Abstract
We constructed a chromosome 9 lambda DNA library from flow-sorted maize chromosomes. Approximately 3 million maize chromosome 9 were collected with high purity by flow cytometric sorting of chromosomes isolated from an oat-maize chromosome 9 addition line based on the cytogram of fluorescent pulse area versus fluorescent pulse width. Chromosome 9 DNA was partially digested withBamH I, dephosphorylated, and ligated with arms ofBamH I-digested lambda DASH vector (Stratagene). A total of 2.0×106 independent recombinants with an average insert size of 15 kb were obtained. For a 99% probability that every sequence of chromosome 9 is represented in at least one chimeric phage, 5.6×104 cloned fragments are needed. This library covers the entire maize chromosome 9. Hybridizing cloned fragments with labeled maize genomic DNA showed that the high, middle, or low copy number DNA sequences presented in the different phage clones. This individual chromosome library is useful in plant genome mapping and gene isolation.
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Abbreviations
- BAC:
-
bacterial artificial chromosome
- DMSO:
-
dimethyl sulfoxide
- FACS:
-
fluorescence-activated cell sorter
- GISH:
-
genomic in situ hybridization
- YAC:
-
yeast artificial chromosome
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Li, L., Arumuganathan, K. Microcloning of maize chromosome 9 by using a flow-sorting technique. Plant Mol Biol Rep 21, 359–364 (2003). https://doi.org/10.1007/BF02772585
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DOI: https://doi.org/10.1007/BF02772585