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Open Access Highly Accessed Methodology article

Sequence based polymorphic (SBP) marker technology for targeted genomic regions: its application in generating a molecular map of the Arabidopsis thaliana genome

Binod B Sahu1, Rishi Sumit12, Subodh K Srivastava1 and Madan K Bhattacharyya12*

Author Affiliations

1 Department of Agronomy, Iowa State University, Ames, Iowa 50011, USA

2 Molecular, Cellular and Developmental Biology Interdepartmental Graduate Program, Iowa State University, Ames, Iowa 5001, USA

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BMC Genomics 2012, 13:20  doi:10.1186/1471-2164-13-20

Published: 13 January 2012

Abstract

Background

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. Here a sequence based polymorphic (SBP) marker technology for generating molecular markers for targeted genomic regions in Arabidopsis is described.

Results

A ~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.

Conclusions

The 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.

Keywords:
Niederzenz; Solexa sequencing; sequence based polymorphic marker; nonhost resistance; Phytophthora sojae; SHORE analysis