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Construction of BAC and BIBAC libraries from sunflower and identification of linkage group-specific clones by overgo hybridization

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Abstract

Complementary BAC and BIBAC libraries were constructed from nuclear DNA of sunflower cultivar HA 89. The BAC library, constructed with BamHI in the pECBAC1 vector, contains 107,136 clones and has an average insert size of 140 kb. The BIBAC library was constructed with HindIII in the plant-transformation-competent binary vector pCLD04541 and contains 84,864 clones, with an average insert size of 137 kb. The two libraries combined contain 192,000 clones and are equivalent to approximately 8.9 haploid genomes of sunflower (3,000 Mb/1C), and provide a greater than 99% probability of obtaining a clone of interest. The frequencies of BAC and BIBAC clones carrying chloroplast or mitochondrial DNA sequences were estimated to be 2.35 and 0.04%, respectively, and insert-empty clones were less than 0.5%. To facilitate chromosome engineering and anchor the sunflower genetic map to its chromosomes, one to three single- or low-copy RFLP markers from each linkage group of sunflower were used to design pairs of overlapping oligonucleotides (overgos). Thirty-six overgos were designed and pooled as probes to screen a subset (5.1×) of the BAC and BIBAC libraries. Of the 36 overgos, 33 (92%) gave at least one positive clone and 3 (8%) failed to hit any clone. As a result, 195 BAC and BIBAC clones representing 19 linkage groups were identified, including 76 BAC clones and 119 BIBAC clones, further verifying the genome coverage and utility of the libraries. These BAC and BIBAC libraries and linkage group-specific clones provide resources essential for comprehensive research of the sunflower genome.

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Acknowledgements

We thank Wei Sun, Lisa Brown, and Leonard Cook for assisting in the array of the libraries, Pilar Rojas and Junfang Chen for their kind help in the lab, and L. G. Campbell and G. J. Seiler for critically reading the manuscript. We are also grateful to Justin Faris for the use of specialized equipment, and to A. A. Schneiter and staff at the Department of Plant Sciences, North Dakota State University, for assistance in administering this grant. Finally, thanks are given to R. Kesseli for sequencing the RFLP markers used in this study. This project was supported by the U.S. Department of Agriculture CRIS project 5442–21000-027-02.

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Authors and Affiliations

  1. Department of Plant Sciences, North Dakota State University, Fargo, ND, 58105, USA

    Jiuhuan Feng

  2. US Department of Agriculture, Agricultural Research Service, Northern Crop Science Laboratory, PO Box 5677, Fargo, ND, 58105, USA

    Brady A. Vick & C. C. Jan

  3. Department of Soil and Crop Sciences and Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, TX, 77843-2123, USA

    Mi-Kyung Lee & Hong-Bin Zhang

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  1. Jiuhuan Feng
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  2. Brady A. Vick
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  5. C. C. Jan
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Correspondence to C. C. Jan.

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Communicated by Q. Zhang

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Feng, J., Vick, B.A., Lee, MK. et al. Construction of BAC and BIBAC libraries from sunflower and identification of linkage group-specific clones by overgo hybridization. Theor Appl Genet 113, 23–32 (2006). https://doi.org/10.1007/s00122-006-0265-7

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