Mutation Scanning in Wheat by Exon Capture and Next-Generation Sequencing.
Mutation Scanning in Wheat by Exon Capture and Next-Generation Sequencing.
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DOI：
10.1371/journal.pone.0137549
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Phillips AL
中科院分区：
文献类型：
--
作者：
King R;Bird N;Ramirez-Gonzalez R;Coghill JA;Patil A;Hassani-Pak K;Uauy C;Phillips AL
基因组靶向诱导局部损伤（TILLING）是一种鉴定基因组中新序列变异的反向遗传学方法，其目的是研究基因功能和/或开发用于育种的有用等位基因。尽管最近在小麦基因组学方面取得了进展，但目前大多数TILLING方法的通量都是低至中等的，其基于靶基因的PCR扩增。我们通过外显子捕获的下一代测序对小麦中的TILLING进行了中试评估。使用覆盖约2 Mbp小麦编码序列的基于谷胱甘肽的富集阵列对含有先前鉴定的TaGA 20 ox 1同源异型基因突变的小麦的三个诱变品系进行外显子捕获和测序。在测试了不同的作图算法和设置后，通过定位到IWGSC小麦染色体调查序列来鉴定候选SNP。在参考中发现所有三种同源物的序列数据的情况下，突变体调用是明确的;然而，在参考缺乏一种或两种同源物的情况下，从这些基因捕获的读取被错误映射到其他同源物，导致变体等位基因频率的稀释或突变分配到错误的同源物。竞争性PCR测定用于验证推定的SNP并估计SNP过滤的截止水平。在三个诱变品系中检测到至少464个高置信度SNP，包括TaGA 20 ox 1中的三个已知等位基因，表明突变率为每Mb约35个SNP，与基于PCR的TILLING估计的突变率相似。这证明了使用外显子捕获进行基因组重测序作为多倍体小麦中突变检测方法的可行性，但准确的突变调用将需要具有更全面的同源物覆盖率的改进的基因组参考。
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.