Repetitive DNA sequences have been widely used in cytogenetic analyses. The use of gene sequences with a low-copy-number, however, is little explored especially in plants. To date, the karyotype details in Brachiaria spp. are limited to the location of rDNA sites. The challenge lies in developing new probes based on incomplete sequencing data for the genus or complete sequencing of related species, since there are no model species with a sequenced genome in Brachiaria spp. The present study aimed at the physical location of conserved genes in chromosomes of Brachiaria ruziziensis, Brachiaria brizantha, and Brachiaria decumbens using RNAseq data, as well as sequences of Setaria italica and Sorghum bicolor through the fluorescent in situ hybridization technique. Five out of approximately 90 selected sequences generated clusters in the chromosomes of the species of Brachiaria studied. We identified genes in synteny with 5S and 45S rDNA sites, which contributed to the identification of chromosome pairs carrying these genes. In some cases, the species of Brachiaria evaluated had syntenic segments conserved across the chromosomes. The use of genomic sequencing data is essential for the enhancement of cytogenetic analyses.

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