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TILLING in the two-rowed barley cultivar 'Barke' reveals preferred sites of functional diversity in the gene HvHox1.

Gottwald S, Bauer P, Komatsuda T, Lundqvist U, Stein N - BMC Res Notes (2009)

Bottom Line: The ethyl methanesulphonate (EMS)mutagenesis efficiency was studied by recording and relating the mutagenesis-dependent effects found in the three mutant generations (M(1)-M(3)).Three of the newly identified mutants exhibited either a six-rowed or an intermedium-spike phenotype, and one mutant displayed a significantly altered spikelet morphology compared to that of the 'Barke' wild type.Pilot screening demonstrated a similar or even slightly higher mutation frequency when compared to previously published barley TILLING populations that should allow for the identification of diverse allelic variation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, D-06466 Gatersleben, Germany.

ABSTRACT

Background: The economic importance of cereals such as barley, and the demand for improved yield and quality require a better understanding of the genetic components that modulate biologically and commercially relevant traits. While Arabidopsis thaliana is the premiere model plant system, the spectrum of its traits cannot address all of the fundamental questions of crop plant development. Unlike Arabidopsis, barley is both a crop and a model system for scientific research, and it is increasingly being used for genetic and molecular investigations into the conserved biological processes of cereals. A common challenge in genetic studies in plants with large genomes arises from the very time-consuming work of associating mutant phenotypes with gene sequence information, especially if insertion mutagenesis is not routine, as in barley. Reverse genetics based on chemical mutagenesis represents the best solution to this obstacle.

Findings: In barley, we generated a new TILLING (Targeting Local Lesions IN Genomes) resource comprising 10,279 M(2 )mutants in the two-rowed malting cultivar 'Barke,' which has been used in the generation of other genomic resources in barley (~150,000 ESTs, DH mapping population). The value of this new resource was tested using selected candidate genes. An average frequency of approximately one mutation per 0.5 Mb was determined by screening ten fragments of six different genes. The ethyl methanesulphonate (EMS)mutagenesis efficiency was studied by recording and relating the mutagenesis-dependent effects found in the three mutant generations (M(1)-M(3)). A detailed analysis was performed for the homeodomain-leucine-zipper (HD-ZIP) gene HvHox1. Thirty-one mutations were identified by screening a 1,270-bp fragment in 7,348 M(2 )lines. Three of the newly identified mutants exhibited either a six-rowed or an intermedium-spike phenotype, and one mutant displayed a significantly altered spikelet morphology compared to that of the 'Barke' wild type. Our results indicate a bias in the frequency of independent functional mutations at specific base pair (bp) positions within the gene HvHox1.

Conclusions: A new TILLING population was developed as a resource for high-throughput gene discovery in an alternative barley germplasm. Pilot screening demonstrated a similar or even slightly higher mutation frequency when compared to previously published barley TILLING populations that should allow for the identification of diverse allelic variation. Partial phenotypic evaluation of the M(2 )and M(3 )generations has revealed the presence of a wide spectrum of morphological diversity that highlights the great potential of this resource for use in forward genetic screens. Altogether, our study shows the efficiency of screening and the applicability of the new TILLING population for genetic studies in the barley crop model system.

No MeSH data available.


Mutation frequency in relation to EMS concentration. The relative SNP rates for EMS treatments between 20 and 50 mM were calculated as particular sub-population specific frequencies. Data are based on the mutation screening of eight gene fragments in 7,389 M2 lines. The white and black bars show the accumulated synonymous and non-synonymous (AA changes and truncation) mutations detected for each sub-population. The relationship between SNP frequencies and EMS dosages was calculated based on a non-parametric Spearman rank correlation (coefficient of rs = 0.94 with a significance at p ≤ 0.01).
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Figure 1: Mutation frequency in relation to EMS concentration. The relative SNP rates for EMS treatments between 20 and 50 mM were calculated as particular sub-population specific frequencies. Data are based on the mutation screening of eight gene fragments in 7,389 M2 lines. The white and black bars show the accumulated synonymous and non-synonymous (AA changes and truncation) mutations detected for each sub-population. The relationship between SNP frequencies and EMS dosages was calculated based on a non-parametric Spearman rank correlation (coefficient of rs = 0.94 with a significance at p ≤ 0.01).

Mentions: Since the TILLING population was derived from seed batches treated with different EMS concentrations, the results could be correlated to EMS dosage [mutation frequency [EMS] = ∑ SNPs per gene/∑ screened Mb per gene]. The single base pair substitution rate was positively correlated (rs = 0.94) with increasing EMS concentration (Figure 1) and ranged (based on TILLING of 8 PCR-derived DNA fragments from 5 genes) from 1 mutation/0.8 Mb at 20 mM EMS to 1 mutation/0.20 Mb and 1 mutation/0.14 Mb after treatment with 40 and 50 mM EMS, respectively. Likewise, an increase in the respective levels of missense mutations was observed between 20 mM- and 40 mM-plants, while there were not any non-synonymous mutations observed with the highest treatment (Figure 1).


TILLING in the two-rowed barley cultivar 'Barke' reveals preferred sites of functional diversity in the gene HvHox1.

Gottwald S, Bauer P, Komatsuda T, Lundqvist U, Stein N - BMC Res Notes (2009)

Mutation frequency in relation to EMS concentration. The relative SNP rates for EMS treatments between 20 and 50 mM were calculated as particular sub-population specific frequencies. Data are based on the mutation screening of eight gene fragments in 7,389 M2 lines. The white and black bars show the accumulated synonymous and non-synonymous (AA changes and truncation) mutations detected for each sub-population. The relationship between SNP frequencies and EMS dosages was calculated based on a non-parametric Spearman rank correlation (coefficient of rs = 0.94 with a significance at p ≤ 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803498&req=5

Figure 1: Mutation frequency in relation to EMS concentration. The relative SNP rates for EMS treatments between 20 and 50 mM were calculated as particular sub-population specific frequencies. Data are based on the mutation screening of eight gene fragments in 7,389 M2 lines. The white and black bars show the accumulated synonymous and non-synonymous (AA changes and truncation) mutations detected for each sub-population. The relationship between SNP frequencies and EMS dosages was calculated based on a non-parametric Spearman rank correlation (coefficient of rs = 0.94 with a significance at p ≤ 0.01).
Mentions: Since the TILLING population was derived from seed batches treated with different EMS concentrations, the results could be correlated to EMS dosage [mutation frequency [EMS] = ∑ SNPs per gene/∑ screened Mb per gene]. The single base pair substitution rate was positively correlated (rs = 0.94) with increasing EMS concentration (Figure 1) and ranged (based on TILLING of 8 PCR-derived DNA fragments from 5 genes) from 1 mutation/0.8 Mb at 20 mM EMS to 1 mutation/0.20 Mb and 1 mutation/0.14 Mb after treatment with 40 and 50 mM EMS, respectively. Likewise, an increase in the respective levels of missense mutations was observed between 20 mM- and 40 mM-plants, while there were not any non-synonymous mutations observed with the highest treatment (Figure 1).

Bottom Line: The ethyl methanesulphonate (EMS)mutagenesis efficiency was studied by recording and relating the mutagenesis-dependent effects found in the three mutant generations (M(1)-M(3)).Three of the newly identified mutants exhibited either a six-rowed or an intermedium-spike phenotype, and one mutant displayed a significantly altered spikelet morphology compared to that of the 'Barke' wild type.Pilot screening demonstrated a similar or even slightly higher mutation frequency when compared to previously published barley TILLING populations that should allow for the identification of diverse allelic variation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, D-06466 Gatersleben, Germany.

ABSTRACT

Background: The economic importance of cereals such as barley, and the demand for improved yield and quality require a better understanding of the genetic components that modulate biologically and commercially relevant traits. While Arabidopsis thaliana is the premiere model plant system, the spectrum of its traits cannot address all of the fundamental questions of crop plant development. Unlike Arabidopsis, barley is both a crop and a model system for scientific research, and it is increasingly being used for genetic and molecular investigations into the conserved biological processes of cereals. A common challenge in genetic studies in plants with large genomes arises from the very time-consuming work of associating mutant phenotypes with gene sequence information, especially if insertion mutagenesis is not routine, as in barley. Reverse genetics based on chemical mutagenesis represents the best solution to this obstacle.

Findings: In barley, we generated a new TILLING (Targeting Local Lesions IN Genomes) resource comprising 10,279 M(2 )mutants in the two-rowed malting cultivar 'Barke,' which has been used in the generation of other genomic resources in barley (~150,000 ESTs, DH mapping population). The value of this new resource was tested using selected candidate genes. An average frequency of approximately one mutation per 0.5 Mb was determined by screening ten fragments of six different genes. The ethyl methanesulphonate (EMS)mutagenesis efficiency was studied by recording and relating the mutagenesis-dependent effects found in the three mutant generations (M(1)-M(3)). A detailed analysis was performed for the homeodomain-leucine-zipper (HD-ZIP) gene HvHox1. Thirty-one mutations were identified by screening a 1,270-bp fragment in 7,348 M(2 )lines. Three of the newly identified mutants exhibited either a six-rowed or an intermedium-spike phenotype, and one mutant displayed a significantly altered spikelet morphology compared to that of the 'Barke' wild type. Our results indicate a bias in the frequency of independent functional mutations at specific base pair (bp) positions within the gene HvHox1.

Conclusions: A new TILLING population was developed as a resource for high-throughput gene discovery in an alternative barley germplasm. Pilot screening demonstrated a similar or even slightly higher mutation frequency when compared to previously published barley TILLING populations that should allow for the identification of diverse allelic variation. Partial phenotypic evaluation of the M(2 )and M(3 )generations has revealed the presence of a wide spectrum of morphological diversity that highlights the great potential of this resource for use in forward genetic screens. Altogether, our study shows the efficiency of screening and the applicability of the new TILLING population for genetic studies in the barley crop model system.

No MeSH data available.