New classes of repetitive DNA elements were effectively identified by isolating small fragments of the elements from the wheat genome. A wheat A genome library was constructed from Triticum monococcum by degenerate cl...New classes of repetitive DNA elements were effectively identified by isolating small fragments of the elements from the wheat genome. A wheat A genome library was constructed from Triticum monococcum by degenerate cleavage with EcoO1091, the recognition sites of which consisted of 5＇-PuGGNCCPy-3＇ multi-sequences. Three novel repetitive sequences pTm6, pTm69 and pTm58 derived from the A genome were screened and tested for high copy number using a blotting approach, pTm6 showed identity with integrase domains of the barley Tyl-Copia-retrotransposon BARE-1 and pTm58 showed similarity to the barley Ty3-gypsy-like retrotransposon Romani. pTm69, however, constituted a tandem array with useful genomic specificities, but did not share any identity with known repetitive elements. This study also sought to isolate wheat D-genome-specific repetitive elements regardless of the level of methylation, by genomic subtraction. Total genomic DNA of Aegilops tauschii was cleaved into short fragments with a methylation-insensitive 4 bp cutter, Mbol, and then common DNA sequences between Ae. tauschii and Triticum turgidum were subtracted by annealing with excess T. turgidum genomic DNA. The D genome repetitive sequence pAt1 was isolated and used to identify an additional novel repetitive sequence family from wheat bacterial artificial chromosomes with a size range of 1 395-1 850 bp. The methods successfully led pathfinding of two unique repetitive families.展开更多
基金Supported by Grants-in-Aid for Scientific Research (01760004 and 04760006) from the Japanese Ministry of Education,Culture,Sports,Science and Technology (MEXT)
文摘New classes of repetitive DNA elements were effectively identified by isolating small fragments of the elements from the wheat genome. A wheat A genome library was constructed from Triticum monococcum by degenerate cleavage with EcoO1091, the recognition sites of which consisted of 5＇-PuGGNCCPy-3＇ multi-sequences. Three novel repetitive sequences pTm6, pTm69 and pTm58 derived from the A genome were screened and tested for high copy number using a blotting approach, pTm6 showed identity with integrase domains of the barley Tyl-Copia-retrotransposon BARE-1 and pTm58 showed similarity to the barley Ty3-gypsy-like retrotransposon Romani. pTm69, however, constituted a tandem array with useful genomic specificities, but did not share any identity with known repetitive elements. This study also sought to isolate wheat D-genome-specific repetitive elements regardless of the level of methylation, by genomic subtraction. Total genomic DNA of Aegilops tauschii was cleaved into short fragments with a methylation-insensitive 4 bp cutter, Mbol, and then common DNA sequences between Ae. tauschii and Triticum turgidum were subtracted by annealing with excess T. turgidum genomic DNA. The D genome repetitive sequence pAt1 was isolated and used to identify an additional novel repetitive sequence family from wheat bacterial artificial chromosomes with a size range of 1 395-1 850 bp. The methods successfully led pathfinding of two unique repetitive families.
