AN INTEGRATED DATABASE AND BIOINFORMATICS RESOURCE FOR SMALL GRAINS
Location: Genomics and Gene Discovery
Title: ConservedPrimers 2.0: A high-throughput pipeline for comparative genome referenced intron-flanking PCR primer design and its application in wheat SNP discovery
Submitted to: BMC Bioinformatics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 13, 2009
Publication Date: October 13, 2009
Citation: You, F., Huo, N., Gu, Y.Q., Lazo, G.R., Dvorak, J., Anderson, O.D. 2009. ConservedPrimers 2.0: A high-throughput pipeline for comparative genome referenced intron-flanking PCR primer design and its application in wheat SNP discovery. BMC Bioinformatics. 10:331.
Single nucleotide polymorphisms (SNPs) are a valuable marker system with many genetic and genomics applications. Wheat (Triticum aestivum L., 2n = 6x = 42) is an hexaploid with the A, B and D genomes. It was formed through hybridization of three diploid species, Triticum urartu (AA), Aegilops tauschii (DD), and a wild diploid species thought to be extinct and closely related to Aegilops speltoides (SS). For SNP discovery in wheat using a PCR-based approach, the first step is to design PCR primers that can be used to amplify specific genomic regions. In this work, a web-based tool, ConservedPrimers 2.0, was develop to facilitate the identification of conserved regions for primer design, therefore, increasing the success of PCR amplification. The web-based tool will be practical applications in laboratories working on molecular marker development.
In some genomic applications it is necessary to design large numbers of PCR primers in exons flanking one or several introns on the basis of orthologous gene sequences in related species. The primer pairs designed by this target gene approach are called "intron-flanking primers" or because they are located in exonic sequences which are usually conserved between related species, "conserved primers". They are useful for large scale single nucleotide polymorphism (SNP) discovery and marker development, especially in species, such as wheat, for which a large number of ESTs are available but for which genome sequences and intron/exon boundaries are not available. To date, no suitable high-throughput tool is available for this purpose. We have developed, the ConservedPrimers 2.0 pipeline, for designing intron-flanking primers for large scale SNP discovery and marker development, and demonstrated its utility in wheat. This tool uses non-redundant wheat EST sequences, such as wheat contigs and singleton ESTs, and related genomic sequences, such as those of rice, as inputs. It aligns the ESTs to the genomic sequences to identify unique colinear exon blocks and predicts intron lengths. Intron-flanking primers are then designed based on the intron/exon information using the Primer3 core program or BatchPrimer3. Finally, a tab-delimited file containing intron-flanking primer pair sequences and their primer properties is generated for primer ordering and their PCR applications. Using this tool, 1,922 bin mapped wheat ESTs (31.8% of the 6,045 in total) were found to have unique colinear exon blocks suitable for primer design and 1,821 primer pairs were designed from these single- or low-copy genes for PCR amplification and SNP discovery. With these primers and subsequently designed genome-specific primers, a total of 1,527 loci were found to contain one or more genome-specific SNPs. The ConservedPrimers 2.0 pipeline for designing intron-flanking primers was developed and its utility demonstrated. The tool can be used for SNP discovery, genetic variation assays and marker development for any target genome that has abundant ESTs and a related reference genome that has been fully sequenced. The ConservedPrimers 2.0 pipeline has been implemented as a command-line tool as well as a web application. Both versions are freely available at http://wheat.pw.usda.gov/demos/ConservedPrimers/.