BMC Genomics 2008, 9:237 http://www.biomedcentral.com/1471-2164/9/237 collinear along its length with the wheat chromosomes of homoeologous group 1 [5]. Conclusion Here we present a strategy that enables sorting of short arms of several chromosomes of rye. Using flow-sorted chromosomes, we have constructed a deep coverage BAC library specific for the short arm of chromosome 1R (1RS). The library is the first subgenomic BAC library available for rye and we demonstrate its potential for posi- tional gene cloning. We expect that the library will facili- tate the analysis of molecular structure of 1RS, evolution of Triticeae genomes, and cloning important genes located on this arm. The library is available for academic users by reimbursement of duplication costs. Academic and commercial users interested in a copy of the BAC library should contact the corresponding author. Methods Plant material Seeds of rye (Secale cereale L., 2n = 2x = 14) cv. 'Imperial' were obtained from Prof. A. J. Lukaszewski (University of California, Riverside, USA). Original seeds of 1RS wheat- rye ditelosomic addition line, derived from rye cv. 'Impe- rial' in the background of wheat (Triticum aestivum L., 2n = 6x = 42) cv. 'Chinese Spring' (Driscoll and Sears 1971), were provided by Dr. B. Friebe (Kansas State University, Manhattan, USA). For cell cycle synchronization, seeds from cytologically selected plants were germinated in the dark at 25 ± 0.5°C on moistened filter paper in glass Petri dishes for 2–3 days to achieve optimal root length (2–3 cm). Preparation of chromosome suspensions and sorting of chromosomes Cell-cycle synchronization, accumulation of metaphases in root tips and preparation of chromosome suspensions were performed according to Kubaláková et al. (2003). Briefly, chromosome suspensions were prepared by mechanical homogenization of 30 formaldehyde-fixed root tips (~1 mm) in 1 ml ice-cold isolation buffer (IB, Šimková et al. 2003). Chromosomes in suspension were stained by 2 µg/ml DAPI (4',6-diamidino-2-phenylin- dole) and analyzed using a FACSVantage SE flow cytome- ter (Becton Dickinson, San José, USA). Sort windows were set on a dot plot of fluorescence pulse area versus fluores- cence pulse width. The purity in sorted fractions was checked regularly by fluorescence in situ hybridization with chromosomes sorted onto a microscope slide. For BAC library construction, chromosome arm 1RS was sorted in aliquots of 1 × 105, 2 × 105 and 4 × 105 copies into 160 µl, 320 µl and 640 µl 1.5 × IB, respectively. Fluorescence in situ hybridization (FISH) 2000 particles were flow-sorted from a peak assumed to represent 1RS into 15-µl drop of PRINS buffer (Kubaláko- vá et al. 2001), which was supplemented with 5% sucrose, onto a microscope slide. After air-drying, flow-sorted chromosomes were identified using FISH with probes for 5S rDNA, 45S rDNA, pSc119.2 and pSc200 repeats, which provide chromosome-specific fluorescent banding pat- terns (Kubaláková et al. 2003). The chromosomes were counterstained with DAPI, and the preparations were evaluated using Olympus BX60 microscope (Olympus, Tokyo, Japan) with CCD camera interfaced to a PC run- ning the ISIS software (Metasystems, Altlussheim, Ger- many). BAC library construction Preparation of high-molecular-weight DNA and construc- tion of BAC library from 1RS were performed as described by Šimková et al. (2003) and Šafář et al. (2004). Briefly, each batch of flow-sorted 1RS arm was pelleted at 200 or 400 g and mixed with low-melting-point agarose to form 15-µl agarose mini-plugs. Proteinase-treated DNA in the mini-plugs was partially digested either with HindIII or BamHI and size-selected by pulsed field gel electrophore- sis (PFGE, 6V/cm, switch time 1–40 s, for 12 h and switch time 2.5–5.5 s for 6 h at 14°C). Fragments of 50–300 kb were subdivided into smaller fractions and isolated from the agarose gel by electroelution. The size-selected DNA was ligated to dephosphorylated vector pIndigoBAC-5 (Epicentre, Madison, USA) in molar ratio of 1 to 5 (in BAC vector excess). Four ligations corresponding to frag- ment size of 50–75 kb (H01fraction), 75–120 kb (H02 fraction), 120–170 (H1 fraction) and 170–300 kb (H2 fraction), respectively, were prepared from HindIII- digested DNA and three ligations, corresponding to 75– 115 kb (C2 fraction), 115–175 kb (C3 fraction) and 175– 300 kb (C4 fraction), respectively, were prepared from the BamHI-digested DNA. The ligation mixtures were desalted and used to transform Escherichia coli ElectroMAX DH10B competent cells (Gibco BRL, Gaithersburg, USA) by elec- troporation. The library was ordered in 384-well plates filled with freezing medium (Woo et al. 1994) and stored at -80°C. Characterization of BAC inserts Three hundred and ninety two randomly selected BAC clones proportionally representing all size fractions from both libraries were used to estimate the average insert size. The DNA was isolated using standard alkaline lysis method and digested with NotI (0.4 U/20 µl). The obtained DNA fragments were separated in 1% agarose gel in 0.5 × TBE buffer by PFGE at 6V/cm, switch time ramp 1–40 sec, 14°C for 14 h. The size of the fragments was estimated by comparing with two size markers: Page 7 of 9 (page number not for citation purposes)