Mutation Scanning in Wheat by Exon Capture and Next-Generation Sequencing

PloS One
Robert KingAndrew L Phillips

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...Continue Reading

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Citations

Jan 18, 2017·Proceedings of the National Academy of Sciences of the United States of America·Ksenia V KrasilevaJorge Dubcovsky
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Feb 7, 2021·Nature Communications·Heïdi SerraPierre Sourdille
Jul 22, 2021·Plant Biotechnology Journal·Michael C U Hammond-KosackKim E Hammond-Kosack

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Datasets Mentioned

BETA
PRJEB9959

Methods Mentioned

BETA
PCR
genotyping

Software Mentioned

Perl script
Klustercaller
Bowtie
Ensembl
KASP
TILLING
BLAST
BWA
PolyMarker
MEM

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