Hard Winter Wheat Genetics Research Unit, United States Department of Agriculture - Agricultural Research Service, Manhattan, Kansas, United States of America.
PLoS One. 2012;7(2):e32253. doi: 10.1371/journal.pone.0032253. Epub 2012 Feb 28.
Advancements in next-generation sequencing technology have enabled whole genome re-sequencing in many species providing unprecedented discovery and characterization of molecular polymorphisms. There are limitations, however, to next-generation sequencing approaches for species with large complex genomes such as barley and wheat. Genotyping-by-sequencing (GBS) has been developed as a tool for association studies and genomics-assisted breeding in a range of species including those with complex genomes. GBS uses restriction enzymes for targeted complexity reduction followed by multiplex sequencing to produce high-quality polymorphism data at a relatively low per sample cost. Here we present a GBS approach for species that currently lack a reference genome sequence. We developed a novel two-enzyme GBS protocol and genotyped bi-parental barley and wheat populations to develop a genetically anchored reference map of identified SNPs and tags. We were able to map over 34,000 SNPs and 240,000 tags onto the Oregon Wolfe Barley reference map, and 20,000 SNPs and 367,000 tags on the Synthetic W9784 × Opata85 (SynOpDH) wheat reference map. To further evaluate GBS in wheat, we also constructed a de novo genetic map using only SNP markers from the GBS data. The GBS approach presented here provides a powerful method of developing high-density markers in species without a sequenced genome while providing valuable tools for anchoring and ordering physical maps and whole-genome shotgun sequence. Development of the sequenced reference genome(s) will in turn increase the utility of GBS data enabling physical mapping of genes and haplotype imputation of missing data. Finally, as a result of low per-sample costs, GBS will have broad application in genomics-assisted plant breeding programs.
下一代测序技术的进步使得许多物种的全基因组重测序成为可能，从而以前所未有的方式发现和描述了分子多态性。然而，对于具有大而复杂基因组的物种，如大麦和小麦，下一代测序方法存在局限性。基因型测序（GBS）已被开发为一种用于关联研究和基因组辅助育种的工具，适用于包括复杂基因组在内的多种物种。GBS 使用限制性内切酶进行靶向复杂性降低，然后进行多重测序，以相对较低的每个样本成本产生高质量的多态性数据。在这里，我们提出了一种用于目前缺乏参考基因组序列的物种的 GBS 方法。我们开发了一种新的双酶 GBS 方案，并对双亲和小麦群体进行了基因型分析，以开发鉴定 SNP 和标签的遗传锚定参考图谱。我们能够将超过 34000 个 SNP 和 240000 个标签映射到俄勒冈沃尔夫大麦参考图谱上，将 20000 个 SNP 和 367000 个标签映射到合成 W9784×Opata85（SynOpDH）小麦参考图谱上。为了进一步评估 GBS 在小麦中的应用，我们还仅使用 GBS 数据中的 SNP 标记构建了一个从头遗传图谱。这里提出的 GBS 方法为没有测序基因组的物种提供了一种开发高密度标记的强大方法，同时为锚定和排序物理图谱和全基因组鸟枪法序列提供了有价值的工具。测序参考基因组的开发反过来又将增加 GBS 数据的实用性，从而实现基因的物理作图和缺失数据的单倍型推断。最后，由于每个样本的成本低，GBS 将在基因组辅助植物育种计划中得到广泛应用。
