BMC Genomics 2008, 9:237 http://www.biomedcentral.com/1471-2164/9/237 s n f e o t r u N m n f m e e s t u o N b r 40 8 4 0 0 2 0 0 Results Flow cytometric analysis of mitotic metaphase chromo- III somes isolated from rye cv. 'Imperial' resulted in a histo- 1 6 0 gram of relative fluorescence intensities (flow karyotypes) with a composite peak representing chromosomes 2R – I 7R and a small peak of chromosome 1R (Figure 1). This is ve1 2 0 II 1RS the smallest chromosome in the karyotype and the only one that could be sorted individually. The chromosome is characterized by a secondary constriction separating the satellite from the short arm and the 5S rDNA locus on the 3B satellite, and its identity in the sorted fraction was con- firmed by FISH with probes for 5S rDNA and pSc119.2 (Figure 1, insert). 0 0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 Although chromosome 1R could easily be sorted, we con- Relative DNA content sidered 1,000 Mbp still too large fraction of the rye genome (~12.7%). Therefore, to simplify the analysis of lTFinihgeeufrloew2karyotype of the wheat-rye 1RS telosome addition 1RS, we targeted only this particular region and tested the The flow karyotype of the wheat-rye 1RS telosome suitability of 1RS wheat-rye ditelosomic addition line for addition line. The karyotype contains four peaks represent- ing the chromosomes of wheat (labeled I, II, III, and 3B) and a sorting 1RS. Flow karyotype obtained from this line was peak of the telocentric chromosome 1RS. Insert: Images of complex and consisted of four peaks representing various the flow-sorted chromosome 1RS after FISH with probes for wheat chromosomes and a peak of telocentric chromo- telomeric sequences (red color) and pSc200 (green color) some 1RS (Figure 2). The telosome could be sorted and DNA sequences. The chromosomes were counterstained the identity of sorted 1RS was determined by FISH with with DAPI (blue color). probes for pSc200 and telomeric repeats (Figure 2, insert). The contamination of sorted 1RS fractions ranged from 5 to 15% depending on the speed of analysis, with low speeds (below 500 particles/sec) resulting in higher reso- lution of the 1RS peak and higher purities. The contami- nation was not due to specific chromosomes, and various 200 wheat chromosomes and their fragments were observed in the sorted fraction of 1RS. 2R - 7R 160 In order to produce enough seeds for the 1RS BAC library construction, seeds from cytologically controlled 1RS ve120 ditelosomic wheat-rye addition plants were used. Whereas monotelosomic plants produce only ca. 40% monotelosomic progeny, with the remaining plants not eb 80 1R containing any 1RS chromosome, the progeny of ditelos- omic plants are up to 60% ditelosomic, with the remain- ing plants being monotelosomic. Only the use of such material permitted preparation of chromosome suspen- sions with sufficient concentration of chromosomes to 0 achieve good resolution of the 1RS arm on a flow karyo- 0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 type and high yields during flow sorting. Relative DNA content tcFHyhiipgrsetouo'm)rgeoroabs1mtoamioneefdrseualsfatpteeivrnefsliofoluwnocoryeftsSocemecnaeclteericicenrateenanallseyits'Iyims(op'ffleoDrwiaAlk'PaI-rsytoa-ined Histogram of relative fluorescence intensity ('flow karyotype') obtained after flow cytometric analysis of DAPI-stained chromosome suspension of Secale cere- ale 'Imperial'. The flow karyotype consists of a composite peak representing chromosomes 2R – 7R, and a peak repre- senting chromosome 1R. Insert: Images of flow-sorted chro- mosome 1R after FISH with probes for 5S rDNA (green color) and pSc119 (red color) DNA sequences. The chromo- somes were counterstained with DAPI (blue color). In total, 10.3 × 106 1RS chromosome arms were sorted from 540 samples that were prepared from 11,600 seeds. To avoid excessive sorting times, the samples were run at ~1000 particles/sec and the average sort rate was 25 chro- mosomes/sec. The sorting took 60 working days and the average purity in the sorted fractions estimated by FISH using probes for 45S rDNA and pSc200 was 86%. The first batch of 5.1 × 106 chromosomes were embedded in 30 agarose miniplugs, each containing 1 × 105 or 2 × 105 chromosomes, and used for construction of a HindIII Page 3 of 9 (page number not for citation purposes)