Development of a high quality reference sequence is a daunting task in crops like wheat with large (~17Gb), highly repetitive (>80%) and polyploid genome. To achieve complete sequence assembly of such genomes, development of a high quality physical map is a necessary first step. However, due to the lack of recombination in certain regions of the chromosomes, genetic mapping, which uses recombination frequency to map marker loci, alone is not sufficient to develop high quality marker scaffolds for a sequence ready physical map. Radiation hybrid (RH) mapping, which uses radiation induced chromosomal breaks, has proven to be a successful approach for developing marker scaffolds for sequence assembly in animal systems. Here, the development and characterization of a RH panel for the mapping of D-genome of wheat progenitor Aegilops tauschii is reported.
Radiation dosages of 350 and 450 Gy were optimized for seed irradiation of a synthetic hexaploid (AABBDD) wheat with the D-genome of Ae. tauschii accession AL8/78. The surviving plants after irradiation were crossed to durum wheat (AABB), to produce pentaploid RH1s (AABBD), which allows the simultaneous mapping of the whole D-genome. A panel of 1,510 RH1 plants was obtained, of which 592 plants were generated from the mature RH1 seeds, and 918 plants were rescued through embryo culture due to poor germination (<3%) of mature RH1 seeds. This panel showed a homogenous marker loss (2.1%) after screening with SSR markers uniformly covering all the D-genome chromosomes. Different marker systems mostly detected different lines with deletions. Using markers covering known distances, the mapping resolution of this RH panel was estimated to be <140kb. Analysis of only 16 RH lines carrying deletions on chromosome 2D resulted in a physical map with cM/cR ratio of 1:5.2 and 15 distinct bins. Additionally, with this small set of lines, almost all the tested ESTs could be mapped. A set of 399 most informative RH lines with an average deletion frequency of ~10% were identified for developing high density marker scaffolds of the D-genome.
The RH panel reported here is the first developed for any wild ancestor of a major cultivated plant species. The results provided insight into various aspects of RH mapping in plants, including the genetically effective cell number for wheat (for the first time) and the potential implementation of this technique in other plant species. This RH panel will be an invaluable resource for mapping gene based markers, developing a complete marker scaffold for the whole genome sequence assembly, fine mapping of markers and functional characterization of genes and gene networks present on the D-genome.
Via Biswapriya Biswavas Misra