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Construction and utility of 10-kb libraries for efficient clone-gap closure for rice genome sequencing

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Abstract

Rice is an important crop and a model system for monocot genomics, and is a target for whole genome sequencing by the International Rice Genome Sequencing Project (IRGSP). The IRGSP is using a clone by clone approach to sequence rice based on minimum tiles of BAC or PAC clones. For chromosomes 10 and 3 we are using an integrated physical map based on two fingerprinted and end-sequenced BAC libraries to identifying a minimum tiling path of clones. In this study we constructed and tested two rice genomic libraries with an average insert size of 10 kb (10-kb library) to support the gap closure and finishing phases of the rice genome sequencing project. The HaeIII library contains 166,752 clones covering approximately 4.6× rice genome equivalents with an average insert size of 10.5 kb. The Sau3AI library contains 138,960 clones covering 4.2× genome equivalents with an average insert size of 11.6 kb. Both libraries were gridded in duplicate onto 11 high-density filters in a 5 × 5 pattern to facilitate screening by hybridization. The libraries contain an unbiased coverage of the rice genome with less than 5% contamination by clones containing organelle DNA or no insert. An efficient method was developed, consisting of pooled overgo hybridization, the selection of 10-kb gap spanning clones using end sequences, transposon sequencing and utilization of in silico draft sequence, to close relatively small gaps between sequenced BAC clones. Using this method we were able to close a majority of the gaps (up to approximately 50 kb) identified during the finishing phase of chromosome-10 sequencing. This method represents a useful way to close clone gaps and thus to complete the entire rice genome.

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Acknowledgements

This work was supported by grants from the U.S. Department of Agriculture CREES, the National Science Foundation, and the Department of Energy and postdoctoral fellowship program from Korea Science and Engineering Foundation (KOSEF). Any opinions, findings, and conclusions or recommendations expressed herein are those of the authors and do not necessarily reflect the views of the U.S. Department of Agriculture, the National Science Foundation or the Department of Energy. We thank Monsanto for providing access to their draft sequence data.

Author information

Correspondence to Rod A. Wing.

Additional information

Communicated by Q. Zhang

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Yang, T., Yu, Y., Nah, G. et al. Construction and utility of 10-kb libraries for efficient clone-gap closure for rice genome sequencing. Theor Appl Genet 107, 652–660 (2003). https://doi.org/10.1007/s00122-003-1302-4

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Keywords

  • Rice genome sequencing
  • 10-kb Library
  • Gap closure