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TILLING to detect induced mutations in soybean.

Cooper JL, Till BJ, Laport RG, Darlow MC, Kleffner JM, Jamai A, El-Mellouki T, Liu S, Ritchie R, Nielsen N, Bilyeu KD, Meksem K, Comai L, Henikoff S - BMC Plant Biol. (2008)

Bottom Line: Soybean (Glycine max L.The NMU-treated population and one EMS mutagenized population had similar mutation density (approximately 1/140 kb), while another EMS population had a mutation density of approximately 1/250 kb.The remaining population had a mutation density of approximately 1/550 kb.

View Article: PubMed Central - HTML - PubMed

Affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98107, USA. jlcooper@fhcrc.org

ABSTRACT

Background: Soybean (Glycine max L. Merr.) is an important nitrogen-fixing crop that provides much of the world's protein and oil. However, the available tools for investigation of soybean gene function are limited. Nevertheless, chemical mutagenesis can be applied to soybean followed by screening for mutations in a target of interest using a strategy known as Targeting Induced Local Lesions IN Genomes (TILLING). We have applied TILLING to four mutagenized soybean populations, three of which were treated with ethyl methanesulfonate (EMS) and one with N-nitroso-N-methylurea (NMU).

Results: We screened seven targets in each population and discovered a total of 116 induced mutations. The NMU-treated population and one EMS mutagenized population had similar mutation density (approximately 1/140 kb), while another EMS population had a mutation density of approximately 1/250 kb. The remaining population had a mutation density of approximately 1/550 kb. Because of soybean's polyploid history, PCR amplification of multiple targets could impede mutation discovery. Indeed, one set of primers tested in this study amplified more than a single target and produced low quality data. To address this problem, we removed an extraneous target by pretreating genomic DNA with a restriction enzyme. Digestion of the template eliminated amplification of the extraneous target and allowed the identification of four additional mutant alleles compared to untreated template.

Conclusion: The development of four independent populations with considerable mutation density, together with an additional method for screening closely related targets, indicates that soybean is a suitable organism for high-throughput mutation discovery even with its extensively duplicated genome.

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Elimination of multiple amplicons. Only the 700 channel is shown. Box indicates a cut DNA strand corresponding to a single nucleotide polymorphism that was identified and sequenced from both undigested and digested templates. A) Filled arrowheads indicate multiple bands in every lane of an eight-fold pool plate. These spurious cut products were derived from CJE digestion of heteroduplexes formed between PCR products from co-amplified targets, presumably homeologs. B) The same template pools from (A) digested with ApaI prior to PCR amplification for TILLING. Ovals denote cut DNA strands corresponding to single nucleotide polymorphisms that were identified only when TILLING from digested template. Open arrowheads show the position of bands from CJE digestion that represent polymorphisms present in more than one member of the population.
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Figure 3: Elimination of multiple amplicons. Only the 700 channel is shown. Box indicates a cut DNA strand corresponding to a single nucleotide polymorphism that was identified and sequenced from both undigested and digested templates. A) Filled arrowheads indicate multiple bands in every lane of an eight-fold pool plate. These spurious cut products were derived from CJE digestion of heteroduplexes formed between PCR products from co-amplified targets, presumably homeologs. B) The same template pools from (A) digested with ApaI prior to PCR amplification for TILLING. Ovals denote cut DNA strands corresponding to single nucleotide polymorphisms that were identified only when TILLING from digested template. Open arrowheads show the position of bands from CJE digestion that represent polymorphisms present in more than one member of the population.

Mentions: Following this observation, subsequent primers were tested by agarose gel analysis and sequencing. Of 27 primer sets tested, 17 primer sets amplified more than one target. Given the high proportion of tested primer sets that amplified more than one target, we wondered whether we could screen for mutations in these targets by eliminating extra templates in the genomic DNA. For example, amplification and CJE digestion with the gmrhg4 primers resulted in multiple bands in every pool (arrowheads, Figure 3A). The multiple bands were still observed when TILLING assays were performed on unpooled DNAs (data not shown), and multiple heterozygous sites were detected upon sequencing individuals (data not shown), consistent with the hypothesis that the primers amplified more than one target. Two sequences were obtained upon cloning the gmrhg4 PCR product; one sequence corresponded to the gmrhg4 target and the other sequence (GenBank EF644646) contained the polymorphisms observed when sequencing the gmrhg4 PCR product.


TILLING to detect induced mutations in soybean.

Cooper JL, Till BJ, Laport RG, Darlow MC, Kleffner JM, Jamai A, El-Mellouki T, Liu S, Ritchie R, Nielsen N, Bilyeu KD, Meksem K, Comai L, Henikoff S - BMC Plant Biol. (2008)

Elimination of multiple amplicons. Only the 700 channel is shown. Box indicates a cut DNA strand corresponding to a single nucleotide polymorphism that was identified and sequenced from both undigested and digested templates. A) Filled arrowheads indicate multiple bands in every lane of an eight-fold pool plate. These spurious cut products were derived from CJE digestion of heteroduplexes formed between PCR products from co-amplified targets, presumably homeologs. B) The same template pools from (A) digested with ApaI prior to PCR amplification for TILLING. Ovals denote cut DNA strands corresponding to single nucleotide polymorphisms that were identified only when TILLING from digested template. Open arrowheads show the position of bands from CJE digestion that represent polymorphisms present in more than one member of the population.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2266751&req=5

Figure 3: Elimination of multiple amplicons. Only the 700 channel is shown. Box indicates a cut DNA strand corresponding to a single nucleotide polymorphism that was identified and sequenced from both undigested and digested templates. A) Filled arrowheads indicate multiple bands in every lane of an eight-fold pool plate. These spurious cut products were derived from CJE digestion of heteroduplexes formed between PCR products from co-amplified targets, presumably homeologs. B) The same template pools from (A) digested with ApaI prior to PCR amplification for TILLING. Ovals denote cut DNA strands corresponding to single nucleotide polymorphisms that were identified only when TILLING from digested template. Open arrowheads show the position of bands from CJE digestion that represent polymorphisms present in more than one member of the population.
Mentions: Following this observation, subsequent primers were tested by agarose gel analysis and sequencing. Of 27 primer sets tested, 17 primer sets amplified more than one target. Given the high proportion of tested primer sets that amplified more than one target, we wondered whether we could screen for mutations in these targets by eliminating extra templates in the genomic DNA. For example, amplification and CJE digestion with the gmrhg4 primers resulted in multiple bands in every pool (arrowheads, Figure 3A). The multiple bands were still observed when TILLING assays were performed on unpooled DNAs (data not shown), and multiple heterozygous sites were detected upon sequencing individuals (data not shown), consistent with the hypothesis that the primers amplified more than one target. Two sequences were obtained upon cloning the gmrhg4 PCR product; one sequence corresponded to the gmrhg4 target and the other sequence (GenBank EF644646) contained the polymorphisms observed when sequencing the gmrhg4 PCR product.

Bottom Line: Soybean (Glycine max L.The NMU-treated population and one EMS mutagenized population had similar mutation density (approximately 1/140 kb), while another EMS population had a mutation density of approximately 1/250 kb.The remaining population had a mutation density of approximately 1/550 kb.

View Article: PubMed Central - HTML - PubMed

Affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98107, USA. jlcooper@fhcrc.org

ABSTRACT

Background: Soybean (Glycine max L. Merr.) is an important nitrogen-fixing crop that provides much of the world's protein and oil. However, the available tools for investigation of soybean gene function are limited. Nevertheless, chemical mutagenesis can be applied to soybean followed by screening for mutations in a target of interest using a strategy known as Targeting Induced Local Lesions IN Genomes (TILLING). We have applied TILLING to four mutagenized soybean populations, three of which were treated with ethyl methanesulfonate (EMS) and one with N-nitroso-N-methylurea (NMU).

Results: We screened seven targets in each population and discovered a total of 116 induced mutations. The NMU-treated population and one EMS mutagenized population had similar mutation density (approximately 1/140 kb), while another EMS population had a mutation density of approximately 1/250 kb. The remaining population had a mutation density of approximately 1/550 kb. Because of soybean's polyploid history, PCR amplification of multiple targets could impede mutation discovery. Indeed, one set of primers tested in this study amplified more than a single target and produced low quality data. To address this problem, we removed an extraneous target by pretreating genomic DNA with a restriction enzyme. Digestion of the template eliminated amplification of the extraneous target and allowed the identification of four additional mutant alleles compared to untreated template.

Conclusion: The development of four independent populations with considerable mutation density, together with an additional method for screening closely related targets, indicates that soybean is a suitable organism for high-throughput mutation discovery even with its extensively duplicated genome.

Show MeSH