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Detection of genomic deletions in rice using oligonucleotide microarrays.

Bruce M, Hess A, Bai J, Mauleon R, Diaz MG, Sugiyama N, Bordeos A, Wang GL, Leung H, Leach JE - BMC Genomics (2009)

Bottom Line: Deletions ranged in size from one gene model to approximately 500 kb and were predicted on all 12 rice chromosomes.The utility of the technique as a tool in forward genetics was demonstrated in combination with an allelic series of mutants to rapidly narrow the genomic region, and eventually identify a candidate gene responsible for a lesion mimic phenotype.This community resource can continue to grow with further hybridizations, allowing researchers to quickly identify mutants that harbor deletions in candidate genomic regions, for example, regions containing QTL of interest.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO, USA. myron.bruce@colostate.edu

ABSTRACT

Background: The induction of genomic deletions by physical- or chemical- agents is an easy and inexpensive means to generate a genome-saturating collection of mutations. Different mutagens can be selected to ensure a mutant collection with a range of deletion sizes. This would allow identification of mutations in single genes or, alternatively, a deleted group of genes that might collectively govern a trait (e.g., quantitative trait loci, QTL). However, deletion mutants have not been widely used in functional genomics, because the mutated genes are not tagged and therefore, difficult to identify. Here, we present a microarray-based approach to identify deleted genomic regions in rice mutants selected from a large collection generated by gamma ray or fast neutron treatment. Our study focuses not only on the utility of this method for forward genetics, but also its potential as a reverse genetics tool through accumulation of hybridization data for a collection of deletion mutants harboring multiple genetic lesions.

Results: We demonstrate that hybridization of labeled genomic DNA directly onto the Affymetrix Rice GeneChip allows rapid localization of deleted regions in rice mutants. Deletions ranged in size from one gene model to approximately 500 kb and were predicted on all 12 rice chromosomes. The utility of the technique as a tool in forward genetics was demonstrated in combination with an allelic series of mutants to rapidly narrow the genomic region, and eventually identify a candidate gene responsible for a lesion mimic phenotype. Finally, the positions of mutations in 14 mutants were aligned onto the rice pseudomolecules in a user-friendly genome browser to allow for rapid identification of untagged mutations http://irfgc.irri.org/cgi-bin/gbrowse/IR64_deletion_mutants/.

Conclusion: We demonstrate the utility of oligonucleotide arrays to discover deleted genes in rice. The density and distribution of deletions suggests the feasibility of a database saturated with deletions across the rice genome. This community resource can continue to grow with further hybridizations, allowing researchers to quickly identify mutants that harbor deletions in candidate genomic regions, for example, regions containing QTL of interest.

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Mutant line d1 contains a ~500 kb deletion on chromosome 5 encompassing the RGA1 gene. a) Gene models in the region show a high percentage of probes with log2(mutant probe intensity/wild type probe intensity) ≤ -0.8, indicating a large deletion. b) PCR confirmation of the deletion of RGA1 (Os05g26890) relative to wild type (indicated by an open arrowhead in part a) and PCR confirmation of the right border of the deletion (Os05g26990) relative to wild type (indicated by a closed arrowhead in part a). The left border was not resolved.
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Figure 2: Mutant line d1 contains a ~500 kb deletion on chromosome 5 encompassing the RGA1 gene. a) Gene models in the region show a high percentage of probes with log2(mutant probe intensity/wild type probe intensity) ≤ -0.8, indicating a large deletion. b) PCR confirmation of the deletion of RGA1 (Os05g26890) relative to wild type (indicated by an open arrowhead in part a) and PCR confirmation of the right border of the deletion (Os05g26990) relative to wild type (indicated by a closed arrowhead in part a). The left border was not resolved.

Mentions: As an example of the process for detecting deletions, we hybridized genomic DNA from a rice dwarf mutant d1 with a known deletion in the single copy RGA1 gene, previously shown to be responsible for the dwarf phenotype [23], to a single array. The mutation was induced by gamma radiation and confirmed using PCR and DNA blot analysis (data not shown). We predicted a deletion on chromosome 5 that contains the gene model Os05g26890, the RGA1 gene (Figure 2). Nine of eleven probes in the Os05g26890 probe set showed a log ratio ≤ -0.8, or a proportion of 0.82, identifying RGA1 as deleted.


Detection of genomic deletions in rice using oligonucleotide microarrays.

Bruce M, Hess A, Bai J, Mauleon R, Diaz MG, Sugiyama N, Bordeos A, Wang GL, Leung H, Leach JE - BMC Genomics (2009)

Mutant line d1 contains a ~500 kb deletion on chromosome 5 encompassing the RGA1 gene. a) Gene models in the region show a high percentage of probes with log2(mutant probe intensity/wild type probe intensity) ≤ -0.8, indicating a large deletion. b) PCR confirmation of the deletion of RGA1 (Os05g26890) relative to wild type (indicated by an open arrowhead in part a) and PCR confirmation of the right border of the deletion (Os05g26990) relative to wild type (indicated by a closed arrowhead in part a). The left border was not resolved.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666768&req=5

Figure 2: Mutant line d1 contains a ~500 kb deletion on chromosome 5 encompassing the RGA1 gene. a) Gene models in the region show a high percentage of probes with log2(mutant probe intensity/wild type probe intensity) ≤ -0.8, indicating a large deletion. b) PCR confirmation of the deletion of RGA1 (Os05g26890) relative to wild type (indicated by an open arrowhead in part a) and PCR confirmation of the right border of the deletion (Os05g26990) relative to wild type (indicated by a closed arrowhead in part a). The left border was not resolved.
Mentions: As an example of the process for detecting deletions, we hybridized genomic DNA from a rice dwarf mutant d1 with a known deletion in the single copy RGA1 gene, previously shown to be responsible for the dwarf phenotype [23], to a single array. The mutation was induced by gamma radiation and confirmed using PCR and DNA blot analysis (data not shown). We predicted a deletion on chromosome 5 that contains the gene model Os05g26890, the RGA1 gene (Figure 2). Nine of eleven probes in the Os05g26890 probe set showed a log ratio ≤ -0.8, or a proportion of 0.82, identifying RGA1 as deleted.

Bottom Line: Deletions ranged in size from one gene model to approximately 500 kb and were predicted on all 12 rice chromosomes.The utility of the technique as a tool in forward genetics was demonstrated in combination with an allelic series of mutants to rapidly narrow the genomic region, and eventually identify a candidate gene responsible for a lesion mimic phenotype.This community resource can continue to grow with further hybridizations, allowing researchers to quickly identify mutants that harbor deletions in candidate genomic regions, for example, regions containing QTL of interest.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO, USA. myron.bruce@colostate.edu

ABSTRACT

Background: The induction of genomic deletions by physical- or chemical- agents is an easy and inexpensive means to generate a genome-saturating collection of mutations. Different mutagens can be selected to ensure a mutant collection with a range of deletion sizes. This would allow identification of mutations in single genes or, alternatively, a deleted group of genes that might collectively govern a trait (e.g., quantitative trait loci, QTL). However, deletion mutants have not been widely used in functional genomics, because the mutated genes are not tagged and therefore, difficult to identify. Here, we present a microarray-based approach to identify deleted genomic regions in rice mutants selected from a large collection generated by gamma ray or fast neutron treatment. Our study focuses not only on the utility of this method for forward genetics, but also its potential as a reverse genetics tool through accumulation of hybridization data for a collection of deletion mutants harboring multiple genetic lesions.

Results: We demonstrate that hybridization of labeled genomic DNA directly onto the Affymetrix Rice GeneChip allows rapid localization of deleted regions in rice mutants. Deletions ranged in size from one gene model to approximately 500 kb and were predicted on all 12 rice chromosomes. The utility of the technique as a tool in forward genetics was demonstrated in combination with an allelic series of mutants to rapidly narrow the genomic region, and eventually identify a candidate gene responsible for a lesion mimic phenotype. Finally, the positions of mutations in 14 mutants were aligned onto the rice pseudomolecules in a user-friendly genome browser to allow for rapid identification of untagged mutations http://irfgc.irri.org/cgi-bin/gbrowse/IR64_deletion_mutants/.

Conclusion: We demonstrate the utility of oligonucleotide arrays to discover deleted genes in rice. The density and distribution of deletions suggests the feasibility of a database saturated with deletions across the rice genome. This community resource can continue to grow with further hybridizations, allowing researchers to quickly identify mutants that harbor deletions in candidate genomic regions, for example, regions containing QTL of interest.

Show MeSH