BMC Genomics 2008, 9:237 http://www.biomedcentral.com/1471-2164/9/237 oFHfiygsbuerlriedecizt5eatdioBnAoCf tchleonmeasraknedr Bth7e6wwhitehathlieneDsraI-digested DNA Hybridization of the marker B76 with the DraI- digested DNA of selected BAC clones and the wheat lines. Lanes 1–13: 1RS BAC clones showing some of the rye fragments missing in deletion mutants. Lane 14: Genomic DNA of Gabo1BL.1RS (wildtype; carrying the 1RS from cv. 'Imperial'); lane 15: interstitial deletion mutant (00.002); lane 16: mutant with chromosome arm loss (00.001). The capital letters (A to Q) show the position of rye fragments ortholo- gous to the barley Mla resistance gene. The corresponding BAC fragments are shown as small letters. Arrows indicate the position of fragments (A, B & L) missing in the interstitial deletion mutant and maybe candidate for the stem rust resistance gene SrR. A detailed picture of the mutants with the loss of 1RS fragments is shown in Mago et al. (2004). All the samples were electrophoresed in the same gel however, two autoradiograms were developed. A 30 min exposure for the BAC clones and an overnight exposure to see the corre- sponding bands in the genomic DNA. chromosomes. Following this work, BAC libraries were prepared from other wheat chromosomes [[13,14], in preparation]. Chromosome-specific BAC libraries facili- tate analysis of molecular structure of targeted genome regions, development of molecular markers from these regions and construction of physical contig maps [30] as well as positional gene cloning [17]. While six chromo- some (arm)-specific BAC libraries have already been cre- ated in hexaploid wheat, until now this system has not been verified in other eukaryotic species. This paper proves that the strategy of constructing BAC libraries from particular chromosome arms is applicable also for rye. Whereas the coverage of the 1RS arm by the whole library was estimated to be 14-fold considering insert size of BAC clones, library screening with a set of DNA markers indicated an 11-fold coverage. Over ten- fold coverage of 1RS was confirmed during the first phase of positional cloning of the SrR gene. All these estimates confirmed the deep coverage of the 1RS arm. The average insert size of the 1RS-specific library is similar to that observed for the 1D, 4D and 6D-specific library (85 kb), and BAC libraries specific for chromosome arms 1BS (82 kb) and 3AS (80 kb), but is lower than the average insert size of the 3B-specific library with 103 kb. As shown in Figure 4, the relatively low insert size is due to presence of a rather abundant class of smaller inserts (40 – 59 kb) rep- resenting 29% of the HindIII library and 19% of the BamHI library. Our protocol for BAC library construction includes only one size selection step prior to cloning due to low amounts of high molecular weight DNA obtained from sorted chromosomes. However, we continue work- ing on improving the protocol to allow for two-step size selection. Nevertheless, the 1RS library contains 56% clones with inserts over 70 kb. These clones represent 11- fold coverage of the 1RS arm (calculated using the insert size), which makes the library suitable for construction of a physical contig map. The isolation of the BACs carrying the three Mla orthologs missing in the deletion mutants demonstrates the value of this library in constructing a DNA sequence contig from the 1RS region identified by deletion mutant analysis as the region containing SrR. It indicates that the 1RS library is an excellent resource for rye and wheat researchers look- ing at the genes encoding important agronomic traits associated with 1RS. For example, as shown in Figure 5, the 13 BACs carry at least 13 of the about 17 Mla-ortholo- gous fragments identified on 1RS. Thus, the 165 BACs identified with B76 probe would indicate over ten-fold coverage of the region. Our previous attempts to isolate the same region of 1RS from ungridded BAC library from a wheat 1BL.1RS translocation line proved unsuccessful (unpublished). Further characterization of the BAC clones is in progress to confirm whether any of the candidates are stem rust SrR gene/s. In addition to development of molecular markers and construction of physical map, the library may find other attractive uses. For example, it has already been used to study the molecular structure of rye chromosome ends [42]. Moreover, its availability provides the opportunity to perform detailed comparative analysis of molecular structure of wheat chromosome group 1 and rye chromo- some 1R. In fact, 1R is the only rye chromosome, which is Page 6 of 9 (page number not for citation purposes)