Protocol abstract
Nature Protocols 2, - 88 - 98 (2007)
Published online: 8 February 2007 | doi:10.1038/nprot.2006.490
Subject Categories: Genetic analysis | Imaging | Immunological techniques | Model organisms | Plant biology
BAC 'landing' on chromosomes of Brachypodium distachyon for comparative genome alignment
Glyn Jenkins1 & Robert Hasterok2
Abstract
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.
- Institute of Biological Sciences, University of Wales Aberystwyth, Penglais, Aberystwyth, Ceredigion SY23 3DA, UK.
- Department of Plant Anatomy and Cytology, Faculty of Biology and Environmental Protection, University of Silesia, 40-032 Katowice, Poland.
Correspondence to: Glyn Jenkins1 e-mail: gmj@aber.ac.uk
Correspondence to: Robert Hasterok2 e-mail: hasterok@us.edu.pl
nature-products
A-Z product listing
- Amberlite monobed mixed resin IRN-150L(Merck)
- Cellulase(Calbiochem)
- Cellulase Onozuka R-10(Serva)
- Cytohelicase(Sigma)
- DAPI(Sigma)
- Dextran sulfate(Sigma)
