Čeština
EnglishConstruction of a subgenomic BAC library specific for chromosomes 1D, 4D and 6D of hexaploid wheat
Janda, Jaroslav; Bartoš, Jan; Šafář, Jan; Kubaláková, Marie; Valárik, Miroslav; Číhalíková, Jarmila; Šimková, Hana; Caboche, M.; Sourdille, P.; Bernard, M.; Chalhoub, B.; Doležel, Jaroslav
THEORETICAL AND APPLIED GENETICS 109: 1337-1345, 2004
Keywords: wheat; subgenomic BAC library; flow cytometry
Abstract: The analysis of the hexaploid wheat genome (Triticum aestium L., 2n = 6x = 42) is hampered by its large size (16,974 Mb / 1C) and presence of three homoeologous genomes (A, B and D). One of the possible strategies is a targeted approach based on subgenomic libraries of large DNA inserts. In this work, we purified by flow cytometry a total of 107 of three wheat D-genome chromosomes: 1D, 4D and 6D. The library (designated as TA-subD) consists of 87,168 clones with an average insert size of 85 kb. Among these clones, 53 % had inserts larger than 100 kb, only 29% of inserts being shorter than 75 kb. The coverage was estimated to be 3.4×, giving a 96.5% probability of identifying a clone corresponding to any sequence on the three chromosomes. Specificity for chromosomes 1D, 4D and 6D was confirmed after screening the library pools with single-locus microsatellite markers. The screening indicated that the library was not biased and gave an estimated coverage of 6-fold. This is the second report on BAC library construction from flow-sorted plant chromosomes, which confirms that dissecting of the complex wheat genome and preparation of subgenomic BAC libraries is possible.
DOI:
IEB authors: Jan Bartoš, Jaroslav Doležel, Jan Šafář, Hana Šimková, Miroslav Valárik
THEORETICAL AND APPLIED GENETICS 109: 1337-1345, 2004
Keywords: wheat; subgenomic BAC library; flow cytometry
Abstract: The analysis of the hexaploid wheat genome (Triticum aestium L., 2n = 6x = 42) is hampered by its large size (16,974 Mb / 1C) and presence of three homoeologous genomes (A, B and D). One of the possible strategies is a targeted approach based on subgenomic libraries of large DNA inserts. In this work, we purified by flow cytometry a total of 107 of three wheat D-genome chromosomes: 1D, 4D and 6D. The library (designated as TA-subD) consists of 87,168 clones with an average insert size of 85 kb. Among these clones, 53 % had inserts larger than 100 kb, only 29% of inserts being shorter than 75 kb. The coverage was estimated to be 3.4×, giving a 96.5% probability of identifying a clone corresponding to any sequence on the three chromosomes. Specificity for chromosomes 1D, 4D and 6D was confirmed after screening the library pools with single-locus microsatellite markers. The screening indicated that the library was not biased and gave an estimated coverage of 6-fold. This is the second report on BAC library construction from flow-sorted plant chromosomes, which confirms that dissecting of the complex wheat genome and preparation of subgenomic BAC libraries is possible.
DOI: