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Digital genotyping of sorghum – a diverse plant species with a large repeat-rich genome

机译：高粱的数字基因分型–具有大量重复序列丰富的基因组的多种植物

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Background Rapid acquisition of accurate genotyping information is essential for all genetic marker-based studies. For species with relatively small genomes, complete genome resequencing is a feasible approach for genotyping; however, for species with large and highly repetitive genomes, the acquisition of whole genome sequences for the purpose of genotyping is still relatively inefficient and too expensive to be carried out on a high-throughput basis. Sorghum bicolor is a C4 grass with a sequenced genome size of ~730?Mb, of which ~80% is highly repetitive. We have developed a restriction enzyme targeted genome resequencing method for genetic analysis, termed Digital Genotyping (DG), to be applied to sorghum and other grass species with large repeat-rich genomes. Results DG templates are generated using one of three methylation sensitive restriction enzymes that recognize a nested set of 4, 6 or 8?bp GC-rich sequences, enabling varying depth of analysis and integration of results among assays. Variation in sequencing efficiency among DG markers was correlated with template GC-content and length. The expected DG allele sequence was obtained 97.3% of the time with a ratio of expected to alternative allele sequence acquisition of >20:1. A genetic map aligned to the sorghum genome sequence with an average resolution of 1.47?cM was constructed using 1,772 DG markers from 137 recombinant inbred lines. The DG map enhanced the detection of QTL for variation in plant height and precisely aligned QTL such as Dw3 to underlying genes/alleles. Higher-resolution NgoMIV-based DG haplotypes were used to trace the origin of DNA on SBI-06, spanning Ma1 and Dw2 from progenitors to BTx623 and IS3620C. DG marker analysis identified the correct location of two miss-assembled regions and located seven super contigs in the sorghum reference genome sequence. Conclusion DG technology provides a cost-effective approach to rapidly generate accurate genotyping data in sorghum. Currently, data derived from DG are used for many marker-based analyses, including marker-assisted breeding, pedigree and QTL analysis, genetic map construction, map-based gene cloning and association studies. DG in combination with whole genome resequencing is dramatically accelerating all aspects of genetic analysis of sorghum, an important genetic reference for C4 grass species.
机译：背景技术快速获取准确的基因分型信息对于所有基于遗传标记的研究都是必不可少的。对于基因组相对较小的物种，完整的基因组重测序是一种可行的基因分型方法。然而，对于具有大型且高度重复的基因组的物种而言，以基因分型为目的的全基因组序列采集仍然相对效率低下，而且成本高昂，无法在高通量的基础上进行。高粱是一种C 4 草，基因组序列大小约为730？Mb，其中约80％具有高度重复性。我们已经开发了一种用于基因分析的限制性内切酶靶向基因组重测序方法，称为数字基因分型（DG），可用于高粱和其他具有大量重复序列丰富的基因组的草种。结果DG模板是使用三种甲基化敏感的限制性内切酶之一生成的，这些酶识别嵌套的4、6或8个bp富含GC的序列，从而能够进行不同深度的分析并整合测定结果。 DG标记之间测序效率的差异与模板GC含量和长度相关。预期的DG等位基因序列在97.3％的时间内获得，预期与替代等位基因序列获取的比率> 20：1。使用来自137个重组自交系的1,772个DG标记构建了与高粱基因组序列对齐的平均分辨率为1.47？cM的遗传图谱。 DG图谱增强了对植物高度变化的QTL的检测，并精确地将QTL（例如Dw3）与基础基因/等位基因对齐。基于高分辨率NgoMIV的DG单倍型被用于追踪SBI-06上DNA的起源，从祖先跨越Ma1和Dw2到BTx623和IS3620C。 DG标记分析确定了两个错配区域的正确位置，并在高粱参考基因组序列中定位了七个超级重叠群。结论DG技术提供了一种经济高效的方法来快速生成高粱中准确的基因分型数据。目前，来自DG的数据用于许多基于标记的分析，包括标记辅助育种，系谱和QTL分析，遗传图谱构建，基于图谱的基因克隆和关联研究。 DG与全基因组重测序相结合极大地促进了高粱遗传分析的各个方面，高粱是C 4 草种的重要遗传参考。

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