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

Discovery of molecular markers has facilitated mapping of both qualitative and quantitative traits. Tightly linked molecular markers facilitate (i) isolation of the genes encoding these traits and (ii) selection of genotypes carrying the desirable alleles. Several molecular marker technologies such as, RFLP, RAPD, DAF, SSR, SSLP, AFLP, CAPS, SNP have been discovered for molecular mapping experiments [1–6]. Fingerprinting of genotypes for restriction fragment length polymorphisms (RFLPs) has been regarded as the most sensitive method of genotyping. This procedure, however, requires a large quantity of genomic DNA and use of radioactive probes. In 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]. In DNA amplification fingerprinting (DAF), many loci are PCR amplified with the aid of a single, short arbitrary primer, as short as 5-nucleotides long [4]. Simple sequence repeat (SSR) markers, also known as microsatellite markers, utilize the variation for tandem repeats such as (CA)_n repeats observed between genotypes [7]. 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. In Arabidopsis, SSLPs are largely based on the (GA)_n repeats [8]. 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].

The high-throughput amplified fragment length polymorphism (AFLP) marker technology combines principles of RFLP and random PCR amplification for rapid identification of molecular loci of the entire genome [1]. AFLP technology is particularly suitable for developing high density molecular marker maps, essential for both map-based cloning of genes and the isolation of molecular markers for selecting desirable genotypes in breeding programs. AFLP technology identifies molecular markers based on a fraction of the restriction fragment length polymorphisms between two genotypes. Restriction site associated DNA (RAD) marker technology, on the other hand, generates markers for all polymorphic sites of a restriction endonuclease between two genotypes; and thus, it is a very sensitive marker technology for developing a high density molecular map [9].

Polymorphisms detected by various marker technologies have been used to generate molecular marker maps of those species that do not have any genome sequences and physical maps. Since assembled genome sequence of many species are available, and the cost of sequencing has declined significantly with advent of the next generation sequencing technologies, single nucleotide polymorphism (SNP) is becoming the most popular molecular marker [10–12]. However, SNP assays are not always simple or flexible. Here, a strategy of using SNPs for rapid generation of molecular markers, termed sequence based polymorphic (SBP) marker technology, is described.

The assembled Arabidopsis thaliana genome sequence is selected for this study [13]. Many of the ecotypes of this species are available and have been used in mapping experiments to conduct genetic and biological studies. SNPs among some of the accessions or ecotypes of this model plant species are available [14-15; at http://www.arabidopsis.org]. Niederzenz-0 (Nd-0), used for mapping the Phytophthora sojae s usceptible (pss) mutants that are infected by the soybean pathogen, P. sojae (R. Sumit, B.B. Sahu and M.K. Bhattacharyya, unpublished), was selected for this study. The pss mutants were created in the pen1-1 mutant of the ecotype, Columbia-0 (Col-0). To facilitate mapping of the putative PSS gene loci conferring nonhost resistance of Arabidopsis against P. sojae, SBP markers were developed as follows. Seventy-five nucleotide long sequencing reads obtained by conducting Solexa sequencing of the Nd-0 genome were compared to Col-0 sequences to identify the SNPs, which were subsequently converted to SBP markers if either of the ecotypes was cut by at least one restriction endonuclease at the SNP sites. By applying this technology, 21 co-dominant SBP markers were generated for the marker-poor regions of the Arabidopsis genome. This novel SBP marker technology should be applicable to any higher eukaryotic species with assembled genome sequences for rapid development of high density molecular marker maps for map-based cloning of genes or identification of suitable molecular markers for selection of desirable genotypes in breeding programs.

The use of molecular markers has gained importance in genetic studies particularly for map based cloning of genes [17]. The relatively low cost of sequencing a genome, with the emergence of high throughput sequencing technology, has facilitated genome wide polymorphism studies [18, 19]. The SBP marker technology can convert most of the single nucleotide polymorphisms to molecular markers for any genomic regions. SBP markers developed based on sequence information are ideal for those species, whose genomes are sequenced and assembled. Reference genome sequence can be utilized to develop SBP markers for a specific genomic region with known physical location. Thus, marker-poor regions can be enriched with SBP markers. In this study, the applicability of the SBP marker technology for generating markers is shown for improving a genetic map that represents polymorphisms between two Arabidopsis ecotypes, Col-0 and Nd-0 (Figure 4). SBP markers were generated from just three genome equivalents Nd-0 genome sequence of 75 bp Solexa reads. The method also has been successfully applied in developing a high density molecular map of the PSS1 gene that confers nonhost resistance against the soybean pathogens, Phytophthora sojae and Fusarium virguliforme (R. Sumit, B.B. Sahu and M.K. Bhattacharyya, unpublished).

The SHORE program used in this study is highly powerful and has been employed successfully in identification of a mutation through analysis of deep sequence data of a bulk of 500 mutant F₂ progenies [20]. If the genome sequencing is not conducted to a higher depth (e.g. ≥ 20 fold), SNPs identified through SHORE analyses can be verified by conducting BLAST analyses. Staggered Solexa sequence reads (Figure 2) containing SNPs are considered for generating SBP markers for such a scenario. Similarly, candidate SNP containing regions of the reference genome should be supported by multiple sequences, such as transcript sequences and/or sequences from more than one BAC clone to avoid any possible sequencing errors (Additional file 4).

If none of the haplotypes of interest are sequenced, then reference genome sequence should be used to define the SNP maps of individual haplotypes by running the SHORE program. The SNP maps then can be compared to determine the SNPs between the haplotypes of interest. Once the candidate SNPs are identified, small PCR amplicons of ~ 200 bp can be amplified and digested with suitable restriction endonuceases to release the restriction length polymorphisms. A significant proportion of the SNPs could be unusable in SBP marker development because they may not be digested with restriction endonucleases in a haplotype- or genotype-specific manner. In such a case, one can apply d erived CAPS (dCAPS) technology to improve the efficiency of SBP marker development [21].

A new molecular marker technology, based on genome sequence and physical map locations, is reported for those species whose assembled genome sequences are available. The technology was applied in identifying 21 SBP markers for some of the marker-poor genomic regions of the Arabidopsis molecular marker map that represent polymorphisms between ecotypes, Col-0 and Nd-0 (Figure 4). The SBP marker technology should be applicable to any genomic regions and will facilitate (i) map-based cloning genes as well as (ii) the development of tightly linked molecular markers for selecting desirable genotypes in animal and plant breeding experiments.

Ease in SBP marker development and application to any genomic regions, and genome-wide abundance of SNPs make this technology suitable for mapping experiments, especially to develop high density molecular maps for positional gene cloning experiments, if the assembled genome sequence and physical maps of the studied species are available. Innumerable SBP markers can be developed rapidly for a genomic region containing a target gene in a map-based cloning experiment. Co-dominant gel-based SBP markers are ideal to identify genetic recombination events between two loci. Such PCR-based markers can be used to screen a large number of segregants to identify informative recombinants of the target gene region. These recombinants will then facilitate the development of high resolution maps of a large number of SBP markers, essential for cloning genes based on their map position. Thus, high-throughput deep sequencing, together with SBP markers, should expedite map-based cloning in higher eukaryotes.