Rye cytogenetics and chromosome genomics

Abstract

Rye (Secale cereale L., 2n = 2x = 14) has a large genome of about 8 Gbp distributed across seven large chromosomes. Although they are easily observable by microscopy, their identification is difficult due to similar morphology. Thus, wheat-rye chromosome addition and substitution lines were originally employed to accomplish this and establish the homology of rye chromosomes with those of Triticinae species. The introduction of differential staining and fluorescence in situ hybridization (FISH) provided an important advance, but chromosome identification was still hampered by polymorphism of chromosome banding patterns. A different approach to identify chromosomes involves crosses of a sample to a tester set of wheat-rye chromosome addition lines and cytological analysis of chromosome pairing during meiosis in F1 hybrids. While FISH enabled the analysis of long-range molecular organization of the chromosomes, genomic in situ hybridization (GISH) using rye genomic DNA as probe allowed identification of rye chromosomes introgressed to wheat, including interspecific chromosome translocations. The analysis of isolated mitotic metaphase chromosomes by flow cytometry enabled identification of chromosome 1R, and, if present, the accessory B chromosome. The two chromosomes could be purified by flow sorting for downstream analyses. Chromosomes 2R–7R could not be discriminated from each other and thus were flow-sorted individually from respective wheat-rye chromosomes addition lines, as was the short arm of 1R (1RS).Flow sorting of rye chromosomes facilitated the development of chromosome-specific molecular markers. Next generation sequencing of flow-sorted B chromosomes provided insights into their molecular organization and origin (Chap. 4). Sequencing each of the seven rye chromosomes resulted in the first draft genome sequence, informing about rye gene complement and evolution, and recently facilitated the assembly of a rye reference genome.