Wheat Radiation Hybrids: Old-Style Meets Next-Gen in Functional Genomics Projects

Radiation hybrid (RH) is a methodology that has been employed since the ‘70s to produce reliable molecular maps in many species, including mammals and plants. In wheat (Triticum species), recent efforts have focused on the molecular characterization of two RH populations generated for chromosome 1D and 3B.  One thousand individuals were screened with 140 3B-specific markers. On the basis of the molecular values, a subset of 320 highly informative lines was selected for saturation genotyping with 2,800 KASPAr assays. The total 0.9M data points were then used to generate a comprehensive map with the scope of providing a solid scaffold for the assembly of the chromosome 3B sequence. Further, these RH lines were phenotypically characterized for a gene(s) controlling chromosomal behavior at diakinesis (desynapsis). QTL analysis pinpointed the location of Qdesw2.ndsu-3B to a region of just 1.4 Mb in size, which was then further refined to just few candidate loci. Similarly, a RH population of ~1,500 individuals for chromosome 1D segregating for the species cytoplasm specific (scs) gene, involved in the compatible interaction between nucleus and cytoplasm, was characterized. We coupled the Illumina platform with bulk segregant analysis of RH lines carrying or missing the scs gene (BulkSeq RH). Among the 1M sites investigated, a total of 3.2 K sequences were found present in the lines carrying the gene, but deleted in the mutant lines. These short pair-ends sequences were then extended to 1-5 Kb in size, employing publicly available 53X survey sequences of Triticum tauschii. Further, these extended sequences were assembled in 556 contigs spanning 1.2 Mb with an N50 of 3 Kb. The ‘WheatZapper’ synteny tool (http://wge.ndsu.nodak.edu/wheatzapper/) identified eight contigs harboring genes in syntenic regions of other grass species. These contigs were then mapped on the RH population, and a rhomboid-like gene was found to cosegregate with the scs locus. The study of its expression behavior proved consistent with what is expected for the scs locus, and the tridimensional structure of its protein presents a functional mutation in an amino acid on chromosome 1D that can potentially affect its catalytic behavior.