Gene mapping is reaching its pinnacle with the progression of whole genome sequence assemblies to the ‘finished’ status. The assemblies will represent ultimate genetic maps in the form of complete, accurate, and annotated nucleotide sequences. The creation of such comprehensive genome sequence assemblies will however remain restricted to a few species in the near future since the recent breakthrough developments in DNA sequencing technology focus on short read lengths, which are more suitable for the efficient re-sequencing of genomes (Bentley 2006; Wheeler et al. 2008). Thus the construction of high-resolution recombination maps and physical maps remains an active area of research (Shifman et al. 2006; Matise et al. 2007; McKay et al. 2007), providing chromosomal locations and intermarker distances, landmarks for locating disease genes and species-specific frameworks for genome sequence assembly (Slonim et al. 1997). Radiation hybrid (RH) mapping has been exploited widely since 1990 (Cox et al. 1990) to construct physical maps. Since then, the technique has been refined and is used to construct high-resolution maps with high throughput techniques (McKay et al. 2007). High-resolution RH maps have proven to be useful for interrogating and amending genome sequence assemblies (Jann et al. 2006; McKay et al. 2007; Karere et al. 2008).One of the limiting factors for RH map construction is the cell culture required to generate sufficient DNA for marker mapping and for sharing of the resource. The propagation of RH clones is especially difficult for non mammalian species like fishes (Senger et al. 2006) and plants (Wardrop et al. 2002) requiring some form of sample amplification (Wardrop et al. 2002). To generate RH clones, chromosomes of donor cells are fragmented by a lethal dose of X-rays. The resulting chromosome fragments are rescued by fusion to a recipient cell (mostly of rodent origin), deficient for a selection gene, for example TK or HRPT, and subsequent integration of the donor genome chromosomal fragments into the recipient karyotype. After culture in selective medium, individual RH clones are isolated and propagated in cell culture in increasing volumes. DNA from the resulting RH clones is genotyped for markers of interest. The random nature of both the X-ray fragmentation and of the integration into the recipient genome enables the generation of genome maps by linkage mapping approaches. However, since RH cell lines are unstable (Graw et al. 1988; Goodfellow 1991; Walter and Goodfellow 1995), results obtained with the same clone can only be combined if DNA originating from the same passage of cells has been tested. Thus, one of the major challenges in the construction of RH maps is the finite amount of DNA available for genotyping. The instability of the RH cell lines further induces strongly varying signal intensities during genotyping since certain donor DNA sequences might only be present in varying fractions of the RH clone cells (Slonim et al. 1997). This problem necessitates duplicate genotyping for the verification of results and diminishes the precision of the RH mapping in cases of persisting ambiguity or induced artifacts. Ambiguous results are statistically very problematic and are often treated differently by the individual RH software packages (Agarwala et al. 2000). To meet the DNA quantity demands, the RH cell lines are typically passaged in multiple stages beginning with the establishment of a clone, passage into 24-well plates, and subsequent expansion in T25 flasks and roller bottles (Chowdhary et al. 2002). In cases where DNA stocks become inadequate, the hybrid cells have to been grown as a new passage. However, the extracted DNA then represents a different population of cells, usually necessitating re-typing of markers. The loss of unselected donor DNA fragments from somatic hybrid clones was already observed in early studies (Goss and Harris 1977) and the process itself is not disputed. However, th
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