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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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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.
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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.

Mentions: In order to confirm the SFPs detected in the Saltol region, we examined the SFP locations by amplicon sequencing. Alignments of the amplicon sequences are shown in Figure 5. For probe set Os.55011.1.S1_x_at, which corresponds to gene model LOC_Os01g20120, one SNP was found in the amplicon sequence at the SFP probe position and the FL478 allele was the same as in the Pokkali genotype. These results confirmed the SFP detection data, which suggested that FL478 contains a Pokkali-derived gene near SKC1 (LOC_Os01g20160). To further examine this region we checked additional genes in the vicinity of LOC_Os01g20120. We found that three additional genes (LOC_Os01g19220, LOC_Os01g19400, and LOC_Os01g20160 [SKC1]) within a < 1 Mb segment of FL478 also are of a non-IR29 origination (Figure 6). Taken together, it appears that FL478 contains a small non-IR29 haplotype block including SKC1 (Figure 4C), which we did not detect previously. We could not detect a SFP in the SKC1 gene in either the previous work or the present study because the expression level from the probe set (Os.30563.1.S1_at) for SKC1 was not "present" in all expression datasets, which is a requirement of our statistical filtering method. The SKC1 sequences are shown in Figure 6C. Surprisingly, in an apparently highly variable region, FL478 contains a haplotype that is not identical to either of the presumed parents. We confirmed this by sequencing amplicons from independent reactions from each genotype, making use of high fidelity Taq polymerase (Platinum pfx DNA polymerase, Invitrogen, USA). The existence in FL478 of an allele that matches neither IR29 nor the genotype which we know as Pokkali could be explained by either parent being genetically not uniform when the crosses to make RILs including FL478 were made. This notion is consistent with records now showing that there are actually at least eight distinct accessions named Pokkali in the germplasm collection at International Rice Research Institute .


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)

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

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

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.
Mentions: In order to confirm the SFPs detected in the Saltol region, we examined the SFP locations by amplicon sequencing. Alignments of the amplicon sequences are shown in Figure 5. For probe set Os.55011.1.S1_x_at, which corresponds to gene model LOC_Os01g20120, one SNP was found in the amplicon sequence at the SFP probe position and the FL478 allele was the same as in the Pokkali genotype. These results confirmed the SFP detection data, which suggested that FL478 contains a Pokkali-derived gene near SKC1 (LOC_Os01g20160). To further examine this region we checked additional genes in the vicinity of LOC_Os01g20120. We found that three additional genes (LOC_Os01g19220, LOC_Os01g19400, and LOC_Os01g20160 [SKC1]) within a < 1 Mb segment of FL478 also are of a non-IR29 origination (Figure 6). Taken together, it appears that FL478 contains a small non-IR29 haplotype block including SKC1 (Figure 4C), which we did not detect previously. We could not detect a SFP in the SKC1 gene in either the previous work or the present study because the expression level from the probe set (Os.30563.1.S1_at) for SKC1 was not "present" in all expression datasets, which is a requirement of our statistical filtering method. The SKC1 sequences are shown in Figure 6C. Surprisingly, in an apparently highly variable region, FL478 contains a haplotype that is not identical to either of the presumed parents. We confirmed this by sequencing amplicons from independent reactions from each genotype, making use of high fidelity Taq polymerase (Platinum pfx DNA polymerase, Invitrogen, USA). The existence in FL478 of an allele that matches neither IR29 nor the genotype which we know as Pokkali could be explained by either parent being genetically not uniform when the crosses to make RILs including FL478 were made. This notion is consistent with records now showing that there are actually at least eight distinct accessions named Pokkali in the germplasm collection at International Rice Research Institute .

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