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

Kim SH, Bhat PR, Cui X, Walia H, Xu J, Wanamaker S, Ismail AM, Wilson C, Close TJ - BMC Plant Biol. (2009)

Bottom Line: Sequencing of putative SFP-containing amplicons from this region and other positions in the genome yielded a validation rate more than 95%.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Botany and Plant Sciences, University of California, Riverside, CA 92521 USA. kshpaulo@yahoo.co.kr

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.

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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.
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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.

Mentions: By applying higher density SFP analysis than previously, a total of 1208 SFP probes were detected in the present analysis (Figure 1, Additional file 1). Plots of the log intensities, affinity differences and individual outlying scores for a representative probe set (Os.33510.1.S2_at) are shown in Figure 2. The intensity differentiation between Pokkali and FL478 is highest at probes 4 and 3, indicating polymorphism at these probe positions. A representative alignment of the amplicon sequences with the target sequence of Os.33510.1.S2_at probe set is shown in Figure 3. Several SNPs were detected, but only probe positions 3 and 4 span a SNP. Probe 4 was selected as a SFP by the RPP method based on a higher outlying score than that of probe 3 (Figure 2).


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

Kim SH, Bhat PR, Cui X, Walia H, Xu J, Wanamaker S, Ismail AM, Wilson C, Close TJ - BMC Plant Biol. (2009)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2697985&req=5

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.
Mentions: By applying higher density SFP analysis than previously, a total of 1208 SFP probes were detected in the present analysis (Figure 1, Additional file 1). Plots of the log intensities, affinity differences and individual outlying scores for a representative probe set (Os.33510.1.S2_at) are shown in Figure 2. The intensity differentiation between Pokkali and FL478 is highest at probes 4 and 3, indicating polymorphism at these probe positions. A representative alignment of the amplicon sequences with the target sequence of Os.33510.1.S2_at probe set is shown in Figure 3. Several SNPs were detected, but only probe positions 3 and 4 span a SNP. Probe 4 was selected as a SFP by the RPP method based on a higher outlying score than that of probe 3 (Figure 2).

Bottom Line: Sequencing of putative SFP-containing amplicons from this region and other positions in the genome yielded a validation rate more than 95%.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Botany and Plant Sciences, University of California, Riverside, CA 92521 USA. kshpaulo@yahoo.co.kr

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.

Show MeSH