Barley allele-specific amplicons useful for identifying wheat-barley recombinant chromosomes

Barley (Hordeum vulgare L.) is potentially a new source of genes for wheat (Triticum aestivum L.) improvement. Wheat-barley chromosome recombinant lines provide a means for introgressing barley genes to wheat genome by chromosome engineering, and since these are expected to occur only rarely in special cytogenetic stocks, an efficient selection skill is necessary to identify them. To convert RFLP markers to barley allele-specific PCR markers useful for effective production of wheat-barley recombinant lines, 91 primer sets derived from RFLP clones which were previously mapped to the barley chromosomes were examined for PCR amplification using 'Chinese Spring' wheat, 'Betzes' barley and the wheat-barley chromosome addition lines. The polymorphisms were detected by an agarose gel electrophoresis of the PCR products without digestion with restriction enzymes. Out of 81 primer sets producing polymorphisms between the wheat and barley genomes, 26 amplified barley chromosome-specific DNAs which were confirmed to be located on the same chromosome as the RFLP markers by using the wheat-barley chromosome addition lines. These amplified DNAs represent barley allele-specific amplicons, which distinguish barley alleles from their wheat homoeologous counterparts. The present investigation revealed a higher probability for obtaining allele-specific amplicons from genomic DNA-derived RFLP markers than from cDNA-derived ones. The barley allele-specific amplicons developed in this study, namely, four for chromosome 2H, two for 3H, seven for 4H, eight for 5H, one for 6H and four for 7H, are suitable for identifying 'Chinese Spring' wheat- 'Betzes' barley recombinant chromosomes. However, one out of eight barley allele-specific amplicons on chromosome 5H did not detect a unique barley band in a 'New Golden' barley chromosome 5H addition line of 'Shinchunaga' wheat, indicating there may be a need to reconstruct allele-specific amplicons with different barley cultivars.     

Development and assessment of simple PCR markers for SNP genotyping in barley

Simple molecular marker assays underpin routine plant breeding and research activities in many laboratories worldwide. With the rapid growth of single nucleotide polymorphism (SNP) resources for many important crop plants, the availability of routine, low-tech marker assays for genotyping SNPs is of increased importance. In this study, we demonstrate that temperature-switch PCR (TSP) supports the rapid development of robust, allele-specific PCR markers for codominant SNP genotyping on agarose gel. A total of 87 TSP markers for assessing gene diversity in barley were developed and used to investigate the efficacy for marker development, assay reliably and genotyping accuracy. The TSP markers described provide good coverage of the barley genome, are simple to use, easy to interpret and score, and are amenable to assay automation. They provide a resource of informative SNP markers for assessing genetic relationships among individuals, populations and gene pools of cultivated barley (Hordeum vulgare L.) and its wild relative H. spontaneum K. Koch. TSP markers provide opportunities to use available SNP resources for marker-assisted breeding and plant genetic research, and to generate information that can be integrated with SNP data from different sources and studies. TSP markers are expected to provide similar advantages for any animal or plant species. M. J. Hayden and T. Tabone contributed equally to this work.     

Improvement of single nucleotide polymorphism genotyping by allele-specific PCR using primers modified with an ENA residue

When we placed an ENA residue into primers at the 3' end, or the n-1, n-2, or n-3 position, which included a single nucleotide polymorphism (SNP) site at the 3' end, only primers containing the ENA residue at the n-2 position were read by Taq DNA polymerase for amplification. The use of the ENA primers avoided the generation of undesired short products, which are thought to be derived from primer-dimers. A greater discrimination of the SNP site by these primers containing the ENA residue was observed compared with that of the corresponding unmodified DNA primers that are often used for allele-specific polymerase chain reaction (AS-PCR). This improvement is probably due to the difficulty of incorporating a nucleotide into the mismatched ENA primer by Taq DNA polymerase in the modified primer-template duplex. These results demonstrate that ENA primer-based AS-PCR would enable a rapid and reliable technique for SNP genotyping.     

Developments of functional markers for <Emphasis Type="Italic">Fom</Emphasis>-<Emphasis Type="Italic">2</Emphasis>-mediated fusarium wilt resistance based on single nucleotide polymorphism in melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.)

Three single nucleotide polymorphism (SNP) sites in which amino acids had changed were detected by sequence analysis within the leucine-rich repeat (LRR) region of the Fom-2 gene. Cleaved amplified polymorphic sequence (CAPS) and allele-specific PCR (AS-PCR) methods were employed to explore the SNP validation linked to fusarium wilt resistance in the F₁ and F₂ generations simultaneously. Homozygous- and heterozygous-resistant genotypes and homozygous-susceptible genotype could be clearly distinguished using the CAPS method, and three detected SNP sites were observed to be linked to fusarium wilt resistance, with a segregation ratio of 1:2:1 in the F₂ generation. In addition, heterozygous-resistant and homozygous-susceptible genotypes could be clearly distinguished in the F₁ generation using the AS-PCR method, showing a 3:1 segregation in terms of resistant and susceptible genotypes in the F₂ generation. We therefore developed SNP-based functional markers (FMs) and identified some melon germplasm resistant to fusarium wilt by FM analysis within melon species. In conclusion, the SNP-based FMs originating from the SNP site of the Fom-2 LRR region were determined to be linked to fusarium wilt resistance and showed promise in the enhancement of breeding in melon.     

Genetic mapping and BAC assignment of EST-derived SSR markers shows non-uniform distribution of genes in the barley genome

A set of 111,090 barley expressed sequence tags (ESTs) was searched for the presence of microsatellite motifs [simple sequence repeat (SSRs)] and yielded 2,823 non-redundant SSR-containing ESTs (SSR–ESTs). From this, a set of 754 primer pairs was designed of which 525 primer pairs yielded an amplicon and as a result, 185 EST-derived microsatellite loci (EST–SSRs) were placed onto a genetic map of barley. The markers show a uniform distribution along all seven linkage groups ranging from 21 (7H) to 35 (3H) markers. Polymorphism information content values ranged from of 0.24 to 0.78 (average 0.48). To further investigate the physical distribution of the EST–SSRs in the barley genome, a bacterial artificial chromosomes (BAC) library was screened. Out of 129 markers tested, BAC addresses were obtained for 127 EST–SSR markers. Twenty-seven BACs, forming eight contigs, were hit by two or three EST–SSRs each. This unexpectedly high incidence of EST–SSRs physically linked at the sub-megabase level provides additional evidence of an uneven distribution of genes and the segmentation of the barley genome in gene-rich and gene-poor regions.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.Primer sequences for developed SSR markers are available upon request from the corresponding author (A. Graner).     

Transfer of sequence tagged site PCR markers between wheat and barley.

Transfer of mapping information between related species has facilitated the development of restriction fragment length polymorphism (RFLP) maps in the cereals. Sequence tagged site (STS) primer sets for use in the polymerase chain reaction may be developed from mapped RFLP clones. For this study, we mapped 97 STS primer sets to chromosomes in wheat and barley to determine the potential transferability of the primer sets and the degree of correspondence between RFLP and STS locations. STS products mapped to the same chromosome group in wheat and barley 75% of the time. RFLP location predicted STS location 69% of the time in wheat and 56% of the time in barley. Southern hybridizations showed that most primer sets amplified sequences homologous to the RFLP clone, although additional sequences were often amplified that did not hybridize to the RFLP clone. Nontarget sequences were often amplified when primer sets were transferred across species. In general, results suggest a good probability of success in transferring STSs between wheat and barley, and that RFLP location can be used to predict STS location. However, transferability of STSs cannot be assumed, suggesting a need for recombinational mapping of STS markers in each species as new primer sets are developed. Key words : sequence tagged sites, PCR, wheat, barley.     

A Unique Primer with an Inosine Chain at the 5′-Terminus Improves the Reliability of SNP Analysis Using the PCR-Amplified Product Length Polymorphism Method

Polymerase chain reaction-amplified product length polymorphism (PCR-APLP) is one of the most convenient and reliable methods for single nucleotide polymorphism (SNP) analysis. This method is based on PCR, but uses allele-specific primers containing SNP sites at the 3′-terminus of each primer. To use this method at least two allele-specific primers and one “counter-primer”, which serves as a common forward or reverse primer of the allele-specific primers, are required. The allele-specific primers have SNP sites at the 3′-terminus, and another primer should have a few non-complementary flaps at the 5′-terminus to detect SNPs by determining the difference of amplicon length by PCR and subsequent electrophoresis. A major disadvantage of the addition of a non-complementary flap is the non-specific annealing of the primer with non-complementary flaps. However, a design principle for avoiding this undesired annealing has not been fully established, therefore, it is often difficult to design effective APLP primers. Here, we report allele-specific primers with an inosine chain at the 5′-terminus for PCR-APLP analysis. This unique design improves the competitiveness of allele-specific primers and the reliability of SNP analysis when using the PCR-APLP method.     

SNP2CAPS: a SNP and INDEL analysis tool for CAPS marker development

With the influx of various SNP genotyping assays in recent years, there has been a need for an assay that is robust, yet cost effective, and could be performed using standard gel-based procedures. In this context, CAPS markers have been shown to meet these criteria. However, converting SNPs to CAPS markers can be a difficult process if done manually. In order to address this problem, we describe a computer program, SNP2CAPS, that facilitates the computational conversion of SNP markers into CAPS markers. 413 multiple aligned sequences derived from barley ESTs were analysed for the presence of polymorphisms in 235 distinct restriction sites. 282 (90%) of 314 alignments that contain sequence variation due to SNPs and InDels revealed at least one polymorphic restriction site. After reducing the number of restriction enzymes from 235 to 10, 31% of the polymorphic sites could still be detected. In order to demonstrate the usefulness of this tool for marker development, we experimentally validated some of the results predicted by SNP2CAPS.     

Molecular haplotype determination using allele-specific PCR and pyrosequencing technology

Haplotyping of single-nucleotide polymorphisms (SNPs) is usually performed statistically by computational analysis or by time-consuming cloning techniques. Here we present a simple molecular approach for reliable haplotype determination on individual samples. The procedure is based on allele-specific PCR (AS-PCR) in combination with Pyrosequencing analysis. AS-PCR primers for each allelic variant of the investigated SNPs were used. A mismatch introduced at the second base from the 3' end dramatically improved allele specificity. Analysis of multiple SNPs on amplified fragments using Pyrosequencing technology allowed determination of haplotypes. Genotyping of heterozygote samples after AS-PCR gave a typical monoallelic pattern at each SNP, in which the identity of the present allele depended on the allele-specific initial amplification. Haplotype determination by the described procedure proved to be highly reliable. The results obtained by Pyrosequencing technology have the benefit of being truly quantitative, enabling detection of any nonspecific allele amplification.     

Enhanced Specificity of TPMT*2 Genotyping Using Unidirectional Wild-Type and Mutant Allele-Specific Scorpion Primers in a Single Tube

Genotyping of thiopurine S-methyltransferase (TPMT) is recommended for predicting the adverse drug response of thiopurines. In the current study, a novel version of allele-specific PCR (AS-PCR), termed competitive real-time fluorescent AS-PCR (CRAS-PCR) was developed to analyze the TPMT*2 genotype in ethnic Chinese. This technique simultaneously uses wild-type and mutant allele-specific scorpion primers in a single reaction. To determine the optimal conditions for both traditional AS-PCR and CRAS-PCR, we used the Taguchi method, an engineering optimization process that balances the concentrations of all components using an orthogonal array rather than a factorial array. Instead of running up to 264 experiments with the conventional factorial method, the Taguchi method achieved the same optimization using only 16 experiments. The optimized CRAS-PCR system completely avoided non-specific amplification occurring in traditional AS-PCR and could be performed at much more relaxed reaction conditions at 1% sensitivity, similar to traditional AS-PCR. TPMT*2 genotyping of 240 clinical samples was consistent with published data. In conclusion, CRAS-PCR is a novel and robust genotyping method, and the Taguchi method is an effective tool for the optimization of molecular analysis techniques.     

Identification of SNPs and development of allele-specific PCR markers for γ-gliadin alleles in Triticum aestivum

The coding regions of 28 entries of hexaploid wheat gamma-gliadin genes, gene fragments or pseudogenes in GenBank were used for nucleotide alignment. These sequences could be divided into nine subgroups based on nucleotide variation. The chromosomal locations of five of the seven unassigned subgroups were identified through subgroup-specific polymerase chain reactions (PCR) using Chinese Spring group-1 nulli-tetrasomic lines. Multiple single nucleotide polymorphisms (SNPs) and small insertions/deletions were identified in each subgroup. With further mining from wheat expressed sequence tag databases and targeted DNA sequencing, two SNPs were confirmed and one SNP was discovered for genes at the Gli-A1, Gli-B1 and Gli-D1 loci. A modified allele-specific PCR procedure for assaying SNPs was used to generate dominant DNA markers based on these three SNPs. For each of these three SNPs, two allele-specific primer sets were used to test Chinese Spring and 52 commercial Australian wheat varieties representing a range of low-molecular-weight (LMW) alleles. PCR results indicated that all were positive with one of the primer sets and negative with the other, with the exception of three varieties containing the 1BL/1RS chromosomal translocation that were negative for both. Furthermore, markers GliA1.1, GliB1.1 and GliD1.1 were found to be correlated with Glu-A3 a, b or c, Glu-B3 b, c, d or e and Glu-D3 a, b or e LMW glutenin alleles, respectively. Markers GliA1.2, GliB1.2 and GliD1.2 were found to be correlated with the Glu-A3 d or e, Glu-B3 a, g or h and Glu-D3 c alleles, respectively. These results indicated that the gamma-gliadin SNP markers could be used for detecting linked LMW glutenin subunit alleles that are important in determining the quality attributes of wheat products.     

High resolution DNA melting markers for identification of <Emphasis Type="Italic">H1</Emphasis>-linked resistance to potato cyst nematode

Although Sequence-Characterized Amplified Region (SCAR) markers linked to the potato H1 locus, which confers resistance to pathotypes Ro1 and Ro4 of the potato cyst nematode (PCN) Globodera rostochiensis, have been reported, robust markers that enable estimation of allele dosage would improve the quality of information obtained from genotyping parental accessions (cultivars/breeding lines) and progeny populations within breeding programmes. With this in mind, we have developed single nucleotide polymorphism (SNP)-based molecular markers flanking the H1 resistance gene, using genomic re-sequence data from five elite tetraploid accessions. The published TG689 and 57R primer sequences were used in a Basic Local Alignment Search Tool (BLAST) examination of the reference potato genome, and SNPs within the vicinity of these primer regions were identified and targeted for designing probe-based High Resolution Melting (HRM) SNP assays. Evaluation of the subsequently developed HRM markers, TG689_1P and 57R_1P, against the publicly available SCAR markers, TG689 and 57R, indicated that the HRM markers enabled more reliable marker-trait association than the SCARs. Additionally, allelic dosage estimates for the H1 locus were also derived using the TG689_1P marker, providing a tool to optimise parental and progeny selections in PCN resistance breeding.     

Identification of mitochondrial DNA sequence variation and development of single nucleotide polymorphic markers for CMS-D8 in cotton

Cytoplasmic male sterility (CMS), which is a maternally inherited trait and controlled by novel chimeric genes in the mitochondrial genome, plays a pivotal role in the production of hybrid seed. In cotton, no PCR-based marker has been developed to discriminate CMS-D8 (from Gossypium trilobum) from its normal Upland cotton (AD1, Gossypium hirsutum) cytoplasm. The objective of the current study was to develop PCR-based single nucleotide polymorphic (SNP) markers from mitochondrial genes for the CMS-D8 cytoplasm. DNA sequence variation in mitochondrial genes involved in the oxidative phosphorylation chain including ATP synthase subunit 1, 4, 6, 8 and 9, and cytochrome c oxidase 1, 2 and 3 subunits were identified by comparing CMS-D8, its isogenic maintainer and restorer lines on the same nuclear genetic background. An allelic specific PCR (AS-PCR) was utilized for SNP typing by incorporating artificial mismatched nucleotides into the third or fourth base from the 3′ terminus in both the specific and nonspecific primers. The result indicated that the method modifying allele-specific primers was successful in obtaining eight SNP markers out of eight SNPs using eight primer pairs to discriminate two alleles between AD1 and CMS-D8 cytoplasms. Two of the SNPs for atp1 and cox1 could also be used in combination to discriminate between CMS-D8 and CMS-D2 cytoplasms. Additionally, a PCR-based marker from a nine nucleotide insertion–deletion (InDel) sequence (AATTGTTTT) at the 59–67 bp positions from the start codon of atp6, which is present in the CMS and restorer lines with the D8 cytoplasm but absent in the maintainer line with the AD1 cytoplasm, was also developed. A SNP marker for two nucleotide substitutions (AA in AD1 cytoplasm to CT in CMS-D8 cytoplasm) in the intron (1,506 bp) of cox2 gene was also developed. These PCR-based SNP markers should be useful in discriminating CMS-D8 and AD1 cytoplasms, or those with CMS-D2 cytoplasm as a rapid, simple, inexpensive, and reliable genotyping tool to assist hybrid cotton breeding.     

Improvement of the dot-blot-SNP technique for efficient and cost-effective genotyping

Although the dot-blot-SNP technique is a laborsaving, cost-effective method for SNP genotyping of a large number of plants, the synthesis of 5′-digoxigenin (DIG)-labeled oligonucleotides for use as probes is still costly. We developed two probe-labeling methods for this technique, one being digoxigenin labeling of oligonucleotides by PCR (PCR-DIG labeling) and the other being hybridization using a bridge probe and a 5′-DIG-labeled oligonucleotide (bridge hybridization). Bridge hybridization detected allele-specific signals under hybridization conditions similar to those for the 5′-DIG-labeled oligonucleotides and biotin-labeled oligonucleotides, while signals were detected only under a lower stringency condition by PCR-DIG labeling. As a method for genotyping using many markers at one time, two methods, i.e., PCR using mixed primer pairs and hybridization using mixed probes, were examined with successful results. Eighty-five SNP markers designed for genotyping of rice cultivars detected allele-specific signals, the genotyping results corresponding to the previously reported ones.     

Dot-blot-SNP analysis for practical plant breeding and cultivar identification in rice

We report dot-blot hybridization with allele-specific oligonucleotides for single nucleotide polymorphisms (SNPs) analysis to be applicable for practical plant breeding and cultivar identification. Competitive hybridization of a digoxigenin-labeled oligonucleotide having the sequence of a mutant allele (or a wild-type allele) together with an unlabeled oligonucleotide having the sequence of a wild-type allele (or a mutant allele) was highly effective to reduce background signals in dot-blot hybridization. All 100 tested genes (200 alleles) in rice having SNPs or insertions/deletions were detected in an allele-specific manner. Genotypes of 43 rice cultivars were identified by this technique, and eight SNP markers were found to be sufficient for distinguishing all the cultivars from each other. Dot-blot analysis was also applied to genotyping of Wx and Sd1 of F₄ plants in a conventional breeding program. Since dot-blot analysis with competitive hybridization provides a highly reliable, simple, and cost-effective technique for SNP analysis of a large number of samples, this technique is expected to realize the practical use of a novel breeding method, in which plants or breeding lines are selected by SNP analyses of many genes in a laboratory.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

A microarray-based high throughput molecular marker genotyping method: the tagged microarray marker (TAM) approach

A microarray-based method has been developed for scoring thousands of DNAs for a co-dominant molecular marker on a glass slide. The approach was developed to detect insertional polymorphism of transposons and works well with single nucleotide polymorphism (SNP) markers. Biotin- terminated allele-specific PCR products are spotted unpurified onto streptavidin-coated glass slides and visualised by hybridisation of fluorescent detector oligonucleotides to tags attached to the allele- specific PCR primers. Two tagged primer oligonucleotides are used per locus and each tag is detected by hybridisation to a concatameric DNA probe labelled with multiple fluorochromes.     

A representative, highly informative ’genotyping set’ of barley SSRs

We have developed a ’genotyping set’ of 48 SSR-based genetic markers for application in genetical studies of barley. The SSRs are a subset of a collection of approximately 600 SSRs available to the barley research community. They have been specifically chosen according to the following criteria: (1) they are single locus; (2) their product quality is good under standard assay conditions; (3) they are distributed across the barley genome; and (4) they exhibit reasonably high polymorphic information content (PIC) values in the cultivated barley gene-pool. To maximise genotyping throughput, one of each SSR primer pair was 5′ end-labelled with either fam, hex or tet fluorochromes to allow automated data capture after running the samples on a DNA sequencer. SSR product sizes were assembled from a reference set of 24 barley genotypes which allowed the construction of ’graphical genotypes’ of each of the individual lines. The graphical genotypes provide a convenient tool for interrogating genetic similarity in the individuals surveyed. The product sizes were compared to those obtained from end-labelling one of the primers with ³³P and separating the products by denaturing PAGE followed by autoradiography. Although inconsistencies in size were common, they could generally be easily resolved. A reference manual for use of the ’genotyping set’ has been produced and is available as a PDF download file at http://www.scri.sari.ac.uk/ssr/pdf. These well-characterised barley SSRs, for the first time, provide a common set of robust PCR-based tools which can be used to integrate and compare information collected from fundamental and/or applied genetic studies on barley in different laboratories across the world. Received: 17 May 2000 / Accepted: 5 September 2000     

Single-reaction for SNP Genotyping on Agarose Gel by Allele-specific PCR in Black Poplar (Populus nigra L.)

The wide development of single nucleotide polymorphism (SNP) markers also in non-model species increases the need for inexpensive methods that do not require sophisticated equipment and time for optimization. This work presents a new method for polymerase chain reaction (PCR) amplification of multiple specific alleles (PAMSA), which allows efficient discrimination of SNP polymorphisms in one reaction tube with standard PCR conditions. This improved PAMSA requires only three unlabeled primers: a common reverse primer and two allele-specific primers having a tail of different length to differentiate the two SNP alleles by the size of amplification products on agarose gel. A destabilizing mismatch within the five bases of the 3′ end is also added to improve the allele specificity. To validate the accuracy of this method, 94 full-sib individuals were genotyped with three SNPs and compared to the genotypes obtained by cleaved amplified polymorphic sequence (CAPS) or derived CAPS. This method is flexible, inexpensive, and well suited for high throughput and automated genotyping.     

Predicting the success of primer extension genotyping assays using statistical modeling

Using an empirical panel of more than 20 000 single base primer extension (SNP-IT) assays we have developed a set of statistical scores for evaluating and rank ordering various parameters of the SNP-IT reaction to facilitate high-throughput assay primer design with improved likelihood of success. Each score predicts either signal magnitude from primer extension or signal noise caused by mispriming of primers and structure of the PCR product. All scores have been shown to correlate with the success/ failure rate of the SNP-IT reaction, based on analysis of assay results. A logistic regression analysis was applied to combine all scored parameters into one measure predicting the overall success/failure rate of a given SNP marker. Three training sets for different types of SNP-IT reaction, each containing about 22000 SNP markers, were used to assign weights to each score and optimize the prediction of the combined measure. c-Statistics of 0.69, 0.77 and 0.72 were achieved for three training sets. This new statistical prediction can be used to improve primer design for the SNP-IT reaction and evaluate the probability of genotyping success for a given SNP based on analysis of the surrounding genomic sequence.     

A multiplexing single nucleotide polymorphism typing method based on restriction-enzyme-mediated single-base extension and capillary electrophoresis

Millions of single nucleotide polymorphisms (SNPs) have been identified in recent years. This provides a great opportunity for large-scale association and population studies. However, many high-throughput SNP typing techniques require expensive and dedicated instruments, which render them out of reach for many laboratories. To meet the need of these laboratories, we here report a method that uses widely available DNA sequencer for SNP typing. This method uses a type II restriction enzyme to create extendable ends at target polymorphic sites and uses single-base extension (SBE) to discriminate alleles. In this design, a restriction site is engineered in one of the two polymerase chain reaction (PCR) primers so that the restriction endonuclease cuts immediately upstream of the targeted SNP site. The digestion of the PCR products generates a 5'-overhang structure at the targeted polymorphic site. This 5'-overhang structure then serves as a template for SBE reaction to generate allele-specific products using fluorescent dye-terminator nucleotides. Following the SBE, the allele-specific products with different sizes can be resolved by DNA sequencers. Through primer design, we can create a series of PCR products that vary in size and contain only one restriction enzyme recognition site. This allows us to load many PCR products in a single capillary/lane. This method, restriction-enzyme-mediated single-base extension, is demonstrated by typing multiple SNPs simultaneously for 44 DNA samples. By multiplexing PCR and pooling multiplexed reactions together, this method has the potential to score 50-100 SNPs/capillary/run if the sizes of PCR products are arranged at every 5-10 bases from 100 to 600 base range.