Fluorescence in situ hybridization (FISH) using bacterial artificial chromosomes (BACs) with large genomic DNA inserts as probes (BAC 'landing') is a powerful means by which eukaryotic genomes can be physically mapped and compared. Here we report a BAC landing protocol that has been developed specifically for the weedy grass species Brachypodium distachyon, which has been adopted recently by the scientific community as an alternative model for the temperate cereals and grasses. The protocol describes the preparation of somatic and meiotic chromosome substrates for FISH, the labeling of BACs, a chromosome mapping strategy, empirical conditions for optimal in situ hybridization and stringency washing, the detection of probes and the capturing and processing of images. The expected outcome of the protocol is the specific assignment of BACs containing single-copy inserts to one of the five linkage groups of the genome of this species. Once somatic or meiotic material is available, the entire protocol can be completed in about 3 d. The protocol has been customized empirically for B. distachyon and its near relatives, but it can be adapted with minor modifications to diverse plant species.
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This work was partially supported by Polish Ministry of Science and Higher Education (grant 2 PO4C 012 30).
The authors declare no competing financial interests.
Preparation of root meristem squashes. The narrated movie shows the cytological procedure for handling and dissecting root tip meristems, their squashing onto slides, and the removal of cover slips (protocol steps 1A viii and 2 to 4). The movie lasts 2.4 min. (MOV 49888 kb)
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Jenkins, G., Hasterok, R. BAC 'landing' on chromosomes of Brachypodium distachyon for comparative genome alignment. Nat Protoc 2, 88–98 (2007). https://doi.org/10.1038/nprot.2006.490
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