Abstract
BackgroundTechnological advances are progressively increasing the application of genomics to a wider array of economically and ecologically important species. High-density maps enriched for transcribed genes facilitate the discovery of connections between genes and phenotypes. We report the construction of a high-density linkage map of expressed genes for the heterozygous genome of Eucalyptus using Single Feature Polymorphism (SFP) markers.ResultsSFP discovery and mapping was achieved using pseudo-testcross screening and selective mapping to simultaneously optimize linkage mapping and microarray costs. SFP genotyping was carried out by hybridizing complementary RNA prepared from 4.5 year-old trees xylem to an SFP array containing 103,000 25-mer oligonucleotide probes representing 20,726 unigenes derived from a modest size expressed sequence tags collection. An SFP-mapping microarray with 43,777 selected candidate SFP probes representing 15,698 genes was subsequently designed and used to genotype SFPs in a larger subset of the segregating population drawn by selective mapping. A total of 1,845 genes were mapped, with 884 of them ordered with high likelihood support on a framework map anchored to 180 microsatellites with average density of 1.2 cM. Using more probes per unigene increased by two-fold the likelihood of detecting segregating SFPs eventually resulting in more genes mapped. In silico validation showed that 87% of the SFPs map to the expected location on the 4.5X draft sequence of the Eucalyptus grandis genome.ConclusionsThe Eucalyptus 1,845 gene map is the most highly enriched map for transcriptional information for any forest tree species to date. It represents a major improvement on the number of genes previously positioned on Eucalyptus maps and provides an initial glimpse at the gene space for this global tree genome. A general protocol is proposed to build high-density transcript linkage maps in less characterized plant species by SFP genotyping with a concurrent objective of reducing microarray costs. HIgh-density gene-rich maps represent a powerful resource to assist gene discovery endeavors when used in combination with QTL and association mapping and should be especially valuable to assist the assembly of reference genome sequences soon to come for several plant and animal species.
Highlights
Technological advances are progressively increasing the application of genomics to a wider array of economically and ecologically important species
Note that in a perfect scenario an expressed transcript would hybridize to all the probes of its corresponding probe set. This indicates that detectable sequence polymorphisms between the short oligonucleotide probes and the hybridized transcripts are the rule rather than the exception
The 103,000-probe Single Feature Polymorphism (SFP)-discovery oligonucleotide array used in this study was built from a relatively small expressed sequence tags (ESTs) data set for current standards: 88,000 phred-20 filtered 5’-Sanger-sequenced ESTs assembled in 21,428 unique sequences
Summary
Technological advances are progressively increasing the application of genomics to a wider array of economically and ecologically important species. We report the construction of a high-density linkage map of expressed genes for the heterozygous genome of Eucalyptus using Single Feature Polymorphism (SFP) markers. High-density linkage maps based on transcribed genes are valuable resources to characterize genome structure, gene space distribution and synteny in species for which reference genome sequences are not yet available. High-throughput genotyping of single-nucleotide polymorphism (SNP) derived from genic sequences have allowed large-scale gene mapping. Current SNP assay methods based on allele-specific primer extension can be of variable robustness in highly heterozygous genomes such as those of forest trees with high nucleotide diversity, frequently above 1% [12]
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