primer dimerization and increases primer specificity by factoring various intrinsic primer properties and employing four primer design strategies. The resulting primer pairs displayed an acceptable qPCR efficiency of between 90% and 110%. When tested on miRNA families, miPrimer-designed primers are capable of discriminating among members of miRNA families, as validated by qPCR assays using Quark Biosciences' platform. Of the 120 miRNA primer pairs tested, 95.6% and 93.3% were successful in amplifying specifically non-family and family miRNA members, respectively, after only one design trial. In summary, miPrimer provides a cost-effective and valuable tool for designing miRNA primers.
A central challenge in the present era of biodiversity loss is to assess and manage human impacts on freshwater ecosystems. Macroinvertebrates are an important group for bioassessment as many taxa ...show specific responses to environmental conditions. However, generating accurate macroinvertebrate inventories based on larval morphology is difficult and error-prone. Here, DNA metabarcoding provides new opportunities. Its potential to accurately identify invertebrates in bulk samples to the species level, has been demonstrated in several case studies. However, DNA based identification is often limited by primer bias, potentially leading to taxa in the sample remaining undetected. Thus, the success of DNA metabarcoding as an emerging technique for bioassessment critically relies on carefully evaluating primers. We used the R package PrimerMiner to obtain and process cytochrome c oxidase I (COI) sequence data for the 15 most globally relevant freshwater invertebrate groups for stream assessment. Using these sequence alignments, we developed four primer combinations optimized for freshwater macrozoobenthos. All primers were evaluated by sequencing ten mock community samples, each consisting of 52 freshwater invertebrate taxa. Additionally, popular metabarcoding primers from the literature and the developed primers were tested in silico against the 15 relevant invertebrate groups. The developed primers varied in amplification efficiency and the number of detected taxa, yet all detected more taxa than standard ‘Folmer’ barcoding primers. Two new primer combinations showed more consistent amplification than a previously tested ribosomal marker (16S) and detected all 42 insect taxa present in the mock community samples. In silico evaluation revealed critical design flaws in some commonly used primers from the literature. We demonstrate a reliable strategy to develop optimized primers using the tool PrimerMiner. The developed primers detected almost all taxa present in the mock samples, and we argue that high base degeneracy is necessary to decrease primer bias as confirmed by experimental results and in silico primer evaluation. We further demonstrate that some primers currently used in metabarcoding studies may not be suitable for amplification of freshwater macroinvertebrates. Therefore, careful primer evaluation and more region / ecosystem specific primers are needed before DNA metabarcoding can be used for routine bioassessment of freshwater ecosystems.
High‐throughput sequencing‐based analysis of microbial diversity has evolved vastly over the last decade. Currently, the go‐to method for studying microbial eukaryotes is short‐read metabarcoding of ...variable regions of the 18S rRNA gene with <500 bp amplicons. However, there is a growing interest in applying long‐read sequencing of amplicons covering the rRNA operon for improving taxonomic resolution. For both methods, the choice of primers is crucial. It determines if community members are covered, if they can be identified at a satisfactory taxonomic level, and if the obtained community profile is representative. Here, we designed new primers targeting 18S and 28S rRNA based on 177,934 and 21,072 database sequences, respectively. The primers were evaluated in silico along with published primers on reference sequence databases and marine metagenomics data sets. We further evaluated a subset of the primers for short‐ and long‐read sequencing on environmental samples in vitro and compared the obtained community profile with primer‐unbiased metagenomic sequencing. Of the short‐read pairs, a new V6‐V8 pair and the V4_Balzano pair used with a simplified PCR protocol provided good results in silico and in vitro. Fewer differences were observed between the long‐read primer pairs. The long‐read amplicons and ITS1 alone provided higher taxonomic resolution than V4. Together, our results represent a reference and guide for selection of robust primers for research on and environmental monitoring of microbial eukaryotes.
Polymerase chain reaction (PCR) is one of the most important laboratory techniques used in molecular biology, genetics and molecular diagnostics. The success of a PCR-based method largely depends on ...the correct nucleic acid sequence analysis in silico prior to a wet-bench experiment. Here, we report the development of an online Java-based software for virtual PCR on linear or circular DNA templates and multiple primer or probe search from large or small databases. Primer or probe sensitivity and specificity are predicted by searching a database to find sequences with an optimal number of mismatches, similarity and stability. The software determines primer location, orientation, efficiency of binding and calculates primer melting temperatures for standard and degenerate oligonucleotides. The software is suitable for batch file processing, which is essential for automation when working with large amounts of data. The online Java software is available for download at http://primerdigital.com/tools/pcr.html.
Accession numbers for the sequences resulting from this study:
EU140956
EU177767
EU867815
EU882730
FJ975775-FJ975780
HM481419
HM481420
KC686837–KC686839
KM262797
•In silico tool for fast primer and probe searching.•Fast primer and probe design and advanced sequence analysis.•Virtual PCR.•Heuristic search algorithm for PCR primers and probes.•In silico DNA fingerprinting.
While high efficiency and cost‐effectiveness are two merits of environmental DNA (eDNA) techniques for detecting aquatic organisms, the difficulty of designing species‐specific primers can result in ...significant expenditure of time and money. During the in silico stage of primer development, primer specificity is predicted with alignment techniques such as BLAST that is based on the number and position of the primer/nontarget template mismatches. However, we speculate that nonspecific amplification is influenced by additional parameters, which lead to inaccuracies of in silico prediction. We performed in vitro specificity tests for 38 species‐specific primers selected for seven fishes and six turtles, using single‐plex conventional PCR (cPCR). A subset of 12 primer pairs were further tested with SYBR Green‐based or TaqMan‐based single‐plex quantitative PCR (qPCR). We disentangle the relative importance of mismatch properties (types and positions), primer properties (length, GC content, and 3′ end stability), PCR conditions (template concentrations and annealing temperatures), and PCR technique (cPCR, TaqMan‐based, or SYBR Green‐based qPCR) in determining the occurrence of amplifications. We then compared the PCR outcomes with the specificity check under two stringency scenarios based on alignment (i.e., BLAST search). We conducted a total of 679 cPCR and 226 qPCR analyses, with 90% of the reactions tested with nontarget templates. Primer pairs predicted by Primer‐BLAST to be specific rarely showed such specificity during the in vitro testing. BLAST searches correctly predicted the outcomes of around 67% of cPCR and qPCR, but had low sensitivity in detection of nontarget amplification (29–57%). Primer specificity increased significantly with total number of mismatches and annealing temperature, but decreased with higher GC content in the primer sequence. Mismatches that consisted of A‐A, G‐A, and C‐C pairings exerted 56% stronger reduction in nonspecific amplification effects than other mismatches. To conclude, we show that the prediction of primer specificity based only on the number and position of mismatches can be misleading. Our findings can be applied to increase the efficiency of the in silico primer selection process to maintain the relatively high efficiency and cost‐effectiveness of eDNA techniques.
msatcommander is a platform-independent program designed to search for microsatellite arrays, design primers, and tag primers using an automated routine. msatcommander accepts as input DNA sequence ...data in single-sequence or concatenated, fasta-formatted files. Search data and locus-specific primers are written to comma-separated value files for subsequent use in spreadsheet or database programs. Binary versions of the graphical interface for msatcommander are available for Apple OS X and Windows XP. Users of other operating systems may run the graphical interface version using the available source code, provided their environment supports at least Python 2.4, Biopython 1.43, and wxPython 2.8. msatcommander is available from http://code.google.com/p/msatcommander/.
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