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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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Identification of a cytochrome P450 family member as a candidate for Spl1. Candidate genes located in the Spl1 region by array hybridization (Figure 4) were screened for SNPs in an EMS-generated mutant showing the spl1 phenotype by TILLING. (a) Detection of heteroduplex by TILLING between DNA for the rice mutant E16923 and wild type parent IR64 PCR products specific for LOC_Os12g16720 (a cytochrome P450 family member). Lanes 1 and 2 are CEL1 treatments of IR64 and E16923 amplicons, respectively. Lane 3 shows the activity of CEL1 enzyme on a heteroduplex generated between IR64 and E16923 amplicons. (b) Sequencing the amplified cytochrome P450 family member from E16923 confirmed the presence of a SNP at position 290 that resulted in a stop codon. Sequence data from two DEB mutants, D1137 and D2943, showing the spl1 phenotype revealed SNPs in LOC_Os12g16720 that caused amino acid changes.
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Figure 5: Identification of a cytochrome P450 family member as a candidate for Spl1. Candidate genes located in the Spl1 region by array hybridization (Figure 4) were screened for SNPs in an EMS-generated mutant showing the spl1 phenotype by TILLING. (a) Detection of heteroduplex by TILLING between DNA for the rice mutant E16923 and wild type parent IR64 PCR products specific for LOC_Os12g16720 (a cytochrome P450 family member). Lanes 1 and 2 are CEL1 treatments of IR64 and E16923 amplicons, respectively. Lane 3 shows the activity of CEL1 enzyme on a heteroduplex generated between IR64 and E16923 amplicons. (b) Sequencing the amplified cytochrome P450 family member from E16923 confirmed the presence of a SNP at position 290 that resulted in a stop codon. Sequence data from two DEB mutants, D1137 and D2943, showing the spl1 phenotype revealed SNPs in LOC_Os12g16720 that caused amino acid changes.

Mentions: Due to their small size, the diepoxybutane-derived (DEB) mutations were not reliably detected by array hybridization. However, they and ethylmethanesulfonate-derived (EMS) mutants were useful for confirming the location of the spl1 gene after delimiting the mutation to a few gene models by array hybridization. TILLING experiments using E16923 (shows spl1 phenotype) focused on gene models within the predicted deletion, and, after sequencing, revealed a point mutation resulting in an in-frame, premature stop codon in the first exon of Os12g16720, a member of the cytochrome P450 gene family (Figure 5). Sequencing of the entire gene from two DEB-derived mutants D1137 and D2943 (confirmed to be spl1 alleles by genetic complementation [25]) also showed single nucleotide polymorphisms (SNPs) in the Os12g16720 gene model. These SNPs were predicted to result in amino acid changes within the gene product that could cause the spl1 lesion mimic phenotype (Figure 5).


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)

Identification of a cytochrome P450 family member as a candidate for Spl1. Candidate genes located in the Spl1 region by array hybridization (Figure 4) were screened for SNPs in an EMS-generated mutant showing the spl1 phenotype by TILLING. (a) Detection of heteroduplex by TILLING between DNA for the rice mutant E16923 and wild type parent IR64 PCR products specific for LOC_Os12g16720 (a cytochrome P450 family member). Lanes 1 and 2 are CEL1 treatments of IR64 and E16923 amplicons, respectively. Lane 3 shows the activity of CEL1 enzyme on a heteroduplex generated between IR64 and E16923 amplicons. (b) Sequencing the amplified cytochrome P450 family member from E16923 confirmed the presence of a SNP at position 290 that resulted in a stop codon. Sequence data from two DEB mutants, D1137 and D2943, showing the spl1 phenotype revealed SNPs in LOC_Os12g16720 that caused amino acid changes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Identification of a cytochrome P450 family member as a candidate for Spl1. Candidate genes located in the Spl1 region by array hybridization (Figure 4) were screened for SNPs in an EMS-generated mutant showing the spl1 phenotype by TILLING. (a) Detection of heteroduplex by TILLING between DNA for the rice mutant E16923 and wild type parent IR64 PCR products specific for LOC_Os12g16720 (a cytochrome P450 family member). Lanes 1 and 2 are CEL1 treatments of IR64 and E16923 amplicons, respectively. Lane 3 shows the activity of CEL1 enzyme on a heteroduplex generated between IR64 and E16923 amplicons. (b) Sequencing the amplified cytochrome P450 family member from E16923 confirmed the presence of a SNP at position 290 that resulted in a stop codon. Sequence data from two DEB mutants, D1137 and D2943, showing the spl1 phenotype revealed SNPs in LOC_Os12g16720 that caused amino acid changes.
Mentions: Due to their small size, the diepoxybutane-derived (DEB) mutations were not reliably detected by array hybridization. However, they and ethylmethanesulfonate-derived (EMS) mutants were useful for confirming the location of the spl1 gene after delimiting the mutation to a few gene models by array hybridization. TILLING experiments using E16923 (shows spl1 phenotype) focused on gene models within the predicted deletion, and, after sequencing, revealed a point mutation resulting in an in-frame, premature stop codon in the first exon of Os12g16720, a member of the cytochrome P450 gene family (Figure 5). Sequencing of the entire gene from two DEB-derived mutants D1137 and D2943 (confirmed to be spl1 alleles by genetic complementation [25]) also showed single nucleotide polymorphisms (SNPs) in the Os12g16720 gene model. These SNPs were predicted to result in amino acid changes within the gene product that could cause the spl1 lesion mimic phenotype (Figure 5).

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