Conditions of the PCR amplification.

In the Japanese hagfish, Eptatretus burgeri, approximately 21% of the genomic DNA in germ cells (2n = 52) consists of 16 chromosomes (eliminated [E]-chromosomes) that are eliminated from presumptive somatic cells (2n = 36). To uncover the eliminated genome (E-genome), we have identified 16 eliminated repetitive DNA families from eight hagfish species, with 11 of these repeats being selectively amplified in the germline genome of E. burgeri. Furthermore, we have demonstrated that six of these sequences, namely EEEb1–6, are exclusively localized on all 16 E-chromosomes. This has led to the hypothesis that the eight pairs of E-chromosomes are derived from one pair of ancestral chromosomes via multiple duplication events over a prolonged evolutionary period. NGS analysis has recently facilitated the re-assembly of two distinct draft genomes of E. burgeri, derived from the testis and liver. This advancement allows for the prediction of not only nonrepetitive eliminated sequences but also over 100 repetitive and eliminated sequences, accomplished through K-mer-based analysis. In this study, we report four novel eliminated repetitive DNA sequences (designated as EEEb7–10) and confirm the relative chromosomal localization of all eliminated repeats (EEEb1–10) by fluorescence in situ hybridization (FISH). With the exception of EEEb10, all sequences were exclusively detected on EEEb1-positive chromosomes. Surprisingly, EEEb10 was detected as an intense signal on EEEb1-positive chromosomes and as a scattered signal on other chromosomes in germ cells. The study further divided the eight pairs of E-chromosomes into six groups based on the signal distribution of each DNA family, and fiber-FISH experiments showed that the EEEb2–10 family was dispersed in the EEEb1-positive extended chromatin fiber. These findings provide new insights into the mechanisms underlying chromosome elimination and the evolution of E-chromosomes, supporting our previous hypothesis.

在蒲氏粘盲鳗（Eptatretus burgeri）中，生殖细胞（二倍体染色体数2n=52）的基因组DNA中约有21%来自16条染色体（即消除型[E-染色体]），这类染色体会在预定体细胞（二倍体染色体数2n=36）中被清除。为解析该消除型基因组（E-genome），我们从8种盲鳗中鉴定出16个消除型重复DNA家族，其中11个家族在蒲氏粘盲鳗的生殖系基因组中被选择性扩增。此外，我们证实其中6个序列（命名为EEEb1–6）仅定位于全部16条E-染色体上。这一结果支撑了如下假说：8对E-染色体起源于1对祖先染色体，经过长期演化过程中的多次重复事件形成。近期，下一代测序（NGS）技术助力完成了蒲氏粘盲鳗的两套独立草稿基因组组装，其样本分别取自精巢与肝脏。这一技术进展不仅支持了非重复消除序列的预测，还通过基于K-mer的分析手段，实现了100余个重复型消除序列的预测。本研究中，我们报道了4个全新的消除型重复DNA序列（命名为EEEb7–10），并通过荧光原位杂交（fluorescence in situ hybridization, FISH）验证了所有消除型重复序列（EEEb1–10）的相对染色体定位。除EEEb10外，其余序列均仅在EEEb1阳性染色体上被检测到。令人意外的是，在生殖细胞中，EEEb10不仅在EEEb1阳性染色体上呈现强烈信号，还在其他染色体上呈现弥散信号。本研究进一步根据每个DNA家族的信号分布模式，将8对E-染色体划分为6个组；纤维荧光原位杂交（fiber-FISH）实验显示，EEEb2–10家族分散分布于EEEb1阳性的伸展染色质纤维中。上述研究结果为染色体消除机制与E-染色体演化提供了新的见解，进一步支撑了我们此前提出的假说。