Marker development for the genetic study of natural variation in Arabidopsis thaliana
1Sainsbury Laboratory, Norwich NR4 7UH, UK and 2Department of Crop and Soil Sciences, Washington State University, Pullman, WA 99164, USA
*To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT 1 INTRODUCTION2 OVERVIEW OF THE...3 EXPERIMENTAL VALIDATION4 DISCUSSION AND CONCLUSIONACKNOWLEDGEMENTREFERENCES |
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Summary: We report AtPRIMER, an application that automates the discovery of new polymorphic markers between ecotypes of Arabidopsis thaliana. On specifying two ecotypes and the genomic region of interest, the script retrieves all corresponding single nucleotide polymorphisms (SNPs) and generates CAPS and/or dCAPS PCR primer sequences. We show that AtPRIMER accurately found specific polymorphic markers for our linkage mapping project. AtPRIMER will therefore be useful for efficient marker development with high density and specificity.
Availability: This PERL/CGI program is available for non-commercial users at http://www.AtPRIMER.tsl.ac.uk. The web-based version is available for public use at the same URL.
Contact: adnane.nemri@tsl.ac.uk
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1 INTRODUCTION |
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TOPABSTRACT1 INTRODUCTION2 OVERVIEW OF THE...3 EXPERIMENTAL VALIDATION4 DISCUSSION AND CONCLUSIONACKNOWLEDGEMENTREFERENCES |
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The recent availability of fully sequenced genomes of organisms such as the model plant Arabidopsis thaliana (At) creates new opportunities for genetic studies, including the study of natural variation. One obvious limitation, however, is that only one reference ecotype is usually fully sequenced. In the case of At, it is the commonly used Columbia-0 (Col-0) accession. For studying the tremendous diversity of traits exhibited within a species, it is necessary to generate and retrieve genetic information relating to natural variants. Single nucleotide polymorphisms (SNPs) are the most abundant type of sequence polymorphism across ecotypes of a same species. By genotyping natural variants based on the sequence of the reference ecotype, it is possible to uncover SNPs between any two individual ecotypes. Increased use of populations derived from crosses between natural variants requires the development of new specific genetic markers. Finding polymorphic markers is nevertheless a tedious process that consumes both time and resources. However, its efficiency can be greatly increased by making use of partial sequence information for a wide range of ecotypes. An automated tool for generating specifically polymorphic markers between non-fully sequenced ecotypes would therefore be useful for plant geneticists.
Here, we exploit genotype data generated by Nordborg and colleagues (2005) on a set of 96 At accessions. This dataset includes 1214 genomic sequence fragments of precise location and distributed over all five chromosomes of At. AtPRIMER uses the sequences to locate SNPs between selected accessions in user-selected regions. In addition to the SNP finder, it integrates a modified dCAPS Finder2.0, a program that generates cleaved amplified polymorphic sequences (CAPS; Konieczny and Ausubel, 1993) and derived CAPS (dCAPS) markers from a SNP (Neff et al., 1998, 2002) and parts of PerlPrimer, a PCR primer design tool (Marshall, 2004).
A CAPS marker is a PCR-based marker in which a restriction site is present in only one of two amplified sequences. This difference can be due to a SNP for example. dCAPS primers differ from CAPS primers in that they contain a mismatch that creates a restriction site in the PCR product of one individual but not the other. After PCR, a step of digestion with a specific restriction endonuclease differentially cleaves the sequences. The differential digestion of both PCR products permits to visualize their polymorphism on agarose gel.
Due to the average size of 
580 bases pairs (bp) of the genotyped fragments and for practical genotyping reasons, the target PCR product size is standardized around 250 bp, while the CAPS or dCAPS primer range from 18 to 24mers. The aim is to get differentially cleaved digestion products of approximately 250 and 230 bp standard size running side by side on agarose gel under standard conditions. These standardized PCR and digestion output sizes enable high-throughput genotyping of the markers generated with AtPRIMER.
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2 OVERVIEW OF THE APPLICATION |
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TOPABSTRACT1 INTRODUCTION2 OVERVIEW OF THE...3 EXPERIMENTAL VALIDATION4 DISCUSSION AND CONCLUSIONACKNOWLEDGEMENTREFERENCES |
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The AtPRIMER tool helps plant geneticists to dissect their trait of interest using natural variation. It facilitates the process of designing polymorphic markers for studying a segregating population derived from a cross. It generates CAPS or dCAPS PCR primer sequences (18–24 nt) designed to amplify a DNA fragment of around 250 bp. On the whole, it aims to increase the efficiency of the generated markers in real lab conditions.
The entry point to the web interface consists of a form in which the user inputs the details of his/her mapping project. The user first selects two different accessions and specifies the genomic region of interest (i.e. the chromosome number and the chromosomal region boundaries). PCR and primer design conditions can be set, including concentrations of reactants and melting temperatures, with standard conditions being selected by default.
A SNP finder script then looks for all sequenced fragments present in the required chromosomal region and selects those that belong to the two chosen accessions. It then compares the two sequences and searches SNPs that are usable for marker design. Location of the SNP on the fragment as well as quality of the surrounding sequence data are important elements of the usable SNPs selection. At this point, the user sees all fragments that are found to be specifically polymorphic in the specified region. The user can then select the fragments that are in particularly interesting locations and/or adjust the marker density to meet his/her mapping objectives.
Upon selection of a fragment, all usable SNPs on the fragment are displayed. The user selects one SNP that the program parses as input to a modified dCAPS Finder2.0. The output of dCAPS Finder2.0 is filtered using a modified PerlPrimer according to primer design specifications. Along the process, CAPS/dCAPS for which the designated enzyme cuts additionally on the predicted PCR primer are also filtered out. Only commercially available enzymes are used by the modified dCAPS Finder2.0. All isoschizomers are also displayed.
Upon choice of a CAPS/dCAPS primer and the cognate restriction enzyme, reverse primers are generated using PerlPrimer. The output is a series of reverse primers ordered based on their GC % and formation of primer-dimers. After the user selects the reverse primer and the corresponding forward primer and enzyme, all three pieces of information and genomic coordinates are passed to a ‘shopping cart’-like window. The user can then go back to other fragments or SNPs on the same fragment to design other markers in the region. The final output is a ‘shopping cart’-like with triplets of forward and reverse primers and restrictions enzymes ordered by their genomic location.
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3 EXPERIMENTAL VALIDATION |
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TOPABSTRACT1 INTRODUCTION2 OVERVIEW OF THE...3 EXPERIMENTAL VALIDATION4 DISCUSSION AND CONCLUSIONACKNOWLEDGEMENTREFERENCES |
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We used AtPRIMER to generate markers polymorphic between accessions Wt-5 and Ct-1, on chromosome 4 on a region spanning from 8.7 to 11.5 Mbp. It retrieved 39 fragments in the region, of which 23 had SNPs usable for generating CAPS/dCAPS. Our goal was to reach a density of one marker every 400 kb in that interval. Based on AtPRIMER results, we ordered nine pairs of primers corresponding to different locations. Eight of the markers amplified successfully from both parents using our default PCR conditions. Out of these, only one failed to appear polymorphic after digestion with the corresponding restriction enzyme. As an example, the marker 9578288_AccII, on fragment 957993 is shown in Figure 1. In total, 7 out of 9 markers could amplify well and be differentially cleaved in our experiment. We later used some of the markers for high-throughput genotyping related to our mapping projects (data not shown). AtPRIMER was run both online on an Opteron-based server (4 GB of RAM) and on a personal computer (Pentium 4 processor, 1 GB of RAM).
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4 DISCUSSION AND CONCLUSION |
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TOPABSTRACT1 INTRODUCTION2 OVERVIEW OF THE...3 EXPERIMENTAL VALIDATION4 DISCUSSION AND CONCLUSIONACKNOWLEDGEMENTREFERENCES |
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AtPRIMER was designed as a user-friendly software where PCR parameters can be set and where all steps of marker design are looked at by the user. This allows the selection of primers and enzymes most efficient in laboratory conditions.
In the post-genomic era, natural variation gains increasing interest. Investigating this variation is of crucial importance for the discovery of new traits and uncovering their genetic basis. For doing so, taking advantage of newly available resources will permit a straightforward and accurate marker design. AtPRIMER was built to help plant geneticists working with reference species A. thaliana through their mapping projects. However, the principle behind AtPRIMER can easily be exported to other plant systems when extensive genotype data on natural variants is generated.
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ACKNOWLEDGEMENT |
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TOPABSTRACT1 INTRODUCTION2 OVERVIEW OF THE...3 EXPERIMENTAL VALIDATION4 DISCUSSION AND CONCLUSIONACKNOWLEDGEMENTREFERENCES |
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This work was supported by the Gatsby Charitable Foundation.
Conflict of Interest: none declared.
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FOOTNOTES |
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Associate Editor: Martin Bishop
Received on August 8, 2007; revised on September 28, 2007; accepted on September 28, 2007
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REFERENCES |
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TOPABSTRACT1 INTRODUCTION2 OVERVIEW OF THE...3 EXPERIMENTAL VALIDATION4 DISCUSSION AND CONCLUSIONACKNOWLEDGEMENTREFERENCES |
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Konieczny A, Ausubel F. A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific markers. Plant J (1993) 4:403–410.[CrossRef][Web of Science][Medline]
Marshall OJ. PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR. Bioinformatics (2004) 20:2471–2472.
Neff MM, et al. dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics. Plant J. (1998) 14:387–392.[CrossRef][Web of Science][Medline]
Neff MM, et al. Web-based primer design for single nucleotide polymorphism analysis. Trends Genet. (2002) 18:613–615.[CrossRef][Web of Science][Medline]
Nordborg M, et al. The pattern of polymorphism in Arabidopsis thaliana. PLoS Biol. (2005) 3:1289–1299.[Web of Science]
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