Plant & Animal Genomes XI Conference Plant & Animal Genomes XI ConferenceJanuary 11-15, 2003
Town & Country Convention Center
San Diego, CA
Poster: Large Insert Libraries
The analysis and mapping the bread wheat (Triticum aestivum, 2n = 6x = 42) genome is hampered by its enormous size and presence of three homeologous genomes. One way to simplify the analysis is to individually purify chromosomes or chromosome arms prior to molecular analysis. We have developed a high-yield protocol for preparation of suspensions of intact wheat chromosomes and their sorting using laser flow cytometry. The chromosomes are classified according to their relative DNA content after staining with DAPI. Due to the lack of sufficient differences in size between individual chromosomes, only chromosome 3B could be sorted from a standard cultivar (Chinese Spring). The analysis of other varieties identified materials from which chromosomes 4B, 4D, 5D and 6D and translocation chromosomes 5BL·7BL and 4AL·4AS-5BL could be sorted. Furthermore, we have found that using various stocks, any single arm of the wheat karyotype, either in the form of ditelosome or isochromosome could be sorted. On average 200,000 chromosomes could be sorted in one working day at purities exceeding 90%. Thus, although originally considered recalcitrant, wheat seems to be suitable for development of flow cytogenetics and the technology is mature to be applied in physical mapping of DNA sequences using PCR or dot-blot hybridization, targeted isolation of molecular makers and construction of chromosome- and arm-specific DNA libraries. These approaches should facilitate the analysis of the complex genome of hexaploid bread wheat.