Abstract
BackgroundGenetic markers are tools that can facilitate molecular breeding, even in species lacking genomic resources. An important class of genetic markers is those based on orthologous genes, because they can guide hypotheses about conserved gene function, a situation that is well documented for a number of agronomic traits. For under-studied species a key bottleneck in gene-based marker development is the need to develop molecular tools (e.g., oligonucleotide primers) that reliably access genes with orthology to the genomes of well-characterized reference species.ResultsHere we report an efficient platform for the design of cross-species gene-derived markers in legumes. The automated platform, named CSGM Designer (URL: http://tgil.donga.ac.kr/CSGMdesigner), facilitates rapid and systematic design of cross-species genic markers. The underlying database is composed of genome data from five legume species whose genomes are substantially characterized. Use of CSGM is enhanced by graphical displays of query results, which we describe as “circular viewer” and “search-within-results” functions. CSGM provides a virtual PCR representation (eHT-PCR) that predicts the specificity of each primer pair simultaneously in multiple genomes. CSGM Designer output was experimentally validated for the amplification of orthologous genes using 16 genotypes representing 12 crop and model legume species, distributed among the galegoid and phaseoloid clades. Successful cross-species amplification was obtained for 85.3% of PCR primer combinations.ConclusionCSGM Designer spans the divide between well-characterized crop and model legume species and their less well-characterized relatives. The outcome is PCR primers that target highly conserved genes for polymorphism discovery, enabling functional inferences and ultimately facilitating trait-associated molecular breeding.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-015-0074-6) contains supplementary material, which is available to authorized users.
Highlights
Genetic markers are tools that can facilitate molecular breeding, even in species lacking genomic resources
Cross-species genic marker (CSGM) Designer focuses on PCR primer design within exon regions, where nucleotide sequence divergence is typically more constrained than non-protein coding regions
To accommodate the need for high rates of polymorphism within crop germplasm, which is of low genetic diversity, CSGM Designer identifies primer pairs that amplify across less constrained intron sequences, enhancing the success rate of marker development
Summary
Genetic markers are tools that can facilitate molecular breeding, even in species lacking genomic resources. For under-studied species a key bottleneck in gene-based marker development is the need to develop molecular tools (e.g., oligonucleotide primers) that reliably access genes with orthology to the genomes of well-characterized reference species. Genetic markers that target orthologous genes are useful, because they permit cross-species inferences (e.g., of structural and conservation), and this approach has been used widely in legumes [3] and other species. On the one hand such information provides the basis of systematic identification of orthologous genes and corresponding tools (e.g., PCR primers), while on the other hand doing so requires organized bioinformatics activities not available for many small research groups working on under-characterized species. Of particular interest are cross-species gene-targeted markers, which have utility in translating marker information across different crop plants, such markers can be technically difficult to design
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