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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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Array-based deletion discovery identifies allelic relationships among spl1 mutants. Hybridization of genomic DNA from two confirmed allelic spl1 mutants (G650 and F1856) and two mutants showing the distinctive spl1 lesion mimic phenotype (G9799 and F2045) identified overlapping deletions in all four lines on chromosome 12. A log ratio cutoff of ≤ -0.8 for 50% or more of probes in a probe set was used. Open arrowheads indicate deletions in gene models confirmed by PCR. Closed arrowheads indicate gene models confirmed to be present by PCR.
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Figure 4: Array-based deletion discovery identifies allelic relationships among spl1 mutants. Hybridization of genomic DNA from two confirmed allelic spl1 mutants (G650 and F1856) and two mutants showing the distinctive spl1 lesion mimic phenotype (G9799 and F2045) identified overlapping deletions in all four lines on chromosome 12. A log ratio cutoff of ≤ -0.8 for 50% or more of probes in a probe set was used. Open arrowheads indicate deletions in gene models confirmed by PCR. Closed arrowheads indicate gene models confirmed to be present by PCR.

Mentions: The rice mutant lines induced by chemical or irradiation strategies likely contained multiple deletions in the genome. We tested if hybridization of DNA from multiple mutant lines that exhibit a phenotype of interest could provide convergent data to identify the mutated region responsible for the phenotype and limit that region to the fewest gene models. Four mutants exhibiting the spl1 lesion mimic phenotype were selected as proof of concept. Two mutants had been genetically confirmed by complementation testing to be allelic at the Spl1 locus (G650 and F1856). Two additional mutants were included that displayed the spl1 phenotype, but had not been genetically confirmed (G9799 and F2045). For the four mutants, we predicted a total of 242 gene models to be deleted throughout the genome (Table 2). However, the four mutants showed overlapping deletions only on chromosome 12, and these deletions were located within the region where the spl1 mutation was previously mapped [24] (Figure 4). Selected candidate gene models predicted to be deleted in this region were validated by PCR. Thus, with a total of four hybridization experiments (one per mutant line), the location of the mutation conferring the phenotype was narrowed to a 70 kb region (21 gene models) on chromosome 12.


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)

Array-based deletion discovery identifies allelic relationships among spl1 mutants. Hybridization of genomic DNA from two confirmed allelic spl1 mutants (G650 and F1856) and two mutants showing the distinctive spl1 lesion mimic phenotype (G9799 and F2045) identified overlapping deletions in all four lines on chromosome 12. A log ratio cutoff of ≤ -0.8 for 50% or more of probes in a probe set was used. Open arrowheads indicate deletions in gene models confirmed by PCR. Closed arrowheads indicate gene models confirmed to be present by PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Array-based deletion discovery identifies allelic relationships among spl1 mutants. Hybridization of genomic DNA from two confirmed allelic spl1 mutants (G650 and F1856) and two mutants showing the distinctive spl1 lesion mimic phenotype (G9799 and F2045) identified overlapping deletions in all four lines on chromosome 12. A log ratio cutoff of ≤ -0.8 for 50% or more of probes in a probe set was used. Open arrowheads indicate deletions in gene models confirmed by PCR. Closed arrowheads indicate gene models confirmed to be present by PCR.
Mentions: The rice mutant lines induced by chemical or irradiation strategies likely contained multiple deletions in the genome. We tested if hybridization of DNA from multiple mutant lines that exhibit a phenotype of interest could provide convergent data to identify the mutated region responsible for the phenotype and limit that region to the fewest gene models. Four mutants exhibiting the spl1 lesion mimic phenotype were selected as proof of concept. Two mutants had been genetically confirmed by complementation testing to be allelic at the Spl1 locus (G650 and F1856). Two additional mutants were included that displayed the spl1 phenotype, but had not been genetically confirmed (G9799 and F2045). For the four mutants, we predicted a total of 242 gene models to be deleted throughout the genome (Table 2). However, the four mutants showed overlapping deletions only on chromosome 12, and these deletions were located within the region where the spl1 mutation was previously mapped [24] (Figure 4). Selected candidate gene models predicted to be deleted in this region were validated by PCR. Thus, with a total of four hybridization experiments (one per mutant line), the location of the mutation conferring the phenotype was narrowed to a 70 kb region (21 gene models) on chromosome 12.

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