Genotyping Eragrostis curvula Mapping Population

E. curvula SSR markers previously developed by Garbus et al. (2017) (link) were used for genotyping of the mapping population; the primers are listed in Supplementary Table S1. PCR was performed in a final volume of 20 μl containing 1X Taq polymerase reaction buffer, 2.5 mM MgCl₂, 0.125 mM of each dNTP, 1 μM of each primer, 50 ng of genomic DNA, and 2 U of Taq polymerase (Invitrogen, Brazil). The PCR program consisted of an initial denaturation at 94°C for 4 min, 35 cycles of 94°C for 30 s, 58°C for 1 min, and 72°C for 5 min, and a final extension at 72°C for 5 min. The PCR was performed in a thermocycler (MJ Research). Samples were mixed (2:1, v/v) with denaturing loading buffer (95% formamide and bromophenol blue), denatured at 95°C for 5 min, chilled on ice, and resolved in 6% (w/v) silver-stained polyacrylamide gels.