Abstract
Targeted Induced Local Lesions in Genomes (TILLING) is a reverse genetics approach to identify novel sequence variation in genomes, with the aims of investigating gene function and/or developing useful alleles for breeding. Despite recent advances in wheat genomics, most current TILLING methods are low to medium in throughput, being based on PCR amplification of the target genes. We performed a pilot-scale evaluation of TILLING in wheat by next-generation sequencing through exon capture. An oligonucleotide-based enrichment array covering ~2 Mbp of wheat coding sequence was used to carry out exon capture and sequencing on three mutagenised lines of wheat containing previously-identified mutations in the TaGA20ox1 homoeologous genes. After testing different mapping algorithms and settings, candidate SNPs were identified by mapping to the IWGSC wheat Chromosome Survey Sequences. Where sequence data for all three homoeologues were found in the reference, mutant calls were unambiguous; however, where the reference lacked one or two of the homoeologues, captured reads from these genes were mis-mapped to other homoeologues, resulting either in dilution of the variant allele frequency or assignment of mutations to the wrong homoeologue. Competitive PCR assays were used to validate the putative SNPs and estimate cut-off levels for SNP filtering. At least 464 high-confidence SNPs were detected across the three mutagenized lines, including the three known alleles in TaGA20ox1, indicating a mutation rate of ~35 SNPs per Mb, similar to that estimated by PCR-based TILLING. This demonstrates the feasibility of using exon capture for genome re-sequencing as a method of mutation detection in polyploid wheat, but accurate mutation calling will require an improved genomic reference with more comprehensive coverage of homoeologues.
Highlights
The introduction of novel sequence variation into crop genomes by induced mutation is a powerful tool for plant breeders: to date, over 3,000 plant varieties developed through the use of PLOS ONE | DOI:10.1371/journal.pone.0137549 September 3, 2015Mutation Scanning in Wheat
At the initiation of this project, little genomic sequence information was available for wheat and the capture array design was based purely on cDNA sequences, with no account taken of intron positions
The capture array consisted of overlapping 120-mer oligonucleotides; to remove baits targeting highly repetitive regions of the genome, the bait sequences were compared by BLAST with unassembled genomic shotgun sequences of bread wheat cv
Summary
The introduction of novel sequence variation into crop genomes by induced mutation is a powerful tool for plant breeders: to date, over 3,000 plant varieties developed through the use of PLOS ONE | DOI:10.1371/journal.pone.0137549 September 3, 2015. Alleles in each homoeologue that are predicted to affect gene function can be combined by crossing to determine the effect on the phenotype The feasibility of this approach was demonstrated nearly ten years ago by Slade et al.[1] who developed a TILLING [2] approach to identify novel loss-of-function alleles in the Waxy (granule-bound starch synthase I) genes within EMS-mutagenized populations of both bread (hexaploid) and durum (tetraploid) wheat. This consists of a large number of relatively small contigs, a large proportion of protein-coding regions are represented providing an opportunity to develop a high-throughput approach to mutation detection and classification in wheat Support of this strategy is shown by Jordan et al[19] who used exome capture in wheat followed by mapping to the IWGSC draft assembly to identify SNPs and insertion/deletion events within a panel of wheat varieties and accessions. When applied across the wheat genome, re-sequencing based on exon capture coupled with improved an genome sequence will enable mutation discovery in the coding regions of the majority of genes within such populations, allowing the development of online mutation resources for this globally important crop
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