Table_4.docx

Saccharum spontaneum is a major Saccharum species that contributed to the origin of modern sugarcane cultivars, and due to a high degree of polyploidy is considered to be a plant species with one of the most complex genetics. Fluorescence in situ hybridization (FISH) is a powerful and widely used tool in genome studies. Here, we demonstrated that FISH based on bacterial artificial chromosome (BAC) clones can be used as a specific cytological marker to identify S. spontaneum individual chromosomes and study the relationship between S. spontaneum and other related species. We screened low-copy BACs as probes from the sequences of a high coverage of S. spontaneum BAC library based on BLAST search of the sorghum genome. In total, we isolated 49 positive BAC clones, and identified 27 BAC clones that can give specific signals on the S. spontaneum chromosomes. Of the 27 BAC probes, 18 were confirmed to be able to discriminate the eight basic chromosomes of S. spontaneum. Moreover, BAC-24, BAC-66, BAC-78, BAC-69, BAC-71, BAC-73, and BAC-77 probes were used to construct physical maps of chromosome 1 and chromosome 2 of S. spontaneum, which indicated synteny in Sb01 between S. spontaneum and sorghum. Furthermore, we found that BAC-14 and BAC-19 probes, corresponding to the sorghum chromosomes 2 and 8, respectively, localized to different arms of the same S. spontaneum chromosome, suggesting that there was an inter-chromosomal rearrangement event between S. spontaneum and sorghum. Our study provides the first set of chromosome-specific cytogenetic markers in Saccharum and is critical for future advances in cytogenetics and genome sequencing studies in Saccharum.

甜根子草（Saccharum spontaneum）是甘蔗属的主要物种之一，其参与了现代甘蔗栽培品种的起源演化；由于其具有高度的多倍体性，被认为是遗传组成最为复杂的植物物种之一。荧光原位杂交（Fluorescence in situ hybridization, FISH）是基因组研究中功能强大且应用广泛的技术手段。本研究证实，基于细菌人工染色体（bacterial artificial chromosome, BAC）克隆的FISH技术可作为特异性细胞学标记，用于鉴定甜根子草的单个染色体，并探究甜根子草与其他近缘物种的演化关系。研究人员基于高粱基因组的BLAST比对，从高覆盖度的甜根子草BAC文库序列中筛选出低拷贝BAC作为探针。最终共获得49个阳性BAC克隆，并鉴定出27个可在甜根子草染色体上产生特异性信号的BAC克隆。在这27个BAC探针中，有18个可有效区分甜根子草的8条基础染色体。此外，使用BAC-24、BAC-66、BAC-78、BAC-69、BAC-71、BAC-73和BAC-77探针，构建了甜根子草第1号和第2号染色体的物理图谱，结果表明甜根子草与高粱在Sb01区域存在共线性关系。进一步研究发现，分别对应高粱第2号和第8号染色体的BAC-14与BAC-19探针，定位在了甜根子草的同一条染色体的不同臂上，这暗示甜根子草与高粱之间曾发生过染色体间的重排事件。本研究首次开发了甘蔗属物种的染色体特异性细胞遗传标记，对后续甘蔗属的细胞遗传学研究及基因组测序工作具有重要的支撑与推动价值。