Efficient protocols for CAPS-based mapping inArabidopsis

Positional cloning continues to be an essential method for gene identification and characterisation. The introduction of PCR-based techniques such as Amplified Fragment Length Polymorphism (AFLP), Simple Sequence Length Polymorphisms (SSLP) and Cleaved Amplified Polymorphic Sequences (CAPS) has greatly increased the efficiency of gene mapping in arabidopsis. To develop the CAPS marker approach further, we have altered several critical mapping parameters. Efficiency was improved by using a small volume of dry seed for DNA extraction instead of the commonly used vegetative tissue. Reproducibility of PCR reactions was enhanced by faster and reduced protocols for PCR and restriction enzyme digestion and optimisation of PCR conditions for over 50 CAPS primer pairs. Finally, the density of genetic markers was increased by providing polymorphic information for all CAPS markers in arabidopsis ecotypes Wassilewskija (Ws), Columbia (Col) and Cape Verde Islands (Cvi).     

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.     

Development of a CAPS marker system for genotyping European pear cultivars harboring 17 <Emphasis Type="Italic">S</Emphasis> alleles

The full-length cDNAs of eight S ribonucleases (S-RNases) were cloned from stylar RNA of European pear cultivars that could not be characterized by the cleaved amplified polymorphic sequences (CAPS) marker system for genotyping European pear cultivars harboring nine S alleles Sa, Sb, Sd, Se, Sh, Sk, Sl, Sq, and Sr. Comparison of the nucleotide sequences between these cDNAs and six putative S-RNase alleles previously amplified by genomic PCR revealed that five corresponded to the putative Sc-, Si-, Sm-, Sn-, and Sp-RNase alleles and the other three corresponded new S-RNase alleles (designated as putative Sg-, Ss-, and St-RNase alleles). Genomic PCR with a new set of primers was used to amplify 17 S-RNase alleles: 1906 bp (Sg), 1642 bp (St), 1414 bp (Sl), ca. 1.3 kb (Sk and Sq), 998 bp (Se), 440 bp (Sb), and ca. 350 bp (Sa, Sc, Sd, Sh, Si, Sm, Sn, Sp, Sr, and Ss). Among them, S-RNase alleles of similar size were discriminated by digestion with 11 restriction endo-nucleases. The PCR amplification of 17 S-RNase alleles following digestion with the restriction endonucleases provided a new CAPS marker system for rapid S-genotyping of European pear cultivars harboring 17 S alleles. Using the CAPS analysis, Sc, Sg, Si, Sm, Sn, Sp, Ss, and St alleles were found in 32 cultivars, which were classified into 23 S-genotypes.     

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.     

A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers

A set of mapping markers have been designed for Arabidopsis thaliana that correspond to DNA fragments amplifed by the polymerase chain reaction (PCR). The ecotype of origin of these amplified fragments can be determined by cleavage with a restriction endo-nuclease. Specifically, 18 sets of PCR primers were synthesized, each of which amplifies a single mapped DNA sequence from the Columbia and Landsberg erecta ecotypes. Also identifed was at least one restriction endonuclease for each of these PCR products that generates ecotype-specific digestion patterns. Using these co-dominant cleaved amplified polymorphic sequences (CAPS), an Arabidopsis gene can be unambiguously mapped to one of the 10 Arabidopsis chromosome arms in a single cross using a limited number of F₂ progeny.     

Detection of multiple β-casein (CASB) alleles by amplification created restriction sites (ACRS)

Direct sequencing of polymerase chain reaction (PCR) amplified DNA has been used to detect the DNA sequences for bovine beta-casein (CASB) A3 and B variants. Based on these sequences we have designed primers which create allele-specific restriction sites in the PCR product. Restriction analysis of PCR product generated in one reaction enable us to identify the A1, A2, A3 and B alleles of CASB rapidly without the use of radioactivity.     

VCF2CAPS–A high-throughput CAPS marker design from VCF files and its test-use on a genotyping-by-sequencing (GBS) dataset

Next-generation sequencing (NGS) is a powerful tool for massive detection of DNA sequence variants such as single nucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs) and insertions/deletions (indels). For routine screening of numerous samples, these variants are often converted into cleaved amplified polymorphic sequence (CAPS) markers which are based on the presence versus absence of restriction sites within PCR products. Current computational tools for SNP to CAPS conversion are limited and usually infeasible to use for large datasets as those generated with NGS. Moreover, there is no available tool for massive conversion of MNPs and indels into CAPS markers. Here, we present VCF2CAPS–a new software for identification of restriction endonucleases that recognize SNP/MNP/indel-containing sequences from NGS experiments. Additionally, the program contains filtration utilities not available in other SNP to CAPS converters–selection of markers with a single polymorphic cut site within a user-specified sequence length, and selection of markers that differentiate up to three user-defined groups of individuals from the analyzed population. Performance of VCF2CAPS was tested on a thoroughly analyzed dataset from a genotyping-by-sequencing (GBS) experiment. A selection of CAPS markers picked by the program was subjected to experimental verification. CAPS markers, also referred to as PCR-RFLPs, belong to basic tools exploited in plant, animal and human genetics. Our new software–VCF2CAPS–fills the gap in the current inventory of genetic software by high-throughput CAPS marker design from next-generation sequencing (NGS) data. The program should be of interest to geneticists involved in molecular diagnostics. In this paper we show a successful exemplary application of VCF2CAPS and we believe that its usefulness is guaranteed by the growing availability of NGS services.

This is a PLOS Computational Biology Software paper.

     

Identification of Orchidaceae species of Northern West of Syria based on chloroplast DNA

The plant family Orchidaceae has a great economic value (ornamental and medical uses, beside the aromatic features). Traditionally, identification of orchid species has relied heavily on morphological features. These features, however, are either not variable enough between species or too plastic to be used for identification at the species level. DNA-based markers could be the alternative strategy towards an accurate and robust identification of those species. Since the chloroplast DNA has a lower level of evolution compared to the nuclear genome, an attempt was made in this study to investigate polymorphism in the chloroplast DNA among orchid species distributed in North-West region of Syria using Cleaved Amplified Polymorphic Sequence (CAPS) technique for developing markers for the diagnosis of targeted species. CAPS analysis was carried out on 34 orchid samples that represent all species observed in the region. Universal primers were used to amplify targeted chloroplast regions. Generated PCR products were digested with various restriction enzymes. CAPS results revealed high polymorphism among species examined. This polymorphism was suffiecient for the diagnosis of all of those species apart from five species (Ophrys fuciflora (one sample), Oph. bornmuelleri, Ophrys sp., scolopax and Oph. argolica). Availability of such species-specific markers would ensure more authentic identification of orchid species compared to morphological characters and can be regarded as a valuable tool to guide in conservation programs of orchid species in Syria. CAPS data generated were converted to an identification key for orchid species studied.     

Application of STS markers for leaf rust resistance genes in near-isogenic lines of spring wheat cv. Thatcher

Sequence tagged site (STS) markers for eight resistance genes against Puccinia recondita f. sp. tritici were used to screen a set of near-isogenic lines of wheat cv. Thatcher containing in total 40 different Lr genes and their alleles. Polymerase chain reaction (PCR) analysis was carried out by using STS, SCAR and CAPS primers specific for the leaf rust resistance genes Lr1, Lr9, Lr10, Lr19, Lr24, Lr28, Lr37 and Lr47. The STS, CAPS and SCAR markers linked to resistance genes Lr9, Lr10, Lr19, Lr24, Lr37 and Lr47 were found to be reliable in diverse genetic backgrounds. The amplification product of the Lr1 gene marker was detected in the susceptible cv. Thatcher and in all of the near-isogenic lines examined except Lr2a, Lr2b, Lr2c and Lr19. The sequence analysis of PCR products amplified in lines Lr1, Lr10, Lr28 and in cv. Thatcher indicated that the near-isogenic lines and cv. Thatcher contained in the targeted chromosome region an allele that differed from the original alleles corresponding to Lr1/6*Thatcher (TLR621) and susceptible Thatcher (TH621). The amplification product specific to the STS marker of the Lr1 gene was amplified in almost all Thatcher near-isogenic lines and in cv. Thatcher because their alleles possessed primer sequences identical to the original allele TLR621. The marker for the Lr28 resistance gene was identified in line Lr28, carrying gene Lr28, and in 21 other near-isogenic lines. The sequencing of PCR products specific to Lr28 and generated in lines Lr1, Lr10 and Lr28 indicated that the lines Lr1, Lr10 and Lr28 are heterozygous in this region.     

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.     

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.     

基于磁珠富集法的绣线菊SSR分子标记分离与筛选

微卫星序列（SSR）具有多态性高、共显性遗传等特点,是一种极具价值的分子遗传标记。采用磁珠富集法从高山绣线菊基因组DNA中分离和筛选SSR标记。高山绣线菊基因组经限制性内切酶Mse I酶切后与接头连接,并与生物素标记SSR探针（AC）15和（AG）15杂交,然后通过链霉亲和素磁珠富集、洗脱、PCR扩增、克隆,完成微卫星文库构建。利用载体通用引物和探针序列引物进行PCR扩增,筛选重组克隆并测序,获得112条序列。随机挑选其中60条序列设计的引物,经初期筛选获得多态性引物16对。用所得16对引物对4个居群92个个体的蒙古绣线菊和高山绣线菊进行PCR扩增。统计分析PCR产物的毛细管电泳结果,发现4个居群的平均等位基因数、平均期望杂合度及平均观测杂合度都比较高。64个数据系列（4个居群×16个位点）中的26个显著偏离HardyWeinberg平衡,推测可能由于无效等位基因的存在所引起。分析显示研究开发的16对多态性SSR引物可以用于后续遗传多样性、物种进化与亲缘关系等方面研究,丰富了绣线菊遗传多样性研究的分子标记。     

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.     

Electrophoretic detection of single-nucleotide polymorphisms

Single-nucleotide polymorphisms (SNPs) represent the most prevalent class of genetic markers available for linkage disequilibrium or cladistic analyses. PCR primers may be labeled with fluorescent dyes and used to rapidly and accurately differentiate among alleles that are defined by a single-nucleotide differences. Here, we describe the primer-mediated detection of SNPs based on primer mismatch during allele-specific amplification of preamplified target sequences. Primers are labeled with different fluors at their 5' nucleotides, with their 3' termini at the transition mutation that defines allelic variation at the target locus. Each primer perfectly matches one of the two available alleles for each locus. Electrophoretic detection permits characterization of the product both by size and fluor. This report demonstrates some of the capabilities of this assay, including heterozygote determination and multiplexed analysis.     

A CAPS marker set for mapping in linkage group III of pea (Pisum sativum L.)

A set of twelve CAPS markers was mapped for linkage group III of pea (Pisum sativum L.). New primers were designed to use a polymerase chain reaction to amplify fragments of sequenced pea genes containing at least one large intron. Amplification products were tested for polymorphism across three pea lines (Chi115, Flagman and WL1238) using eleven four-base restriction endonucleases. Nine STS markers for linkage group III from the literature were also tested for polymorphism, and five of these were used in this mapping study as anchor points. All polymorphic loci were located by genetic analysis of the F(2)population from the cross Chi115 x WL1238, and a map of linkage group III consisting of one morphological and twelve CAPS markers was created. The map covers the full length of the chromosome and is about 162 cM long. All the CAPS markers in a set were used to test for polymorphism among 10 additional pea DNA samples extracted from different marker lines and cultivars.     

The primer generator: a program that facilitates the selection of oligonucleotides for site-directed mutagenesis.

Site-directed mutagenesis is a powerful tool that has enabled molecular biologists to perform functional analysis of altered nucleic acids and proteins. Newer PCR-based mutagenesis techniques have reduced the process of mutagenesis to as little as one day. While each technique has its advantages, both require a strategy to isolate the desired clone from a population that contains mutagenized and wild-type genes. In this report, we describe a World Wide Web-based computer program that facilitates the design of mutagenic primers such that successfully mutagenized clones can be identified by the presence or absence of a unique restriction site.     

Generation of co-dominant PCR-based markers by duplex analysis on high resolution gels

Rapid and efficient procedures for the detection of sequence polymorphisms are essential for chromosomal walking and mutation detection analyses. While DNA chip technology and denaturing high-performance liquid chromatography (DHPLC) are the methods of choice for large scale facilities, small laboratories are dependent on simple ready-to-use techniques. We show that heteroduplex analysis on high resolution gel matrices efficiently detects sequence polymorphism differing as little as a single base pair (e.g. single-nucleotide polymorphism, SNP) with standard laboratory equipment. Furthermore, the matrices also discerned differences between homoduplexes, a prerequisite for co-dominant markers. The markers thus generated are referred to as duplex analysis markers. We designed PCR primers for 36 Arabidopsis thaliana loci ranging in length from 230 bp to 1000 bp. Among three ecotypes, more than half ( n = 19) of the loci examined were polymorphic; five of which contained three different alleles. This simple, high resolution technique can be used to rapidly convert sequence tagged sites into co-dominant PCR-based molecular markers for fine-scale mapping studies and chromosomal walking strategies as well as for the detection of mutations in particular genes.     

Denaturing gradient gel electrophoresis detection of polymorphism in a PCR fragment of sheep epidermal growth factor gene

The ovine map is not yet well-developed, which represents a problem when looking for markers of a region of interest in sheep. A means of circumventing this is to use comparative mapping. In this study primers were determined using consensus sequences for the epidermal growth factor gene of humans, rats and mice, and an ovine epidermal growth factor gene fragment was amplified by polymerase chain reaction (PCR). A new set of specific ovine primers was chosen to study the polymorphism of this DNA fragment by denaturing gradient gel electrophoresis. Eighty-four individuals belonging to seven sheep breeds were studied with this technique and four alleles were detected. The heterozygosity rate was 0.57. Family analysis showed mendelian inheritance of the alleles. Usually, genetic analysis of type-I loci used in the comparative mapping is based on the detection of restriction fragment length polymorphisms in sheep DNA using cDNA probes from other species. Our work shows that another method, based on PCR and denaturing gradient gel electrophoresis techniques, can be efficiently used.     

Web-based primer design for single nucleotide polymorphism analysis

The detection of single nucleotide polymorphisms by PCR is necessary for many types of genetic analysis, from mapping genomes to tracking specific mutations. This technique is most commonly used when polymorphisms alter restriction endonuclease recognition sites. Here we describe a web-based program, dCAPS Finder 2.0, that facilitates the design of mismatched PCR primers to create or remove a restriction endonuclease recognition site relative to the polymorphism being analyzed.     

A molecular approach to the identification of S-alleles in Brassica oleracea

A molecular technique for the identification of S-alleles involved in self-incompatibility has been used to analyse the S-allele reference collection of Brassica oleracea. The reference collection contains nearly 50 different lines each with a different S-allele present in the homozygous state. The technique consists of amplifying by the polymerase chain reaction (PCR) sequences belonging to the S multigene sequence family using a single pair of conserved primers. PCR products are then analysed further by digestion with six restriction enzymes followed by gel electrophoresis of the digestion products. A simple method of estimating the band sizes of the digestion products is described. The S-locus-related sequences can be distinguished from S-locus glycoprotein and S-receptor kinase genes by the restriction patterns. Furthermore, with any one restriction enzyme, several alleles showed the same restriction pattern. Alleles could therefore be grouped together. With two exceptions, each member of the S-allele reference collection showed a unique set of restriction patterns. Investigation of the exceptions using pollen tube growth tests showed that these accessions represented duplications within the collection. This technique therefore provides a simple and useful method for identifying different S-alleles.