Detection and validation of single feature polymorphisms using RNA expression data from a rice genome array

Abstract

Background: A large number of genetic variations have been identified in rice. Such variations must in many cases control phenotypic differences in abiotic stress tolerance and other traits. A single feature polymorphism (SFP) is an oligonucleotide array-based polymorphism which can be used for identification of SNPs or insertion/deletions (INDELs) for high throughput genotyping and high density mapping. Here we applied SFP markers to a lingering question about the source of salt tolerance in a particular rice recombinant inbred line (RIL) derived from a salt tolerant and salt sensitive parent.

Results: Expression data obtained by hybridizing RNA to an oligonucleotide array were analyzed using a statistical method called robustified projection pursuit (RPP). By applying the RPP method, a total of 1208 SFP probes were detected between two presumed parental genotypes (Pokkali and IR29) of a RIL population segregating for salt tolerance. We focused on the Saltol region, a major salt tolerance QTL. Analysis of FL478, a salt tolerant RIL, revealed a small (< 1 Mb) region carrying alleles from the presumed salt tolerant parent, flanked by alleles matching the salt sensitive parent IR29. Sequencing of putative SFP-containing amplicons from this region and other positions in the genome yielded a validation rate more than 95%.

Conclusion: Recombinant inbred line FL478 contains a small (< 1 Mb) segment from the salt tolerant parent in the Saltol region. The Affymetrix rice genome array provides a satisfactory platform for high resolution mapping in rice using RNA hybridization and the RPP method of SFP analysis.

Figure 1

Rice pseudomolecule map showing positions of SFPs detected in this study. SFPs in FL478 detected as Pokkali or IR29 haplotype by RPP method are shown in squares (pink) and triangles (yellow), respectively. Stars and vertical bars indicate the positions of the centromeres and the ends of chromosomes, respectively. Horizontal bar (blue) means the Saltol region.

Figure 2

SFP detection in a probe set by RPP method. (Left panel) Plots of the log intensities (PM, perfect match) for the representative probe set (Os.33510.1.S2_at) from three genotypes. (Middle panel) Plots of the differentiations of average log intensities among three genotypes. (Right panel) Plots of individual outlying scores. P, Pokkali; I, IR29; F, FL478. After Cui et al. (2005) [2].

Figure 3

Nucleotide sequence alignment of amplicon sequences of a probe set. Polymorphic residues are highlighted in gray. Bars 0–10 indicate the positions of eleven probes in the probe set (Os.33510.1.S2_at). The position of SFP probe number 4 detected by the RPP method is double-underlined. Arrows indicate SNPs. P, Pokkali; I, IR29; F, FL478; S, target sequence from SIF.

Figure 4

Chromosome 1 segment associated with a major QTL for salt tolerance. Genetic linkage maps showing the location of Saltol described by Lin et al. (2004) [11] and Bonilla et al. (2002) [8] are shown in (A) and (B), respectively. (C) The segment of pseudomolecule map showing the physical positions of the SKC1 gene [12] and loci with SFPs in the Saltol region. Numbers in parentheses indicate physical positions (Mb) on chromosome 1.

Figure 5

Alignments of SFPs in the Saltol region. Polymorphic residues are highlighted in gray. The locus corresponding to each probe set is indicated in parentheses. Arrows indicate SNPs. Bar, INDEL. P, Pokkali; I, IR29; F, FL478; S, target sequence from SIF.

Figure 6

Alignments of amplicon sequences of genes in a small segment of the Saltol region from the non-IR29 parent. Polymorphic residues of (A) LOC_Os01g19220, (B) LOC_Os01g19400 and (C) LOC_Os01g20160 (SKC1 gene) are highlighted in gray. Arrows indicate SNPs or INDEL. P, Pokkali; I, IR29; F, FL478.