DataSheet1_Extension of Mitogenome Enrichment Based on Single Long-Range PCR: mtDNAs and Putative Mitochondrial-Derived Peptides of Five Rodent Hibernators.zip

Enriching mitochondrial DNA (mtDNA) for sequencing entire mitochondrial genomes (mitogenomes) can be achieved by single long-range PCR. This avoids interference from the omnipresent nuclear mtDNA sequences (NUMTs). The approach is currently restricted to the use of samples collected from humans and ray-finned fishes. Here, we extended the use of single long-range PCR by introducing back-to-back oligonucleotides that target a sequence of extraordinary homology across vertebrates. The assay was applied to five hibernating rodents, namely alpine marmot, Arctic and European ground squirrels, and common and garden dormice, four of which have not been fully sequenced before. Analysis of the novel mitogenomes focussed on the prediction of mitochondrial-derived peptides (MDPs) providing another level of information encoded by mtDNA. The comparison of MOTS-c, SHLP4 and SHLP6 sequences across vertebrate species identified segments of high homology that argue for future experimentation. In addition, we evaluated four candidate polymorphisms replacing an amino acid in mitochondrially encoded subunits of the oxidative phosphorylation (OXPHOS) system that were reported in relation to cold-adaptation. No obvious pattern was found for the diverse sets of mammalian species that either apply daily or multiday torpor or otherwise cope with cold. In summary, our single long-range PCR assay applying a pair of back-to-back primers that target a consensus sequence motif of Vertebrata has potential to amplify (intact) mitochondrial rings present in templates from a taxonomically diverse range of vertebrates. It could be promising for studying novel mitogenomes, mitotypes of a population and mitochondrial heteroplasmy in a sensitive, straightforward and flexible manner.

通过单组长距聚合酶链式反应（single long-range PCR），可实现线粒体DNA（mtDNA）的富集，用于完整线粒体基因组（mitogenomes）的测序。该方法可规避无处不在的核线粒体DNA序列（NUMTs）带来的干扰。目前，此方法仅适用于人类和辐鳍鱼类的样本。
本研究通过引入靶向脊椎动物间极高同源性序列的背向寡核苷酸引物，拓展了单组长距PCR的应用范围。我们将该检测方法应用于五种冬眠啮齿类动物，分别为阿尔卑斯旱獭、北极地松鼠与欧洲地松鼠，以及普通睡鼠和园睡鼠，其中四种的完整线粒体基因组此前尚未被测序。
对新获得的线粒体基因组的分析聚焦于线粒体衍生肽（MDPs）的预测，这为线粒体DNA所编码的信息提供了另一维度的研究内容。通过比对脊椎动物物种间的MOTS-c、SHLP4及SHLP6序列，我们鉴定出了高度同源的区段，这为后续实验研究提供了支撑。
此外，我们评估了四种与冷适应相关的候选多态性位点，这些位点可替换氧化磷酸化（OXPHOS）系统的线粒体编码亚基的氨基酸。针对采用每日蛰伏或多日蛰伏，或以其他方式适应寒冷的多样哺乳动物类群，未发现明显的相关性模式。
综上，我们开发的单组长距PCR检测方法，通过使用一对靶向脊椎动物共有序列基序的背向引物，能够扩增来自分类学上多样的脊椎动物模板中的完整线粒体环状基因组。该方法有望以灵敏、简便且灵活的方式，用于研究新的线粒体基因组、种群线粒体基因型以及线粒体异质性。