J Integr Plant Biol. ›› 2010, Vol. 52 ›› Issue (7): 679-691.DOI: 10.1111/j.1744-7909.2010.00954.x

• Molecular Ecology and Evolution • Previous Articles    

Effective Isolation of Retrotransposons and Repetitive DNA Families from the Wheat Genome

Motonori Tomita* *, Munenori Asao and Aya Kuraki   

  1. Molecular Genetics Laboratory, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan

Abstract:

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 EcoO109I, 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 Ty1-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, MboI, 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.

Tomita M, Asao M, Kuraki A (2010) Effective isolation of retrotransposons and repetitive DNA families from the wheat genome. J. Integr. Plant Biol. 52(7), 679–691.

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