Chromosome identification and reconstruction of evolutionary rearrangements in Brachypodium distachyon, B. stacei and B. hybridum

Abstract

Background and AimsThe Brachypodium genus represents a useful model system to study grass genome organization. Palaeogenomic analyses (e.g. Murat F, Armero A, Pont C, Klopp C, Salse J. 2017. Reconstructing the genome of the most recent common ancestor of flowering plants. Nature Genetics49: 490–496) have identified polyploidization and dysploidy as the prime mechanisms driving the diversity of plant karyotypes and nested chromosome fusions (NCFs) crucial for shaping grass chromosomes. This study compares the karyotype structure and evolution in B. distachyon (genome Bd), B. stacei (genome Bs) and in their putative allotetraploid B. hybridum (genomes BdBs).Methods Brachypodium chromosomes were measured and identified using multicolour fluorescence in situ hybridization (mcFISH). For higher resolution, comparative chromosome barcoding was developed using sets of low-repeat, physically mapped B. distachyon-derived bacterial artificial chromosome (BAC) clones.Key ResultsAll species had rather small chromosomes, and essentially all in the Bs genome were morphometrically indistinguishable. Seven BACs combined with two rDNA-based probes provided unambiguous and reproducible chromosome discrimination. Comparative chromosome barcoding revealed NCFs that contributed to the reduction in the x = 12 chromosome number that has been suggested for the intermediate ancestral grass karyotype. Chromosome Bd3 derives from two NCFs of three ancestral chromosomes (Os2, Os8, Os10). Chromosome Bs6 shows an ancient Os8/Os10 NCF, whilst Bs4 represents Os2 only. Chromosome Bd4 originated from a descending dysploidy that involves two NCFs of Os12, Os9 and Os11. The specific distribution of BACs along Bs9 and Bs5, in both B. stacei and B. hybridum, suggests a Bs genome-specific Robertsonian rearrangement.ConclusionsmcFISH-based karyotyping identifies all chromosomes in Brachypodium annuals. Comparative chromosome barcoding reveals rearrangements responsible for the diverse organization of Bd and Bs genomes and provides new data regarding karyotype evolution since the split of the two diploids. The fact that no chromosome rearrangements were observed in B. hybridum compared with the karyotypes of its phylogenetic ancestors suggests prolonged genome stasis after the formation of the allotetraploid.

Progressive refinement of the karyotyping of Brachypodium genomes

“…(a, b) CP of chromosome Bd4 in B. distachyon (2 n = 10, x = 5) and its homoeologues Bs9 and Bs5 in B. stacei (2 n = 20, x = 10), respectively, using a cocktail of Bd4‐derived, arm‐specific bacterial artificial chromosome (BAC) clones (adapted from Idziak et al , ). (c, d) Identification of all chromosomes in B. hybridum (2 n = 30, x = 5 + 10) using six B. distachyon‐ derived, chromosome‐specific BACs, two rDNA probes and a centromeric probe (adapted from Lusinska et al , ). [Correction added after online publication 3 January 2020: in panel (d) the chromosome designation has been corrected to read Bs1‐Bs10.]…”

“…[Correction added after online publication 3 January 2020: in panel (d) the chromosome designation has been corrected to read Bs1‐Bs10.] (e) An example of the reconstruction of individual chromosomes and the karyotype evolutionary histories using CB with abundance mixture of chromosome‐specific BACs derived from B. distachyon (adapted from Lusinska et al , ). The chromosomes are counterstained with DAPI (blue or grey).…”

“…S, short arm; L, long arm; Os12, Os9 and Os11 indicate syntenic segments defined by ancestral rice chromosome equivalents (ARCEs) constituting a hypothetical 12‐chromosome intermediate ancestral grass karyotype (IBI, ). Nomenclature of Bs chromosomes are as in Lusinska et al ().…”

“…Chromosome barcoding and comparative chromosome barcoding (CCB) refine the approach by using sequence information to select BACs defining specific regions of each chromosome. These are the current methods of choice to identify and compare all chromosomes in somatic complements of both the tractable Brachypodium diploids and more challenging polyploids (Lusinska et al , ), such as B. hybridum (Fig. c,d).…”

“…The CCB approach with high‐density probes has also been applied recently to reconstruct the evolutionary rearrangements of individual chromosomes of the three annual species of Brachypodium (Lusinska et al , ). This has been augmented by sequence comparisons of the probes to the 12 chromosomes (Os1‐Os12) constituting the genome of ancestral rice, the so‐called ancestral rice chromosome equivalents (ARCEs).…”