Abstract
BackgroundMolecular markers facilitate both genotype identification, essential for modern animal and plant breeding, and the isolation of genes based on their map positions. Advancements in sequencing technology have made possible the identification of single nucleotide polymorphisms (SNPs) for any genomic regions. Here a sequence based polymorphic (SBP) marker technology for generating molecular markers for targeted genomic regions in Arabidopsis is described.ResultsA ~3X genome coverage sequence of the Arabidopsis thaliana ecotype, Niederzenz (Nd-0) was obtained by applying Illumina's sequencing by synthesis (Solexa) technology. Comparison of the Nd-0 genome sequence with the assembled Columbia-0 (Col-0) genome sequence identified putative single nucleotide polymorphisms (SNPs) throughout the entire genome. Multiple 75 base pair Nd-0 sequence reads containing SNPs and originating from individual genomic DNA molecules were the basis for developing co-dominant SBP markers. SNPs containing Col-0 sequences, supported by transcript sequences or sequences from multiple BAC clones, were compared to the respective Nd-0 sequences to identify possible restriction endonuclease enzyme site variations. Small amplicons, PCR amplified from both ecotypes, were digested with suitable restriction enzymes and resolved on a gel to reveal the sequence based polymorphisms. By applying this technology, 21 SBP markers for the marker poor regions of the Arabidopsis map representing polymorphisms between Col-0 and Nd-0 ecotypes were generated.ConclusionsThe SBP marker technology described here allowed the development of molecular markers for targeted genomic regions of Arabidopsis. It should facilitate isolation of co-dominant molecular markers for targeted genomic regions of any animal or plant species, whose genomic sequences have been assembled. This technology will particularly facilitate the development of high density molecular marker maps, essential for cloning genes based on their genetic map positions and identifying tightly linked molecular markers for selecting desirable genotypes in animal and plant breeding experiments.
Highlights
Molecular markers facilitate both genotype identification, essential for modern animal and plant breeding, and the isolation of genes based on their map positions
Generation of a global molecular map for the polymorphic loci of the Arabidopsis thaliana ecotypes, Col-0 and Nd-0 Arabidopsis is a nonhost for the soybean pathogen, Phytophthora sojae
In order to map the putative PSS genes that confer nonhost resistance of Arabidopsis against the soybean pathogen, P. sojae, a global map of the Simple sequence length polymorphism (SSLP) and Cleaved amplified polymorphic sequences (CAPS) markers that are polymorphic between ecotypes, Col-0 and Nd-0 was generated
Summary
Molecular markers facilitate both genotype identification, essential for modern animal and plant breeding, and the isolation of genes based on their map positions. Advancements in sequencing technology have made possible the identification of single nucleotide polymorphisms (SNPs) for any genomic regions. A sequence based polymorphic (SBP) marker technology for generating molecular markers for targeted genomic regions in Arabidopsis is described. Linked molecular markers facilitate (i) isolation of the genes encoding these traits and (ii) selection of genotypes the random amplified polymorphic DNA (RAPD) marker technology, multiple random loci of the genomes are PCR amplified with a single, 10 nucleotide long primer of arbitrary sequence [3]. Simple sequence length polymorphism (SSLP) markers, similar to SSR markers, are designed based on a unique segment of genomic DNA sequence that contains a simple tandem repeat that distinguishes the genotypes. Cleaved amplified polymorphic sequences (CAPS) markers are designed based on restriction fragment length polymorphisms of PCR amplified fragments, when sequence information of one of the haplotypes is unknown [6]
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