Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan.
PLoS One. 2021 Sep 30;16(9):e0258028. doi: 10.1371/journal.pone.0258028. eCollection 2021.
Lycoris species have various chromosome numbers and karyotypes, but all have a constant total number of chromosome major arms. In addition to three fundamental types, including metacentric (M-), telocentric (T-), and acrocentric (A-) chromosomes, chromosomes in various morphology and size were also observed in natural populations. Both fusion and fission translocation have been considered as main mechanisms leading to the diverse karyotypes among Lycoris species, which suggests the centromere organization playing a role in such arrangements. We detected several chromosomal structure changes in Lycoris including centric fusion, inversion, gene amplification, and segment deletion by using fluorescence in situ hybridization (FISH) probing with rDNAs. An antibody against centromere specific histone H3 (CENH3) of L. aurea (2n = 14, 8M+6T) was raised and used to obtain CENH3-associated DNA sequences of L. aurea by chromatin immunoprecipitation (ChIP) cloning method. Immunostaining with anti-CENH3 antibody could label the centromeres of M-, T-, and A-type chromosomes. Immunostaining also revealed two centromeres on one T-type chromosome and a centromere on individual mini-chromosome. Among 10,000 ChIP clones, 500 clones which showed abundant in L. aurea genome by dot-blotting analysis were FISH mapped on chromosomes to examine their cytological distribution. Five of these 500 clones could generate intense FISH signals at centromeric region on M-type but not T-type chromosomes. FISH signals of these five clones rarely appeared on A-type chromosomes. The five ChIP clones showed similarity in DNA sequences and could generate similar but not identical distribution patterns of FISH signals on individual chromosomes. Furthermore, the distinct distribution patterns of FISH signals on each chromosome generated by these five ChIP clones allow to identify individual chromosome, which is considered difficult by conventional staining approaches. Our results suggest a different organization of centromeres of the three chromosome types in Lycoris species.
石蒜属植物具有不同的染色体数目和核型，但所有的染色体主要臂的总数是恒定的。除了三种基本类型，包括着丝粒（M-）、端着丝粒（T-）和近端着丝粒（A-）染色体外，在自然种群中还观察到各种形态和大小的染色体。融合和裂变易位都被认为是导致石蒜属植物多样核型的主要机制，这表明着丝粒组织在这种排列中起作用。我们通过使用 rDNA 的荧光原位杂交（FISH）探测，在石蒜属植物中检测到包括着丝粒融合、倒位、基因扩增和片段缺失在内的几种染色体结构变化。针对 L. aurea（2n = 14，8M+6T）的着丝粒特异性组蛋白 H3（CENH3）的抗体被制备出来，并用于通过染色质免疫沉淀（ChIP）克隆方法获得 L. aurea 的 CENH3 相关 DNA 序列。抗 CENH3 抗体的免疫染色可以标记 M-、T-和 A-型染色体的着丝粒。免疫染色还揭示了一个 T-型染色体上的两个着丝粒和一个单个小型染色体上的一个着丝粒。在 10000 个 ChIP 克隆中，通过点印迹分析显示在 L. aurea 基因组中丰富的 500 个克隆被 FISH 映射到染色体上，以检查它们的细胞学分布。这 500 个克隆中的 5 个可以在 M-但不在 T-型染色体的着丝粒区产生强烈的 FISH 信号。这些 5 个克隆的 FISH 信号在 A-型染色体上很少出现。这 5 个 ChIP 克隆在 DNA 序列上具有相似性，并且可以在单个染色体上产生相似但不相同的 FISH 信号分布模式。此外，这些 5 个 ChIP 克隆在每条染色体上产生的独特的 FISH 信号分布模式可以识别单个染色体，这是通过传统染色方法难以做到的。我们的结果表明，石蒜属植物的三种染色体类型的着丝粒具有不同的组织。
