Comparatively Barcoded Chromosomes of Brachypodium Perennials Tell the Story of Their Karyotype Structure and Evolution.

The Brachypodium genus is an informative model system for studying grass karyotype organization. Previous studies of a limited number of species and reference chromosomes have not provided a comprehensive picture of the enigmatic phylogenetic relationships in the genus. Comparative chromosome barcoding, which enables the reconstruction of the evolutionary history of individual chromosomes and their segments, allowed us to infer the relationships between putative ancestral karyotypes of extinct species and extant karyotypes of current species. We used over 80 chromosome-specific BAC (bacterial artificial chromosome) clones derived from five reference chromosomes of B. distachyon as probes against the karyotypes of twelve accessions representing five diploid and polyploid Brachypodium perennials. The results showed that descending dysploidy is common in Brachypodium and occurs primarily via nested chromosome fusions. Brachypodium distachyon was rejected as a putative ancestor for allotetraploid perennials and B. stacei for B. mexicanum. We propose two alternative models of perennial polyploid evolution involving either the incorporation of a putative x = 5 ancestral karyotype with different descending dysploidy patterns compared to B. distachyon chromosomes or hybridization of two x = 9 ancestors followed by genome doubling and descending dysploidy. Details of the karyotype structure and evolution in several Brachypodium perennials are revealed for the first time.

Methods for Cytogenetic Chromosome Barcoding and Chromosome Painting in Brachypodium distachyon and Its Relative Species.

Brachypodium distachyon provides a particularly appealing object for molecular cytogenetic analysis due to its compact genome and low repetitive DNA content, as well as low (x = 5) basic number of chromosomes easily identifiable on the basis of their morphometric features. Some of these features, such as genome compactness, are shared by the other members of the genus, thus making them amenable for comparative cytogenetic mapping. Cytogenetic infrastructure established for B. distachyon was initially based on fluorescence in situ hybridization with various tandemly repeated sequences as probes. The molecular cytogenetic studies advanced greatly with the development of B. distachyon large DNA insert genomic libraries. These resources coupled with the access to the fully sequenced genome of B. distachyon enabled chromosome painting in monocots for the first time. This pioneering work was subsequently extended to other Brachypodium species, allowing insight into grass karyotype evolution. In this protocol we describe the methods of making somatic and meiotic chromosome preparations, probe labeling, FISH with BAC clones, a strategy for chromosome barcoding and chromosome painting in B. distachyon, and comparative chromosome painting in the other Brachypodium species.