National Key Laboratory for Germplasm Innovation and Utilization for Fruit and Vegetable Horticultural Crops, Hubei Hongshan Laboratory, Wuhan, Hubei 430070, China; College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China.
College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China.
J Genet Genomics. 2023 Jun;50(6):410-421. doi: 10.1016/j.jgg.2022.12.003. Epub 2023 Jan 4.
Citrus sinensis is the most cultivated and economically valuable Citrus species in the world, whose genome has been assembled by three generation sequencings. However, chromosome recognition remains a problem due to the small size of chromosomes, and difficulty in differentiating between pseudo and real chromosomes because of a highly heterozygous genome. Here, we employ fluorescence in situ hybridization (FISH) with 9 chromosome painting probes, 30 oligo pools, and 8 repetitive sequences to visualize 18 chromosomes. Then, we develop an approach to identify each chromosome in one cell through single experiment of oligo-FISH and Chromoycin A3 (CMA) staining. By this approach, we construct a high-resolution molecular cytogenetic map containing the physical positions of CMA banding and 38 sequences of FISH including centromere regions, which enables us to visualize significant differences between homologous chromosomes. Based on the map, we locate several highly repetitive sequences on chromosomes and estimate sizes and copy numbers of each site. In particular, we discover the translocation regions of chromosomes 4 and 9 in C. sinensis "Valencia." The high-resolution molecular cytogenetic map will help improve understanding of sweet orange genome assembly and also provide a fundamental reference for investigating chromosome evolution and chromosome engineering for genetic improvement in Citrus.
甜橙是世界上种植面积最广、经济价值最高的柑橘属物种，其基因组已通过三代测序组装完成。然而，由于染色体较小，以及高度杂合的基因组导致真假染色体难以区分，因此染色体识别仍然是一个问题。在这里，我们使用荧光原位杂交（FISH）技术，结合 9 个染色体涂染探针、30 个寡核苷酸池和 8 个重复序列，对 18 条染色体进行可视化。然后，我们开发了一种通过寡核苷酸 FISH 和 Chromoycin A3（CMA）染色的单次实验来识别每个细胞中染色体的方法。通过这种方法，我们构建了一个高分辨率的分子细胞遗传学图谱，包含 CMA 带的物理位置和 38 个 FISH 序列，包括着丝粒区域，这使我们能够可视化同源染色体之间的显著差异。基于该图谱，我们定位了染色体上的几个高度重复序列，并估计了每个位点的大小和拷贝数。特别是，我们在甜橙“瓦伦西亚”中发现了染色体 4 和 9 的易位区域。该高分辨率的分子细胞遗传学图谱将有助于提高对甜橙基因组组装的理解，也为研究柑橘染色体进化和染色体工程以进行遗传改良提供了基本参考。
