SNP‐SCALE: SNP scoring by colour and length exclusion

Here we present SNP‐SCALE, a new single nucleotide polymorphism genotyping protocol based on allele‐specific polymerase chain reaction (AS‐PCR), but with two critical modifications designed to maximize accuracy, efficiency and throughput and minimize costs. The first is the addition of a locked nucleic acid (LNA) at the 3′‐SNP position of each of the two allele‐specific forward primers. The second is the addition of size‐differentiating M13 tails at the 5′ end of each primer, which facilitates the use of universal fluorescent primers. The use of LNAs makes SNP‐SCALE significantly more sensitive and specific than standard AS‐PCR. Moreover, SNP‐SCALE is ideally suited to a touchdown‐like PCR protocol, such that PCRs can be standardized, PCR optimization is straightforward, and medium‐high throughput can be readily achieved. We anticipate that the SNP‐SCALE protocol will be useful to molecular ecologists for a broad range of studies.     

Multiplex restriction amplicon sequencing: a novel next‐generation sequencing‐based marker platform for high‐throughput genotyping

To enable rapid selection of traits in marker‐assisted breeding, markers must be technically simple, low‐cost, high‐throughput and randomly distributed in a genome. We developed such a technology, designated as Multiplex Restriction Amplicon Sequencing (MRASeq), which reduces genome complexity by polymerase chain reaction (PCR) amplification of amplicons flanked by restriction sites. The first PCR primers contain restriction site sequences at 3’‐ends, preceded by 6‐10 bases of specific or degenerate nucleotide sequences and then by a unique M13‐tail sequence which serves as a binding site for a second PCR that adds sequencing primers and barcodes to allow sample multiplexing for sequencing. The sequences of restriction sites and adjacent nucleotides can be altered to suit different species. Physical mapping of MRASeq SNPs from a biparental population of allohexaploid wheat (Triticum aestivum L.) showed a random distribution of SNPs across the genome. MRASeq generated thousands of SNPs from a wheat biparental population and natural populations of wheat and barley (Hordeum vulgare L.). This novel, next‐generation sequencing‐based genotyping platform can be used for linkage mapping to screen quantitative trait loci (QTL), background selection in breeding and many other genetics and breeding applications of various species.     

Single nucleotide polymorphism genotyping using allele-specific PCR and fluorescence melting curves

We present a PCR method for identification of single nucleotide polymorphisms (SNPs), using allele-specific primers designed for selective amplification of each allele. Matching the SNP at the 3' end of the forward or reverse primer, and additionally incorporating a 3' mismatch to prevent amplification of the incorrect allele, results in selectivity of the allele-specific primers. DNA melting analysis with fluorescent SYBR Green affords detection of the PCR products. By incorporating a GC-rich sequence into one of the two allele-specific primers to increase the melting temperature, both alleles can be measured simultaneously at their respective melting temperatures. Applying the DNA melting analysis to SNPs in ApoE and ABCA1 yielded results identical to those obtained with other genotyping methods. This provides a cost-effective, high-throughput method for amplification and scoring of SNPs.     

Validation of loop‐mediated isothermal amplification for fast and portable sex determination across the phylogeny of birds

PCR is a universal tool for the multiplication of specific DNA sequences. For example, PCR‐based sex determination is widely used, and a diversity of primer sets is available. However, this protocol requires thermal cycling and electrophoresis, so results are typically obtained in laboratories and several days after sampling. Loop‐mediated isothermal amplification (LAMP) is an alternative to PCR that can take molecular ecology outside the laboratory. Although its application has been successfully probed for sex determination in three species of a single avian Family (raptors, Accipitridae), its generality remains untested and suitable primers across taxa are lacking. We designed and tested the first LAMP‐based primer set for sex determination across the modern birds (NEO‐W) based on a fragment of the gene chromo‐helicase‐DNA‐binding protein located on the female‐specific W chromosome. As nucleotide identity is expected to increase among more related taxa, taxonomically targeted primers were also developed for the Order Falconiformes and Families Psittacidae, Ciconiidae, Estrildidae and Icteridae as examples. NEO‐W successfully determined sex in a subset of 21 species within 17 Families and 10 Orders and is therefore a candidate primer for all modern birds. Primer sets designed specifically for the selected taxa correctly assigned sex to the evaluated species. A short troubleshooting guide for new LAMP users is provided to identify false negatives and optimize LAMP reactions. This study represents the crucial next step towards the use of LAMP for molecular sex determination in birds and other applications in molecular ecology.     

Simple and rapid bioluminescent detection of two verotoxin genes using allele‐specific PCR of E. coli O157: H7

Allele‐specific PCR for E. coli O157 was conducted with primers specific to verotoxin genes, verotoxin 1 (VT1) and verotoxin 2 (VT2). VT is an important cause of haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS) worldwide. We developed a simple, rapid bioluminescent detection method for E. coli O157. The method is based on the determination of pyrophosphoric acid (PPi) released during allele‐specific PCR. Thus, released PPi is converted to ATP by ATP sulphurylase and the concentration of ATP is determined using the firefly luciferase reaction. As a result, VT1, VT2 and DNA with VT1/VT2 were clearly identified by this method. This protocol, which does not require expensive equipment, can be utilized to monitor the PCR product rapidly. Additionally, this methodology can be used as a high‐throughput approach for measuring PCR products. Copyright © 2003 John Wiley & Sons, Ltd.     

Microdevice‐based solid‐phase polymerase chain reaction for rapid detection of pathogenic microorganisms

We demonstrate the integration of DNA amplification and detection functionalities developed on a lab‐on‐a‐chip microdevice utilizing solid‐phase polymerase chain reaction (SP‐PCR) for point‐of‐need (PON) DNA analyses. First, the polycarbonate microdevice was fabricated by thermal bonding to contain microchambers as reservoirs for performing SP‐PCR. Next, the microchambers were subsequently modified with polyethyleneimine and glutaraldehyde for immobilizing amine‐modified forward primers. During SP‐PCR, the immobilized forward primers and freely diffusing fluorescence‐labeled reverse primers cooperated to generate target amplicons, which remained covalently attached to the microchambers for the fluorescence detection. The SP‐PCR microdevice was used for the direct identifications of two widely detected foodborne pathogens, namely Salmonella spp. and Staphylococcus aureus, and an alga causing harmful algal blooms annually in South Korea, Cochlodinium polykrikoides. The SP‐PCR microdevice would be versatilely applied in PON testing as a universal platform for the fast identification of foodborne pathogens and environmentally threatening biogenic targets.     

Angiotensin-converting enzyme insertion/deletion polymorphism genotyping error: the cause and a possible solution to the problem

Rigat and colleagues were the first ones to develop a rapid PCR-based assay for identifying the angiotensin converting enzyme insertion/deletion (I/D) polymorphism. Due to a big difference between the length of the wild-type and mute alleles the PCR method is prone to mistyping because of preferential amplification of the D allele causing depicting I/D heterozygotes as D/D homozygotes. The aim of this study was to investigate whether this preferential amplification can be repressed by amplifying a longer DNA fragment in a so called Long PCR protocol. We also aimed to compare the results of genotyping using five different PCR protocols and to estimate the mistyping rate. The study included 200 samples which were genotyped using standard method used in our laboratory, a stepdown PCR, PCR protocol with the inclusion of 4 % DMSO, PCR with the use of insertion specific primers and new Long PCR method. The results of this study have shown that accurate ACE I/D polymorphism genotyping can be accomplished with the standard and the Long PCR method. Also, as of our results, accurate ACE I/D polymorphism genotyping can be accomplished regardless of the method used. Therefore, if the standard method is optimized more cautiously, accurate results can be obtained by this simple, inexpensive and rapid PCR protocol.     

SNP discovery based on CATS and genotyping in the finless porpoise (Neophocaena phocaenoides)

Single nucleotide polymorphisms (SNPs) discovery and genotyping were performed for the finless porpoises (Neophocaena phocaenoides). About 202 comparative anchor tagged sequence primers derived from genomes of human, mouse and some other mammals were used to screen the finless porpoise population. Of the 51 SNPs discovered, 25 were further characterized with ideal genotyping primers and using fragment length discrepant allele specific PCR assay. This is the first report of SNP loci for the finless porpoise, which is helpful to provide some novel molecular markers and new genetic information relevant to the conservation and management of this endangered species.     

A simple procedure for the analysis of single nucleotide polymorphisms facilitates map-based cloning in Arabidopsis

We developed a modified allele-specific PCR procedure for assaying single nucleotide polymorphisms (SNPs) and used the procedure (called SNAP for single-nucleotide amplified polymorphisms) to generate 62 Arabidopsis mapping markers. SNAP primers contain a single base pair mismatch within three nucleotides from the 3' end of one allele (the specific allele) and in addition have a 3' mismatch with the nonspecific allele. A computer program called SNAPER was used to facilitate the design of primers that generate at least a 1,000-fold difference in the quantity of the amplification products from the specific and nonspecific SNP alleles. Because SNAP markers can be readily assayed by electrophoresis on standard agarose gels and because a public database of over 25,000 SNPs is available between the Arabidopsis Columbia and Landsberg erecta ecotypes, the SNAP method greatly facilitates the map-based cloning of Arabidopsis genes defined by a mutant phenotype.     

Design of character‐based DNA barcode motif for species identification: A computational approach and its validation in fishes

The DNA barcodes are generally interpreted using distance‐based and character‐based methods. The former uses clustering of comparable groups, based on the relative genetic distance, while the latter is based on the presence or absence of discrete nucleotide substitutions. The distance‐based approach has a limitation in defining a universal species boundary across the taxa as the rate of mtDNA evolution is not constant throughout the taxa. However, character‐based approach more accurately defines this using a unique set of nucleotide characters. The character‐based analysis of full‐length barcode has some inherent limitations, like sequencing of the full‐length barcode, use of a sparse‐data matrix and lack of a uniform diagnostic position for each group. A short continuous stretch of a fragment can be used to resolve the limitations. Here, we observe that a 154‐bp fragment, from the transversion‐rich domain of 1367 COI barcode sequences can successfully delimit species in the three most diverse orders of freshwater fishes. This fragment is used to design species‐specific barcode motifs for 109 species by the character‐based method, which successfully identifies the correct species using a pattern‐matching program. The motifs also correctly identify geographically isolated population of the Cypriniformes species. Further, this region is validated as a species‐specific mini‐barcode for freshwater fishes by successful PCR amplification and sequencing of the motif (154 bp) using the designed primers. We anticipate that use of such motifs will enhance the diagnostic power of DNA barcode, and the mini‐barcode approach will greatly benefit the field‐based system of rapid species identification.     

ddradseqtools: a software package for in silico simulation and testing of double‐digest RADseq experiments

Double‐digested RADseq (ddRADseq) is a NGS methodology that generates reads from thousands of loci targeted by restriction enzyme cut sites, across multiple individuals. To be statistically sound and economically optimal, a ddRADseq experiment has a preliminary design stage that needs to consider issues related to the selection of enzymes, particular features of the genome of the focal species, possible modifications to the library construction protocol, coverage needed to minimize missing data, and the potential sources of error that may impact upon the coverage. We present ddradseqtools , a software package to help ddRADseq experimental design by (i) the generation of in silico double‐digested fragments; (ii) the construction of modified ddRADseq libraries using adapters with either one or two indexes and degenerate base regions (DBRs) to quantify PCR duplicates; and (iii) the initial steps of the bioinformatics preprocessing of reads. ddradseqtools generates single‐end (SE) or paired‐end (PE) reads that may bear SNPs and/or indels. The effect of allele dropout and PCR duplicates on coverage is also simulated. The resulting output files can be submitted to pipelines of alignment and variant calling, to allow the fine‐tuning of parameters. The software was validated with specific tests for the correct operability of the program. The correspondence between in silico settings and parameters from ddRADseq in vitro experiments was assessed to provide guidelines for the reliable performance of the software. ddradseqtools is cost‐efficient in terms of execution time, and can be run on computers with standard CPU and RAM configuration.     

Conditions for rapid sex determination in 47 avian species by PCR of genomic DNA from blood,shell‐membrane blood vessels,and feathers

The ability to rapidly and reliably determine the sex of birds is very important for successful captive‐bird breeding programs, as well as for field research. Visual inspection of adult birds is sufficient for sexually dimorphic species, but nestlings and monomorphic species are difficult, if not impossible, to sex by sight only. A method for rapid extraction of gDNA from blood, shell‐membrane blood vessels, and fully grown feathers, using Chelex, and the PCR conditions for determination of sex‐specific bands in 47 species (39 genera, 21 families, and 10 orders) are described. The PCR primers used amplify a length of DNA spanning an intron in the CHD‐1 gene, which is present on both the W and Z chromosomes. The intron differs in size between the two sex chromosomes, resulting in PCR products that separate into two bands for females and a single band for males in most avian species (except ratites). Because this simple technique uses Chelex, a rapid gDNA isolation protocol, and sets of PCR primers independent of restriction enzyme digestion, birds can be accurately sexed within 5 hr of sample collection. Zoo Biol 22:561–571, 2003. © 2003 Wiley‐Liss, Inc.     

Development of a sequence‐characterized amplified region marker using inter‐simple sequence repeats for detection of Puccinia striiformis f. sp. tritici

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is the most devastating wheat disease in China. Early and accurate detection of the pathogens would facilitate effective control of the diseases. DNA‐based methods now provide essential tools for accurate plant disease diagnosis. In this study, inter‐simple sequence repeats (ISSR) technique has been successfully applied to develop a sequence‐characterized amplified region (SCAR) marker for diagnosis of stripe rust of wheat and detection of Pst. In this study, one fragment unique to Pst was identified by ISSR and then sequenced. Based on the specific fragment, a pair of SCAR primers (616AF/616AR) was designed to amplify a 299‐bp DNA fragment within the sequenced region. The primers can amplify a unique DNA fragment for all tested isolates of Pst but not for the other pathogens of wheat leaves and the uninfected leaves. The polymerase chain reaction (PCR) assay could detect as low as 0.1 ng of genomic DNA in a 25.0 μl PCR reaction mixture and detect the pathogen from asymptomatic wheat leaves inoculated with Pst under glasshouse conditions.     

T-track PCR fingerprinting for the rapid detection of genetic polymorphism

The diversity of DNA sequences can be analyzed by comparing randomly amplified polymorphic DNA, or restriction fragment length polymorphism fragments of DNA. Such analyses are dependent on the selection of appropriate restriction enzyme(s) and/or primers. We have investigated a simpler approach to providing sensitive and specific genotyping. Cyclic extension of target sequences with dideoxythymidine generates PCR products with variable lengths. We analyzed these variable PCR products by scoring the number of variable bands and comparing the scores (numerical profiles) to establish similarities. We found that the polymorphic lengths of the PCR products were comparable among serologically defined strains. It suggests that this single PCR reaction followed by a one-step electrophoresis yields easily analyzable data that can be compared with data from other gels.     

Development of amplified fragment length polymorphism (AFLP)‐derived specific primer for the detection of Fusarium solani aetiological agent of peanut brown root rot

Aims

The objective of this work was to design an amplified fragment length polymorphism (AFLP)‐derived specific primer for the detection of Fusarium solani aetiological agent of peanut brown root rot (PBRR) in plant material and soil.

Methods and Results

Specific primers for the detection of the pathogen were designed based on an amplified region using AFLPs. The banding patterns by AFLPs showed that isolates from diseased roots were clearly distinguishable from others members of the F. solani species complex. Many bands were specific to F. solani PBRR, one of these fragments was selected and sequenced. Sequence obtained was used to develop specific PCR primers for the identification of pathogen in pure culture and in plant material and soil. Primer pair FS1/FS2 amplified a single DNA product of 175 bp. Other fungal isolates occurring in soil, included F. solani non‐PBRR, were not detected by these specific primers. The assay was effective for the detection of pathogen from diseased root and infected soils.

Conclusions

The designed primers for F. solani causing PBRR can be used in a PCR diagnostic protocol to rapidly and reliably detect and identify this pathogen.

Significance and Impact of the Study

These diagnostic PCR primers will aid the detection of F. solani causing PBRR in diseased root and natural infected soils. The method developed could be a helpful tool for epidemiological studies and to avoid the spread of this serious disease in new areas.     

Substantial differences in bias between single‐digest and double‐digest RAD‐seq libraries: A case study

The trade‐offs of using single‐digest vs. double‐digest restriction site‐associated DNA sequencing (RAD‐seq) protocols have been widely discussed. However, no direct empirical comparisons of the two methods have been conducted. Here, we sampled a single population of Gulf pipefish (Syngnathus scovelli) and genotyped 444 individuals using RAD‐seq. Sixty individuals were subjected to single‐digest RAD‐seq (sdRAD‐seq), and the remaining 384 individuals were genotyped using a double‐digest RAD‐seq (ddRAD‐seq) protocol. We analysed the resulting Illumina sequencing data and compared the two genotyping methods when reads were analysed either together or separately. Coverage statistics, observed heterozygosity, and allele frequencies differed significantly between the two protocols, as did the results of selection components analysis. We also performed an in silico digestion of the Gulf pipefish genome and modelled five major sources of bias: PCR duplicates, polymorphic restriction sites, shearing bias, asymmetric sampling (i.e., genotyping fewer individuals with sdRAD‐seq than with ddRAD‐seq) and higher major allele frequencies. This combination of approaches allowed us to determine that polymorphic restriction sites, an asymmetric sampling scheme, mean allele frequencies and to some extent PCR duplicates all contribute to different estimates of allele frequencies between samples genotyped using sdRAD‐seq versus ddRAD‐seq. Our finding that sdRAD‐seq and ddRAD‐seq can result in different allele frequencies has implications for comparisons across studies and techniques that endeavour to identify genomewide signatures of evolutionary processes in natural populations.     

Gel‐free species identification using melt‐curve analysis

DNA‐based identification of organisms is an important tool in biosecurity, ecological monitoring and wildlife forensics. Current methods usually involve post‐polymerase chain reaction (PCR) manipulations (e.g. restriction digest, gel electrophoresis), which add to the expense and time required for processing samples, and may introduce error. We developed a method of species identification that uses species‐specific primers and melt‐curve analysis, and avoids post‐PCR manipulation of samples. The method was highly accurate when trialled on DNA from six large carnivore species from Tasmania, Australia. Because of its flexibility and cost‐effectiveness, this method should find wide use in many areas of applied biological science.