Kim Jeong-Soon, Childs Kevin L, Islam-Faridi M Nurul, Menz Monica A, Klein Robert R, Klein Patricia E, Price H James, Mullet John E, Stelly David M
Department of Soil and Crop Sciences, Texas A&M University, College Station 77843, USA.
Genome. 2002 Apr;45(2):402-12. doi: 10.1139/g01-141.
The reliability of genome analysis and proficiency of genetic manipulation are increased by assignment of linkage groups to specific chromosomes, placement of centromeres, and orientation with respect to telomeres. We have endeavored to establish means to enable these steps in sorghum (Sorghum bicolor (L.) Moench), the genome of which contains ca. 780 Mbp spread across n = 10 chromosomes. Our approach relies on fluorescence in situ hybridization (FISH) and integrated structural genomic resources, including large-insert genomic clones in bacterial artificial chromosome (BAC) libraries. To develop robust FISH probes, we selected sorghum BACs by association with molecular markers that map near the ends of linkage groups, in regions inferred to be high in recombination. Overall, we selected 22 BACs that encompass the 10 linkage groups. As a prelude to development of a multiprobe FISH cocktail, we evaluated BAC-derived probes individually and in small groups. Biotin- and digoxygenin-labeled probes were made directly from the BAC clones and hybridized in situ to chromosomes without using suppressive unlabelled C0t-1 DNA. Based on FISH-signal strength and the relative degree of background signal, we judged 19 BAC-derived probes to be satisfactory. Based on their relative position, and collective association with all 10 linkage groups, we chose 17 of the 19 BACs to develop a 17-locus probe cocktail for dual-color detection. FISH of the cocktail allowed simultaneous identification of all 10 chromosomes. The results indicate that linkage and physical maps of sorghum allow facile selection of BAC clones according to position and FISH-signal quality. This capability will enable development of a high-quality molecular cytogenetic map and an integrated genomics system for sorghum, without need of chromosome flow sorting or microdissection. Moreover, transgeneric FISH experiments suggest that the sorghum system might be applicable to other Gramineae.
通过将连锁群分配到特定染色体、确定着丝粒位置以及相对于端粒的方向，基因组分析的可靠性和基因操作的熟练度得以提高。我们致力于建立在高粱（Sorghum bicolor (L.) Moench）中实现这些步骤的方法，高粱基因组包含约780兆碱基对，分布在n = 10条染色体上。我们的方法依赖于荧光原位杂交（FISH）和整合的结构基因组资源，包括细菌人工染色体（BAC）文库中的大插入基因组克隆。为了开发强大的FISH探针，我们通过与位于连锁群末端附近、推断重组率高的区域的分子标记相关联来选择高粱BAC。总体而言，我们选择了22个涵盖10个连锁群的BAC。作为开发多探针FISH混合物的前奏，我们单独和分组评估了BAC衍生的探针。生物素和地高辛标记的探针直接从BAC克隆制备，并原位杂交到染色体上，无需使用抑制性未标记的C0t-1 DNA。根据FISH信号强度和背景信号的相对程度，我们判断19个BAC衍生的探针是令人满意的。基于它们的相对位置以及与所有10个连锁群的集体关联，我们从19个BAC中选择了17个来开发用于双色检测的17位点探针混合物。该混合物的FISH允许同时鉴定所有10条染色体。结果表明，高粱的连锁图谱和物理图谱允许根据位置和FISH信号质量轻松选择BAC克隆。这种能力将使高粱能够开发高质量的分子细胞遗传图谱和整合基因组系统，而无需染色体流式分选或显微切割。此外，跨属FISH实验表明高粱系统可能适用于其他禾本科植物。
