Data from: The HypoMethylated Partial Restriction (HMPR) method reduces the repetitive content of genomic libraries in Norway spruce (Picea abies)

To evaluate the usefulness of Reduced Representation Libraries (RRL) in species with large and highly repetitive genomes such as conifers, we employed Hypomethylated Partial Restriction (HMPR) on the genome of Norway spruce (Picea abies). The HMPR method preferentially removes the repetitive fraction of the genome, which is commonly hypermethylated. Hence, RRLs should be enriched for the hypomethylated gene space. For comparison a standard shotgun library was constructed and samples of the respective libraries were obtained through Sanger sequencing. We obtained a nine-fold gene enrichment, a value which is slightly higher than for other plant species. The higher relative efficiency of HMPR is probably a consequence of the large Norway spruce genome size since the probability of finding genes in a standard shotgun library is very small. The amount of repetitive DNA was reduced by 45% in the RRLs, demonstrating the ability to efficiently remove hypermethylated DNA. Annotating sequences in an uncharacterized genome remains challenging and a large number of sequences could not be classified as either repetitive DNA or as belonging to the gene space. Upon further investigation, we found that some of these uncharacterized fragments were expressed, and in most cases the expression was spatially differentiated, indicating that they might have a function. Full-length transcripts of a subset of expressed fragments also revealed that these could be long non-coding RNAs. Long non-coding RNAs have been shown to be involved in gene regulation and deserve future attention. In conclusion, our study shows that the HMPR method is effective in constructing libraries enriched for the genic fraction of the genome, while simultaneously reducing the repetitive fraction, in P. abies and may prove a valuable tool for the discovery, validation and assessment of genetic markers in population studies when combined with next generation sequencing technology.

为评估简化代表文库（Reduced Representation Libraries，RRL）在针叶树等大型高重复基因组物种中的应用价值，我们针对挪威云杉（Picea abies）的基因组采用了低甲基化部分酶切（Hypomethylated Partial Restriction，HMPR）技术。该方法可优先去除基因组中普遍呈高甲基化状态的重复序列组分，因此RRL应能富集低甲基化的基因空间区域。为设置对照，我们同时构建了标准鸟枪法文库，并通过桑格测序（Sanger sequencing）获取了两类文库的测序样本。本实验实现了9倍的基因富集效果，该数值略高于其他植物物种的相关报道。HMPR相对效率更高的原因，大概率与挪威云杉庞大的基因组尺寸相关——在标准鸟枪法文库中获取基因序列的概率本就极低。RRL中重复DNA的占比降低了45%，证实该方法可高效去除高甲基化DNA序列。对未特征化基因组的序列进行注释仍存在较大挑战，大量序列无法被归类为重复DNA或基因空间组分。经进一步研究发现，部分未特征化的片段具有表达活性，且多数呈现空间表达分化特征，提示其可能具备生物学功能。对部分表达片段的全长转录本分析显示，这类序列可归类为长链非编码RNA（long non-coding RNAs）。已有研究证实，长链非编码RNA参与基因调控过程，值得后续深入关注。综上，本研究证实，HMPR方法可在挪威云杉中有效构建富集基因组基因组分、同时降低重复序列占比的文库；若结合下一代测序技术（next generation sequencing technology），该方法可作为种群研究中遗传标记发现、验证与评估的实用工具。