A Robust, Simple Genotyping-by-Sequencing (GBS) Approach for High Diversity Species

Advances in next generation technologies have driven the costs of DNA sequencing down to the point that genotyping-by-sequencing (GBS) is now feasible for high diversity, large genome species. Here, we report a procedure for constructing GBS libraries based on reducing genome complexity with restriction enzymes (REs). This approach is simple, quick, extremely specific, highly reproducible, and may reach important regions of the genome that are inaccessible to sequence capture approaches. By using methylation-sensitive REs, repetitive regions of genomes can be avoided and lower copy regions targeted with two to three fold higher efficiency. This tremendously simplifies computationally challenging alignment problems in species with high levels of genetic diversity. The GBS procedure is demonstrated with maize (IBM) and barley (Oregon Wolfe Barley) recombinant inbred populations where roughly 200,000 and 25,000 sequence tags were mapped, respectively. An advantage in species like barley that lack a complete genome sequence is that a reference map need only be developed around the restriction sites, and this can be done in the process of sample genotyping. In such cases, the consensus of the read clusters across the sequence tagged sites becomes the reference. Alternatively, for kinship analyses in the absence of a reference genome, the sequence tags can simply be treated as dominant markers. Future application of GBS to breeding, conservation, and global species and population surveys may allow plant breeders to conduct genomic selection on a novel germplasm or species without first having to develop any prior molecular tools, or conservation biologists to determine population structure without prior knowledge of the genome or diversity in the species.

下一代测序技术的进步已将DNA测序成本降至极低水平，使得基因型测序（genotyping-by-sequencing，GBS）如今可应用于高多样性、大基因组物种。本研究报道了一种基于限制性内切酶（restriction enzymes，REs）降低基因组复杂度的GBS文库构建方法。该方法操作简便、快速高效，特异性极强且重现性优异，可覆盖序列捕获技术难以触及的基因组重要区域。通过使用甲基化敏感型限制性内切酶，可规避基因组重复区域，并以2~3倍的更高效率靶向低拷贝区域。这极大简化了高遗传多样性物种中极具计算挑战性的序列比对问题。本研究以玉米（IBM）和大麦（俄勒冈沃尔夫大麦，Oregon Wolfe Barley）的重组自交系群体验证了该GBS流程，分别成功定位了约20万个和2.5万个序列标签。对于缺乏完整基因组序列的大麦这类物种，其优势在于仅需围绕限制性内切酶识别位点构建参考图谱，且该过程可与样本基因型分型同步完成。在此类场景下，序列标签位点间的读段聚类共识序列即可作为参考。此外，在缺乏参考基因组的情况下开展亲缘关系分析时，可直接将序列标签作为显性标记使用。未来将GBS应用于育种、物种保护以及全球物种与种群调查后，植物育种者可直接对新种质或新物种开展基因组选择，无需预先开发任何分子工具；而保护生物学家也可在无需预先知晓物种基因组或多样性信息的前提下，确定种群结构。