Table_3_Trypanosoma cruzi Genomic Variability: Array Comparative Genomic Hybridization Analysis of Clone and Parental Strain.xlsx

Trypanosoma cruzi, the etiological agent of Chagas disease, exhibits extensive inter- and intrastrain genetic diversity. As we have previously described, there are some genetic differences between the parental G strain and its clone D11, which was isolated by the limiting dilution method and infection of cultured mammalian cells. Electrophoretic karyotyping and Southern blot hybridization of chromosomal bands with specific markers revealed chromosome length polymorphisms of small size with additional chromosomal bands in clone D11 and the maintenance of large syntenic groups. Both G strain and clone D11 belong to the T. cruzi lineage TcI. Here, we designed intraspecific array-based comparative genomic hybridization (aCGH) to identify chromosomal regions harboring copy-number variations between clone D11 and the G strain. DNA losses were more extensive than DNA gains in clone D11. Most alterations were flanked by repeated sequences from multigene families that could be involved in the duplication and deletion events. Several rearrangements were detected by chromoblot hybridization and confirmed by aCGH. We have integrated the information of genomic sequence data obtained by aCGH to the electrophoretic karyotype, allowing the reconstruction of possible recombination events that could have generated the karyotype of clone D11. These rearrangements may be explained by unequal crossing over between sister or homologous chromatids mediated by flanking repeated sequences and unequal homologous recombination via break-induced replication. The genomic changes detected by aCGH suggest the presence of a dynamic genome that responds to environmental stress by varying the number of gene copies and generating segmental aneuploidy.

Trypanosoma cruzi，克氏锥虫病（Chagas disease）的病原体，展现出广泛的种内和种间遗传多样性。正如我们之前所述，亲本菌株G与克隆D11之间存在一些遗传差异，克隆D11是通过限制稀释法从培养的哺乳动物细胞中分离出来的。通过电泳核型分析和Southern印迹杂交特定标记染色体带，揭示了克隆D11中的小型染色体长度多态性以及额外染色体带的维持，同时保持了大型同源连锁群的稳定。G菌株和克隆D11均属于克氏锥虫的TcI谱系。在此，我们设计了基于比较基因组杂交（aCGH）的种内阵列，以识别克隆D11与G菌株之间携带拷贝数变异的染色体区域。克隆D11中的DNA丢失比DNA获得更为广泛。大多数改变均由多基因家族的重复序列所包围，这些序列可能与复制和删除事件有关。通过染色质印迹杂交检测到几处重排，并通过aCGH得到证实。我们将aCGH获得的基因组序列数据整合到电泳核型中，从而重建了可能产生克隆D11核型的重组事件。这些重排可能由旁侧重复序列介导的姐妹染色单体或同源染色单体的不等交换以及通过断裂诱导复制的不等同源重组引起。aCGH检测到的基因组变化表明存在一个动态的基因组，它通过改变基因拷贝数和产生片段性非整倍体来响应环境压力。