Sequence composition and gene content of the short arm of rye (Secale cereale) chromosome 1 as revealed by 454 sequencing
More than 10,000 seeds from controlled plants of 1RS          wheat-rye ditelosomic addition line, derived from rye (Secale cereale L.) cv. 'Imperial'          in the background of wheat (Triticum aestivum L.) cv. 'Chinese Spring' were germinated up          to an optimal root length of 2–3 cm. After cell cycle synchronization to accumulate metaphases          in root tips preparation of liquid chromosome suspensions were performed.          Chromosomes in suspension were stained by 2 microg/ml DAPI (4',6-diamidino-2-phenylindole)          and analyzed at rate of about 800-1200 chromosomes/sec using a FACSVantage SE flow cytometer          (Becton Dickinson, San Jose, USA). Telosome 1RS was sorted at rates of 10/sec          in batches of 30,000 chromosome arms into 50 microl deionized water in a PCR tube.          The purity in sorted fractions was checked by fluorescence in situ hybridization          with chromosomes sorted onto a microscope slide using probes for pSc200 and telomeric repeats.          Chromosomal DNA was isothermally amplified. In short, flow-sorted chromosomes were treated with          proteinase K, purified using a Microcon YM-100 column (Millipore Corporate, Billerica, USA)          and the purified chromosomal DNA was amplified using illustra GenomiPhi V2 DNA Amplification          Kit (GE Healthcare, Chalfont St. Giles, United Kingdom) in a 20 microl reaction for 1.5 hours.          The samples were lyophilized and shipped for 454 sequencing.