Mutation Scanning in Wheat by Exon Capture and Next-Generation Sequencing.
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.
Computational and Systems Biology Department, Rothamsted Research, Harpenden AL5 2JQ, United Kingdom.
Robert
R
King
John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom.
Nicholas
N
Bird
John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom; The Genome Analysis Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom.
Ricardo
R
Ramirez-Gonzalez
University of Bristol Transcriptomics Facility, School of Biological Sciences, Bristol BS8 1UG, United Kingdom.
Jane A
JA
Coghill
Plant Biology and Crop Science Department, Rothamsted Research, Harpenden AL5 2JQ, United Kingdom.
Archana
A
Patil
Computational and Systems Biology Department, Rothamsted Research, Harpenden AL5 2JQ, United Kingdom.
Keywan
K
Hassani-Pak
John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom.
Cristobal
C
Uauy
Plant Biology and Crop Science Department, Rothamsted Research, Harpenden AL5 2JQ, United Kingdom.
Andrew L
AL
Phillips
D000483:Alleles; D004252:DNA Mutational Analysis; D018744:DNA, Plant; D005091:Exons; D018745:Genome, Plant; D059014:High-Throughput Nucleotide Sequencing; D009154:Mutation; D010641:Phenotype; D011123:Polyploidy; D014908:Triticum / Q000235:genetics*
D018744:DNA, Plant