BMC Genomics 2008, 9:237 http://www.biomedcentral.com/1471-2164/9/237 Lambda Ladder PFG Marker and MidRange Marker I (New England Biolabs, Beverly, USA). Estimation of 1RS coverage using molecular markers BAC clones were grown overnight in 384-well plates con- taining 2YT medium supplemented with chlorampheni- col (12.5 µg/ml). Clones from each of the 384-well plates were pooled, pelleted and resuspended in 4 ml of TE (10 mM Tris, 1 mM EDTA) buffer. In order to prepare DNA pools, the bacterial suspensions were lysed at 95°C for 30 min, pelleted at 3,000 g for 60 min and the supernatant was diluted 25-fold with deionized water. PCR reaction mix (10 µl) consisted of 1 µl template DNA of diluted pool, 1× Buffer for DyNAzyme DNA Polymerase (Finnzymes, Espoo, Finland), 0.2 mM dNTPs, 1 µM prim- ers (Table 1), and DyNAzyme II DNA Polymerase (Finnzymes). PCR reaction was performed as follows: denaturation for 4 min at 94°C followed by 30–35 cycles consisting of denaturation at 94°C for 30 s, annealing at appropriate temperature (58°–62°C) for 40 s, extension at 72°C for 40 s, and final extension at 72°C for 10 min. Presence of PCR products was checked by electrophoresis in 1% agarose gels run in 0.5 × TBE buffer. Screening of high-density filters and BAC DNA isolation High-density colony filters were prepared by spotting BAC clones in duplicate in a 4 × 4 gridding pattern on Hybond N+ nylon membranes (Amersham Biosciences, Piscata- way, USA) using the GeneTAC G3 robot (Genomic Solu- tions, Ann Arbor, USA). 18,432 individual colonies (36,864 spots in total) were arrayed on one filter. DNA probes for hybridization to screen the BAC library or blot- ted DNA digests were labeled with [32P]dCTP using Meg- aprime DNA Labeling System (Amersham). The probes derived from the barley Mla gene that were used for screening the library were described earlier (Mago et al. 2004). For high-throughput isolation of DNA from selected BAC clones, the alkaline lysis protocol was adapted for 96-well plate format. BAC clones were grown overnight in 300 µl LB with chloramphenicol (12.5 µg/ ml) in 96-well plates. Cells were harvested by centrifuga- tion at 2,000 rpm for 20 min. The cells were resuspended in 50 µl GTE with RNaseA and lysozyme. 100 µl of lysis buffer was added and mixed by gentle vortexing followed by addition of 75 µl of chilled potassium acetate and mixed. The plate was centrifuged for 30 min at 3,000 rpm. 200 µl of supernatant was transferred to a fresh plate and DNA precipitated by using 0.6 volume of cold isopropa- nol. After centrifugation for 30 min at 3,000 rpm, the DNA was washed with 70% ethanol, air dried and resus- pended in 25 µl sterile water. The DNA was used directly for restriction digestion. For individual BAC clones, DNA was isolated using the modified alkaline lysis protocol (Sinnett et al. 1998). Authors' contributions HŠ, JŠ and JJ constructed the 1RS BAC library and drafted the manuscript. JB screened the library with chromosome- specific markers. PK and PS performed flow cytometric analyses and sorting. JЖ prepared samples for flow cytometry. MK performed FISH on flow-sorted fractions. RM used the library to isolate SrR gene. TL selected cyto- logically the parents providing the seeds for sorting and together with JD conceived and supervised the project. JD prepared a final version of the manuscript. All authors read and approved the final manuscript. Acknowledgements We are grateful to Prof. A. J. Lukaszewski (University of California, River- side, USA) for seeds of rye cv. 'Imperial' and to Dr. B. Friebe (Kansas State University, Manhattan, USA) for seeds of the 1RS ditelosomic wheat-rye addition line. We thank our colleagues, Bc. Romana Nováková, Bc. Michaela Sekerová, Radka Tušková, and Pavlína Hylmarová, for excellent technical assistance. This work was supported by research grants from the Czech Science Foundation (grant awards 521/04/0607, 521/05/P257 and 521/05/ H013), Ministry of Education, Youth and Sports of the Czech Republic (grant award LC06004), work by Rohit Mago was supported by grants from Grains Research and Development Corporation (GRDC), Australia (CSP242, CSP0017). References 1. Bennett MD, Leitch IJ: Nuclear DNA amounts in angiosperms. Ann Bot 1995, 76:113-176. 2. Carver BF, Rayburn AL: Comparison of related wheat stocks possessing 1B or T1BL-1RS chromosomes – grain and flour quality. Crop Science 1995, 35:1316-1321. 3. Clarke L, Carbon J: A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome. Cell 1976, 9:91-99. 4. Conner RL, Thomas JB, Whelan EDP: Comparison of mite resist- ance for control of wheat streak mosaic. Crop Science 1991, 31:315-318. 5. 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