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Brachypodium hybridum  (2n = 30) is a natural allopolyploid with highly divergent sub-genomes derived from two extant diploid species,  B .  distachyon  (2n = 10) and  B .  stacei  (2n = 20) that differ in chromosome evolution and number. We created synthetic  B .  hybridum  allotetraploids by hybridizing various lines of  B .  distachyon  and  B .  stacei . The initial amphihaploid F1 interspecific hybrids were obtained at low frequencies when  B .  distachyon  was used as the maternal parent (0.15% or 0.245% depending on the line used) and were sterile. No hybrids were obtained from reciprocal crosses or when autotetraploids of the parental species were crossed. Colchicine treatment was used to double the genome of the F1 amphihaploid lines leading to allotetraploids. The genome-doubled F1 plants produced a few S1 (first selfed generation) seeds after self-pollination. S1 plants from one parental combination (Bd3-1×Bsta5) were fertile and gave rise to further generations whereas those of another parental combination (Bd21×ABR114) were sterile, illustrating the importance of the parental lineages crossed. The synthetic allotetraploids were stable and resembled the natural  B .  hybridum  at the phenotypic, cytogenetic and genomic levels. The successful creation of synthetic  B .  hybridum  offers the possibility to study changes in genome structure and regulation at the earliest stages of allopolyploid formation in comparison with the parental species and natural  B .  hybridum .

Recreating Stable Brachypodium hybridum Allotetraploids by Uniting the Divergent Genomes of B. distachyon and B. stacei