Abstract
BackgroundForward genetic approaches have limited use for agronomic traits that can’t be reliably scored on a single plant basis. Thus, mutants in wheat and other crops are more useful for gene function studies by reverse genetic approach. With a long-term goal to develop a sequence-based mutation detection resource in hexaploid wheat, we conducted a feasibility study to accurately differentiate induced mutations from the homoeologs’ sequence variations present among the three wheat genomes.ResultsA reduced representation ApeKI library consisting of 21 Ethylmethane Sulfonate (EMS) induced mutants and two wild type cv. Indian plants was developed using individual barcode adapters and sequenced. A novel bioinformatics pipeline was developed to identify sequence variants using 178,464 wheat unigenes as a reference wheat transcriptome. In total, 14,130 mutational changes [Single Nucleotide Polymorphisms (SNPs) and Insertions/Deletions (INDELs)] and 150,511 homoeologous sequence changes were detected. On an average, 662 SNPs (ranging from 46 to 1,330) and 10 small INDELs (ranging from 0 to 23) were identified for each of the mutants. A mutation frequency of one per 5 Kb was observed with 70 % being transitions and 30 % transversions. The pipeline was tested using the known sequence changes in the three wheat genes. Genes present in the distal regions of the chromosomes were found to be more prone to EMS compared to genes present in the proximal regions. Redefined parameters identified a total of 28,348 mutational changes (1,349/plant).ConclusionsWe conclude that sequencing based mutation detection is a valuable method to identify induced mutations at large.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2112-1) contains supplementary material, which is available to authorized users.
Highlights
Forward genetic approaches have limited use for agronomic traits that can’t be reliably scored on a single plant basis
About 40 % of the unigenes were longer than 500 bp whereas the length of the remaining 60 % ranged from 103 bp to 500 bp
The unigene set covered about 110 million basepairs (Mbp) of the genic part of the wheat genome
Summary
Forward genetic approaches have limited use for agronomic traits that can’t be reliably scored on a single plant basis. Mutants in wheat and other crops are more useful for gene function studies by reverse genetic approach. With a long-term goal to develop a sequence-based mutation detection resource in hexaploid wheat, we conducted a feasibility study to accurately differentiate induced mutations from the homoeologs’ sequence variations present among the three wheat genomes. The development of functional genomic resources in wheat is critical for determining function, especially for genes controlling agronomic traits. The creation of such resources involves disruption of genes either by mutagenesis or gene knockout. Used, mutagenesis creates a large and discrete spectrum of lesions that are subsequently detected by forward or reverse genetic approaches.
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