Institute of Botany and Landscape Ecology, University of Greifswald, Soldmannstr. 15, Greifswald, 17489, Germany.
Department of Vertebrate Zoology, Saint Petersburg State University, Universitetskaya nab. 7/9, Saint Petersburg, 199034, Russia.
Mol Biol Rep. 2023 Dec 29;51(1):36. doi: 10.1007/s11033-023-08913-4.
BACKGROUND: Ellobius talpinus is a subterranean rodent representing an attractive model in population ecology studies due to its highly special lifestyle and sociality. In such studies, mitochondrial DNA (mtDNA) is widely used. However, if nuclear copies of mtDNA, aka NUMTs, are present, they may co-amplify with the target mtDNA fragment, generating misleading results. The aim of this study was to determine whether NUMTs are present in E. talpinus. METHODS AND RESULTS: PCR amplification of the putative mtDNA CytB-D-loop fragment using 'universal' primers from 56 E. talpinus samples produced multiple double peaks in 90% of the sequencing chromatograms. To reveal NUMTs, molecular cloning and sequencing of PCR products of three specimens was conducted, followed by phylogenetic analysis. The pseudogene nature of three out of the seven detected haplotypes was confirmed by their basal positions in relation to other Ellobius haplotypes in the phylogenetic tree. Additionally, 'haplotype B' was basal in relation to other E. talpinus haplotypes and found present in very distant sampling sites. BLASTN search revealed 195 NUMTs in the E. talpinus nuclear genome, including fragments of all four PCR amplified pseudogenes. Although the majority of the NUMTs studied were short, the entire mtDNA had copies in the nuclear genome. The most numerous NUMTs were found for rrnL, COXI, and D-loop. CONCLUSIONS: Numerous NUMTs are present in E. talpinus and can be difficult to discriminate against mtDNA sequences. Thus, in future population or phylogenetic studies in E. talpinus, the possibility of cryptic NUMTs amplification should always be taken into account.
背景:艾鼬是一种穴居啮齿动物,因其高度特殊的生活方式和社会性,成为种群生态学研究中极具吸引力的模式生物。在这些研究中,线粒体 DNA(mtDNA)被广泛应用。然而,如果存在核拷贝的 mtDNA,即 NUMTs,它们可能会与目标 mtDNA 片段一起扩增,产生误导性的结果。本研究旨在确定艾鼬是否存在 NUMTs。
方法和结果:使用来自 56 个艾鼬样本的“通用”引物对假定的 mtDNA CytB-D-loop 片段进行 PCR 扩增,90%的测序图谱中出现了多个双峰。为了揭示 NUMTs,对三个标本的 PCR 产物进行了分子克隆和测序,然后进行了系统发育分析。在所检测到的七个单倍型中,有三个是假基因,这是通过它们在系统发育树中与其他艾鼬单倍型的相对基础位置来证实的。此外,“单倍型 B”在与其他艾鼬单倍型的关系中处于基础位置,并且在非常遥远的采样地点发现。BLASTN 搜索显示,艾鼬的核基因组中有 195 个 NUMTs,包括所有四个扩增的假基因片段。尽管所研究的 NUMTs 大多数较短,但整个 mtDNA 都有核基因组的副本。在 rrnL、COXI 和 D-loop 中发现了最多的 NUMTs。
结论:艾鼬中存在大量的 NUMTs,并且很难与 mtDNA 序列区分开来。因此,在未来的艾鼬种群或系统发育研究中,应该始终考虑到隐匿的 NUMTs 扩增的可能性。
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