Cross-species bacterial artificial chromosome (BAC) library screening via overgo-based hybridization and BAC-contig mapping of a yield enhancement quantitative trait locus (QTL) yld1.1 in the Malaysian wild rice Oryza rufipogon

Song, Beng-Kah and Nadarajah, Kalaivani and Romanov, Michael N and Ratnam, Wickneswari (2005) Cross-species bacterial artificial chromosome (BAC) library screening via overgo-based hybridization and BAC-contig mapping of a yield enhancement quantitative trait locus (QTL) yld1.1 in the Malaysian wild rice Oryza rufipogon. Cellular & Molecular Biology Letters, 10 (3). pp. 425-437. ISSN 1425-8153. (Access to this publication is currently restricted. You may be able to access a copy if URLs are provided)

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Abstract

The construction of BAC-contig physical maps is an important step towards a partial or ultimate genome sequence analysis. Here, we describe our initial efforts to apply an overgo approach to screen a BAC library of the Malaysian wild rice species, Oryza rufipogon. Overgo design is based on repetitive element masking and sequence uniqueness, and uses short probes (approximately 40 bp), making this method highly efficient and specific. Pairs of 24-bp oligos that contain an 8-bp overlap were developed from the publicly available genomic sequences of the cultivated rice, O. sativa, to generate 20 overgo probes for a 1-Mb region that encompasses a yield enhancement QTL yld1.1 in O. rufipogon. The advantages of a high similarity in melting temperature, hybridization kinetics and specific activities of overgos further enabled a pooling strategy for library screening by filter hybridization. Two pools of ten overgos each were hybridized to high-density filters representing the O. rufipogon genomic BAC library. These screening tests succeeded in providing 69 PCR-verified positive hits from a total of 23,040 BAC clones of the entire O. rufipogon library. A minimal tilling path of clones was generated to contribute to a fully covered BAC-contig map of the targeted 1-Mb region. The developed protocol for overgo design based on O. sativa sequences as a comparative genomic framework, and the pooled overgo hybridization screening technique are suitable means for high-resolution physical mapping and the identification of BAC candidates for sequencing.

Item Type: Article
Uncontrolled keywords: overgo; BAC library screening; Oryza rufipogon; Oryza sativa; pooled probes hybridization; yield QTL yld1.1; BAC-contig physical mapping
Subjects: Q Science > QH Natural history > QH426 Genetics
Divisions: Faculties > Sciences > School of Biosciences > Biomedical Research Group
Depositing User: M. Romanov
Date Deposited: 12 Dec 2013 16:48 UTC
Last Modified: 26 Apr 2018 10:02 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/37552 (The current URI for this page, for reference purposes)
Romanov, Michael N: https://orcid.org/0000-0003-3584-4644
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