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首页> 外文期刊>BMC Genomics >A hybrid next generation transcript sequencing-based approach to identify allelic and homeolog-specific single nucleotide polymorphisms in allotetraploid white clover
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A hybrid next generation transcript sequencing-based approach to identify allelic and homeolog-specific single nucleotide polymorphisms in allotetraploid white clover

机译:基于杂种下一代转录本测序的方法,用于识别等位四倍体白三叶草中的等位基因和同源物特异性单核苷酸多态性

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Background White clover (Trifolium repens L.) is an allotetraploid species possessing two highly collinear ancestral sub-genomes. The apparent existence of highly similar homeolog copies for the majority of genes in white clover is problematic for the development of genome-based resources in the species. This is especially true for the development of genetic markers based on single nucleotide polymorphisms (SNPs), since it is difficult to distinguish between homeolog-specific and allelic variants. Robust methods for categorising single nucleotide variants as allelic or homeolog-specific in large transcript datasets are required. We illustrate one potential approach in this study. Results We used 454-pyrosequencing sequencing to generate ~760,000 transcript sequences from an 8th generation white clover inbred line. These were assembled and partially annotated to yield a reference transcript set comprising 71,545 sequences. We subsequently performed Illumina sequencing on three further white clover samples, generating 14 million transcript reads from a mixed sample comprising 24 divergent white clover genotypes, and 50 million reads on two further eighth generation white clover inbred lines. Mapping these reads to the reference transcript set allowed us to develop a significant SNP resource for white clover, and to partition the SNPs from the inbred lines into categories reflecting allelic or homeolog-specific variation. The potential for using haplotype reconstruction and progenitor genome comparison to assign haplotypes to specific ancestral sub-genomes of white clover is demonstrated for sequences corresponding to genes encoding dehydration responsive element binding protein and acyl-coA oxidase. Conclusions In total, 208,854 independent SNPs in 31,715 reference sequences were discovered, approximately three quarters of which were categorised as representing allelic or homeolog-specific variation using two inbred lines. This represents a significant resource for white clover genomics and genetics studies. We discuss the potential to extend the analysis to identify a “core set” of ancestrally derived homeolog specific variants in white clover.
机译:背景技术白三叶草(Trifolium repens L.)是一种具有四个高度共线祖先亚基因组的异源四倍体物种。白三叶草中大多数基因的高度相似的同源同源物拷贝的明显存在对于物种中基于基因组的资源的开发是有问题的。对于基于单核苷酸多态性(SNP)的遗传标记开发,尤其如此,因为很难区分同源同源物特异性和等位基因变异。需要在大笔录数据集中将单核苷酸变体分类为等位基因或同源同源物的可靠方法。我们说明了这项研究中的一种潜在方法。结果我们使用454焦磷酸测序法从第8代白三叶草自交系产生了约760,000个转录物序列。这些被组装并部分注释以产生包含71,545个序列的参考转录物组。随后,我们对另外三个白三叶草样品进行了Illumina测序,从包含24个不同白三叶草基因型的混合样品中产生了1400万个转录物读数,并在另外两个第八代白三叶草自交系上获得了5000万个读数。将这些读段映射到参考转录本集可以使我们为白三叶草开发重要的SNP资源,并将自交系中的SNP划分为反映等位基因或同源同源物特异性变异的类别。对于与编码脱水响应元件结合蛋白和酰基辅酶A氧化酶的基因相对应的序列,证明了使用单倍型重建和祖细胞基因组比较将单倍型分配给白三叶草的特定祖先亚基因组的潜力。结论总共在31,715个参考序列中发现了208,854个独立的SNP,其中大约四分之三被归类为代表具有两个自交系的等位基因或同源同源物特异性变异。这代表了白三叶草基因组学和遗传学研究的重要资源。我们讨论了扩展分析以识别白三叶草祖先衍生的同源同源物特定变体的“核心集”的潜力。

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