BMC Genomics 2008, 9:237 http://www.biomedcentral.com/1471-2164/9/237 library. This number of chromosomes corresponded to approximately 4.6 µg of DNA. For a BamHI library, 5.2 × 106 chromosomes were sorted in batches of 4 × 105 and embedded in 13 agarose miniplugs. This amount repre- sented about 4.7 µg of DNA. Chromosomal DNA partially digested either with HindIII or BamHI was then used to construct two 1RS-specific BAC libraries. The first BAC library (SccImp1RShA) con- structed using HindIII consisted of 66,816 clones obtained from four size fractions of DNA: H01 (con- structed from the 50–75 kb fraction) consisted of 8,064 clones, H02 (75–120 kb) contained 31,488 clones, H1 (120–170 kb) was represented by 15,360 clones and H2 (170–300 kb) by 11,904 clones. As the insert size did not differ significantly among the particular fractions (data not shown), the average insert size was estimated for the complete HindIII library (Figure 3). Based on analysis of 191 clones, the mean insert size of this library was found to be 72 kb (Figure 4) with a median at 70 kb. The small- est insert was 15 kb while the largest was 160 kb. As the purity of the sorted fraction estimated by FISH showed to be 86% and the percentage of empty clones was 6%, the HindIII library was estimated to cover the 1RS chromo- some arm 8.8-fold. The second library (SccImp1RSbA) was constructed from BamHI-digested DNA and comprised 36,864 clones obtained from three size fractions: C2 (75–115 kb), C3 (115–175 kb) and C4 (175–300 kb). Similarly to the Hin- dIII library, the insert sizes resembled each other in all three fractions thus they were not analyzed separately. Based on analysis of 201 clones the observed mean insert size reached 75 kb with a median at 80 kb (Figure 4). The smallest insert was 15 kb while the largest was 160 kb. Considering the purity of the sorted fraction of 86% and FAingaulyrseis3of inserts in the 1RS BAC library Analysis of inserts in the 1RS BAC library. 24 randomly selected BAC clones were digested with NotI, run on PFGE and stained with ethidium bromide. M = molecular size marker Lambda Ladder. The arrow indicates the vector. IFnisgeurrtesiz4e distribution in the 1RS BAC library Insert size distribution in the 1RS BAC library. 191 random clones from the HindIII sub-library (SccImp1RShA) and 201 random clones from the BamHI sub-library (SccImp1RSbA) were isolated and sized using PFGE. Grey bars: HindIII sub-library; white bars: BamHI sub-library; hatched bars: Complete 1RS BAC library. 3% empty clones, the coverage of the BamHI library was calculated to be 5.2-fold. The complete 1RS-specific BAC library consisted of 103,680 clones ordered in 270 × 384-well microtitre plates. Almost one third of the 1RS library (30%) had inserts larger than 100 kb, while 26% inserts were distrib- uted from 70 to 100 kb, and 44% of inserts were shorter than 70 kb. With the average insert size of 73 kb, the library represents 14 equivalents of the 1RS chromosome arm. Using the formula of Clarke and Carbon [3], the probability of finding any sequence from the short arm of chromosome 1R in the library was 99.9%. The chromosome arm specificity and predicted coverage of the 1RS arm, was confirmed by screening the BAC library by PCR with a set of molecular markers. This was done on 270 DNA pools, each of them prepared from one 384-well plate. The pools were screened with one SSR marker and five STS markers specific for the short arm of chromosome 1R. The number of pools that gave PCR products of the expected lengths with particular markers ranged from 6 to 14 (Table 1). Based on these results, we estimated the coverage of the complete 1RS BAC library to be at least 11-fold. To test the utility of the library for isolating candidates for the SrR gene, BAC filters from both BamHI and HindIII sub-libraries were used for hybridization with the B76 probe, derived from the LRR region of the Mla gene [26] and a total of 165 positive clones were identified. Previ- ously, using hybridization of wheat 1RS.1BL genomic DNA with the B76 probe, we identified ~20 Mla-ortholo- gous DraI-digested fragments on 1RS of rye cv. 'Imperial' Page 4 of 9 (page number not for citation purposes)