Construction and characterization of a rice YAC library for physical mapping

• Published in: Molecular breeding 1995-03, Vol.1 (1), p.79-89
• Main Authors: Umehara, Y, Inagaki, A, Tanoue, H, Yasukochi, Y, Nagamura, Y, Saji, S, Otsuki, Y, Fujimura, T, Kurata, N, Minobe, Y
• Format: Article
• Language: English
• Subjects: chromosome mapping; chromosomes; DNA libraries; genetic markers; linkage groups; molecular genetics; Oryza sativa; restriction fragment length polymorphism; Saccharomyces cerevisiae; yeast artificial chromosomes
• ISSN: 1380-3743; 1572-9788
• DOI: 10.1007/bf01682091

Genomic libraries of rice, Oryza sativa L. cv. Nipponbare, in yeast artificial chromosomes were prepared for construction of a rice physical map. High-molecular-weight genomic DNA was extracted from cultured suspension cells embedded in agarose plugs. After size fractionation of the Eco RI- and Not I-digested DNA fragments, they were ligated with pYAC4 and pYAC55, respectively, and used to transform Saccharomyces cerevisiae AB1380. A total of 6932 clones were obtained containing on average ca. 350 kb DNA. The YAC library was estimated to contain six haploid genome equivalents. The YACs were examined for their chimerism by mapping both ends on an RFLP linkage map. Most YACs with Eco RI fragments below 400 kb were intact colinear clones. About 40% of clones were chimeric. Genetic mapping of end clones from large size YACs revealed that the physical distance corresponding to 1 cM genetic distance varies from 120 to 1000 kb, depending on the chromosome region. To select and order YAC clones for making contig maps, high-density colony hybridization using ECL was applied. With several probes, at least one and at most ten YAC clones could be selected in this library. The library size and clone insert size indicate that this YAC library is suitable for physical map construction and map-based cloning.