BMC Genomics 2008, 9:237 http://www.biomedcentral.com/1471-2164/9/237 Table 1: Results of 1RS BAC library screening on pools of 384-well plates with the 1RS-specific DNA markers Marker code Reference Primers Number of positive pools scm9 bcd1434 1 mwg2062 2 psr109 3 psr949 4 IB-262 Average SSR markers Saal and Wricke 1999 TGACAACCCCCTTTCCCTCGTTCATCGACGCTAAGGAGGACCC Loarce et al. 1996 Korzun et al. 1998 Masojc et al. 2001 Masojc et al. 2001 Mago et al. 2002 STS markers* CCACCTTCTCGCTGTTGAAT CTGCTGTCCAGCCAGAAAAT TCTCGCTGGTATTCAGGGTCC AAACGATAGCAAGAGGAACCG GACTACCTGATCCGCTCCAA ACTTGCACTTTAGGCCTTGC GCCCTGCCATATAACTTCCA GCATCATGTCTCCCTTTACCA GTAGGTAATGTATCAGAGTTGTAC GTCTTTGTGCTCGGTAGCTCC * developed from RFLP markers 1 primers designed from bcd1434-3 sequence (GrainGenes http://wheat.pw.usda.gov/GG2/index.shtml) 2 primer sequences downloaded from GrainGenes http://wheat.pw.usda.gov/GG2/index.shtml 3 primers designed from psr109 sequence (GrainGenes http://wheat.pw.usda.gov/GG2/index.shtml) 4 primers designed from psr949-Forward sequence (GrainGenes http://wheat.pw.usda.gov/GG2/index.shtml) 10 11 11 6 16 14 11.3 [26] of these at least three were missing in interstitial dele- tion mutants. DNA from the 165 BAC clones was digested with DraI to identify the BACs carrying these three frag- ments missing in the interstitial deletions. Figure 5 shows hybridization of the B76 probe to wheat genomic DNA and selected BACs digested with DraI. At least three BAC clones (no. 3, 8 and 10) were confirmed to carry two of the fragments (B and L) missing in interstitial deletion mutants based on fragment size. This was also confirmed using a PCR marker developed from BACs no. 3 and 10 which amplified the expected product from the wildtype but not the deletion mutant indicating that the BAC is from the expected region (data not shown). In total 19 BAC clones were identified carrying the three fragments missing in the mutants. Discussion This work focuses on the short arm of rye chromosome 1 (1RS), which represents an important part of not only the rye genome but also the genomes of many cultivars of wheat. However, while the 1RS arm contains several genes of agronomic importance such as disease resistance, yield enhancement, its genetic makeup has a negative impact on bread making quality. Since, rye chromosomes do not recombine regularly with wheat chromosomes, getting rid of the negative characters is not easy by conventional recombination and (standard genetic) segregation. It is hoped that advanced tools of genomics will assist in clon- ing useful genes from 1RS. Flow cytometric analysis of chromosomes prepared from rye cv. 'Imperial' confirmed our previous observation [18] according to which only chromosome 1R can be discrim- inated and sorted from this cultivar. Moreover, until now we have not identified other rye cultivars from which chromosome 1R could be sorted. The ability to sort only one chromosome would greatly limit the application of flow cytogenetics in rye to dissect its nuclear genome. In order to overcome this limitation, we adopted the strategy based on using wheat-rye chromosome addition lines [18]. Since the rye chromosomes are larger than any of the wheat chromosomes, chromosomes 2R – 7R can be sorted from appropriate cytogenetic stocks. These chromosomes represent 13.2% – 16.6% of the rye genome [36]. This paper expands the potential of flow cytogenetics for genomics of rye by demonstrating a possibility to sort sin- gle chromosome arms. Our system is similar to that described above and to that used by Suchánková et al. [41] to sort chromosome arms of barley, and relies on using wheat-rye telosomic addition lines. We demonstrate that using this system, it is possible to purify the short arm of chromosome 1R (1RS). Considering the relative lengths of rye [36] and wheat [11] chromosomes and the genome sizes of these species [1], it may be predicted that, except 2RS, short arms of all rye chromosomes can be discrimi- nated and sorted from respective wheat-rye telosome addition lines. However, it should be noted that only 3RS, 4RS, 5RS and 7RS wheat-rye telosomic addition lines are currently available [29] and that the 5RS line is unstable (B. Friebe, pers. comm.). On the other hand, peaks of long arms of most of rye chromosomes would overlap with the peak I representing wheat chromosomes 1D, 4D and 6D, and thus only 1RL and 3RL would be sortable. Flow-sorted chromosomes were shown to be suitable in a number of applications in plant genome analysis and mapping, with the construction of chromosome (arm)- specific BAC libraries being the most attractive [7,8]. Šafář et al. [35] were the first to show that it was possible to con- struct BAC libraries from small amounts of high molecu- lar weight DNA that can be prepared from flow-sorted Page 5 of 9 (page number not for citation purposes)