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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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Type and distribution of induced mutations discovered in seven amplicons. Orange boxes correspond to exons, lines to introns. Homology to proteins in the BLOCKS database [38] is indicated by the green boxes above gmppck4 and gmrhg4b. The other amplicons did not contain regions of BLOCKS homology. Arrowheads indicate approximate position of missense changes, upside down arrowheads indicate silent changes, asterisks indicate nonsense mutations, boxes indicate deletions. Hollow arrowheads = A population; red = B population; gray = C population; black = D population. The number of mutations discovered in each amplicon per population is indicated on the right.
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Figure 2: Type and distribution of induced mutations discovered in seven amplicons. Orange boxes correspond to exons, lines to introns. Homology to proteins in the BLOCKS database [38] is indicated by the green boxes above gmppck4 and gmrhg4b. The other amplicons did not contain regions of BLOCKS homology. Arrowheads indicate approximate position of missense changes, upside down arrowheads indicate silent changes, asterisks indicate nonsense mutations, boxes indicate deletions. Hollow arrowheads = A population; red = B population; gray = C population; black = D population. The number of mutations discovered in each amplicon per population is indicated on the right.

Mentions: We discovered 116 mutations: 32 in A, 12 in B, 25 in C, and 47 in D (Figure 2 and Additional File 1). Two individual lines, one from the A population and the other from C, had more than one base change detected in an amplicon. Because these changes were homozygous and not the expected G/C to A/T EMS-induced transitions, we considered the individual lines to be likely cultivar contaminants, and we excluded them from the analysis. Mutation density was estimated as the total number of mutations divided by the total number of base pairs screened (amplicon size × individuals screened). For each target, 200 bp is subtracted from the amplicon size to adjust for the 100 bp regions at the top and bottom of TILLING gel images that are difficult to analyze [20]. The A and D populations showed similar mutation densities (~1/140 kb for both). Mutation density in the population designated C was ~1/250 kb and ~1/550 kb in the B population.


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)

Type and distribution of induced mutations discovered in seven amplicons. Orange boxes correspond to exons, lines to introns. Homology to proteins in the BLOCKS database [38] is indicated by the green boxes above gmppck4 and gmrhg4b. The other amplicons did not contain regions of BLOCKS homology. Arrowheads indicate approximate position of missense changes, upside down arrowheads indicate silent changes, asterisks indicate nonsense mutations, boxes indicate deletions. Hollow arrowheads = A population; red = B population; gray = C population; black = D population. The number of mutations discovered in each amplicon per population is indicated on the right.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Type and distribution of induced mutations discovered in seven amplicons. Orange boxes correspond to exons, lines to introns. Homology to proteins in the BLOCKS database [38] is indicated by the green boxes above gmppck4 and gmrhg4b. The other amplicons did not contain regions of BLOCKS homology. Arrowheads indicate approximate position of missense changes, upside down arrowheads indicate silent changes, asterisks indicate nonsense mutations, boxes indicate deletions. Hollow arrowheads = A population; red = B population; gray = C population; black = D population. The number of mutations discovered in each amplicon per population is indicated on the right.
Mentions: We discovered 116 mutations: 32 in A, 12 in B, 25 in C, and 47 in D (Figure 2 and Additional File 1). Two individual lines, one from the A population and the other from C, had more than one base change detected in an amplicon. Because these changes were homozygous and not the expected G/C to A/T EMS-induced transitions, we considered the individual lines to be likely cultivar contaminants, and we excluded them from the analysis. Mutation density was estimated as the total number of mutations divided by the total number of base pairs screened (amplicon size × individuals screened). For each target, 200 bp is subtracted from the amplicon size to adjust for the 100 bp regions at the top and bottom of TILLING gel images that are difficult to analyze [20]. The A and D populations showed similar mutation densities (~1/140 kb for both). Mutation density in the population designated C was ~1/250 kb and ~1/550 kb in the B population.

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