Rapid Genome-wide Single Nucleotide Polymorphism Discovery in Soybean and Rice via Deep Resequencing of Reduced Representation Libraries with the Illumina Genome Analyzer
Rapid Genome-wide Single Nucleotide Polymorphism Discovery in Soybean and Rice via Deep Resequencing of Reduced Representation Libraries with the Illumina Genome Analyzer
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DOI：
10.3835/plantgenome2009.09.0026
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发表时间：
2010-07-01
期刊：
影响因子：
4.2
通讯作者：
Campbell, Matthew A.
中科院分区：
文献类型：
--
作者：
Deschamps, Stephane;la Rota, Mauricio;Campbell, Matthew A.
大规模并行测序平台允许快速发现物种内相关基因型之间的单核苷酸多态性（SNP）。我们描述了减少代表性文库（RRL）的创建使用的甲基化敏感的限制性内切酶，然后由二次消化与4 bp-限制性内切酶Dpn II的核基因组DNA的初始消化。该策略允许富集低甲基化的基因组DNA，其已被证明富含基因序列，并且用Dpn II消化用于增加个体之间重新测序的共同基因座的数量。用Illumina基因组分析仪对这些RRL进行深度重测序，鉴定出水稻中的2618个SNP和大豆中的1682个SNP，用于每个物种中的两个代表性基因型。通过桑格测序验证了这些SNP的一个子集，在水稻（Oryza sativa）和大豆（Glycine max）中的验证率分别为96.4%和97.0%。相对于参考基因组组装体中注释基因的读段分布的比较分析表明，RRL策略主要是在两个物种的基因区域内取样。用于全基因组SNP发现的甲基化敏感性RRL的大规模平行测序可以应用于具有足够参考基因组序列的广泛植物物种。
Massively parallel sequencing platforms have allowed for the rapid discovery of single nucleotide polymorphisms (SNPs) among related genotypes within a species. We describe the creation of reduced representation libraries (RRLs) using an initial digestion of nuclear genomic DNA with a methylation-sensitive restriction endonuclease followed by a secondary digestion with the 4bp-restriction endonuclease DpnII. This strategy allows for the enrichment of hypomethylated genomic DNA, which has been shown to be rich in genic sequences, and the digestion with DpnII serves to increase the number of common loci resequenced between individuals. Deep resequencing of these RRLs performed with the Illumina Genome Analyzer led to the identification of 2618 SNPs in rice and 1682 SNPs in soybean for two representative genotypes in each of the species. A subset of these SNPs was validated via Sanger sequencing, exhibiting validation rates of 96.4 and 97.0%, in rice (Oryza sativa) and soybean (Glycine max), respectively. Comparative analysis of the read distribution relative to annotated genes in the reference genome assemblies indicated that the RRL strategy was primarily sampling within genic regions for both species. The massively parallel sequencing of methylation-sensitive RRLs for genome-wide SNP discovery can be applied across a wide range of plant species having sufficient reference genomic sequence.