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Genomic Subtraction Recovers Rye-Specific DNA Elements Enriched in the Rye Genome

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Abstract

Repetitive DNA sequence families have been identified in methylated relic DNAs of rye. This study sought to isolate rye genome-specific repetitive elements regardless of the level of methylation, using a genomic subtraction method. The total genomic DNAs of rye-chromosome-addition-wheat lines were cleaved to short fragments with a methylation-insensitive 4-bp cutter, MboI, and then common DNA sequences between rye and wheat were subtracted by annealing with excess wheat genomic DNA. Four classes of rye-specific repetitive elements were successfully isolated from both the methylated and non-methylated regions of the genome. Annealing of the DNA mixture at a ratio of the enzyme-restricted fragments:the sonicated fragments (1:3–1:5) was key to this success. Two classes of repetitive elements identified here belong to representative repetitive families: the tandem 350-family and the dispersed R173 family. Southern blot hybridization patterns of the two repetitive elements showed distinct fragments in methylation-insensitive EcoO109I digests, but continuous smear signals in the methylation-sensitive PstI and SalI digests, indicating that both of the known families are contained in the methylated regions. The subtelomeric tandem 350-family is organized by multimers of a 380-bp-core unit defined by the restriction enzyme EcoO109I. The other two repetitive element classes had new DNA sequences (444, 89 bp) and different core-unit sizes, as defined by methylation-sensitive enzymes. The EcoO109I recognition sites consisting of PyCCNGGPu-multi sequences existed with high frequency in the four types of rye repetitive families and might be a useful tool for studying the genomic organization and differentiation of this species.

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Acknowledgment

This study was supported by Grants-in-Aid for Scientific Research (No. 01760004 and No. 04760006) from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT).

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  1. Molecular Genetics Laboratory, Faculty of Agriculture, Tottori University, 101, Minami 4-chome, Koyama-cho, Tottori, 680-8553, Japan

    Motonori Tomita, Keiichi Akai & Takayoshi Morimoto

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  1. Motonori Tomita
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Correspondence to Motonori Tomita.

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Tomita, M., Akai, K. & Morimoto, T. Genomic Subtraction Recovers Rye-Specific DNA Elements Enriched in the Rye Genome. Mol Biotechnol 42, 160–167 (2009). https://doi.org/10.1007/s12033-009-9151-2

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