The heterochromatin banding patterns as well as the physical locations of 45S and 5S rDNA of the mitotic metaphase chromosomes in Fagopyrum esculentum Moench were analyzed for the first time using orcein staining and double-color fluorescent in situ hybridization, respectively. These techniques led to the successful identification of the full set of F. esculentum trisomics from 25 trisomic lines. The results showed the abundant heterochromatin banding of 27 bands, which included 9 centromeric bands, 12 terminal bands, 5 intercalary bands, and 1 satellite band. These highly polymorphic bands exhibited significant differences in the heterochromatin banding among eight pairs of homologous chromosomes. The resulting rDNA physical map showed five pairs of 45S DNA loci located on IS, IIS, IIIS, IVS, and VIS, as well as two pairs of 5S rDNA loci located on IL and VS. The eight distinct pairs of homologous F. esculentum chromosomes can be differentiated based on the locations of the 45S and 5S rDNA as well as the karyotypic characteristics of the mitotic metaphase chromosomes. The full set of F. esculentum trisomics from the 25 trisomic lines include T10, T7, and T24 as T1H ch ; T13 and T14 as T2H ch ; T15, T16, and T23 as T3H ch ; T4 and T18 as T4H ch ; T3, T17, and T19 as T5H ch ; T1, T6, and T20 as T6H ch ; T5, T8, T21, T22, and T25 as T7H ch ; and T9 and T11 as T8H ch .
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