*4.2. SSR Marker Selection and Genotyping*

A total of 853 randomly selected SSR markers, including 200 DPL, 310 MonCGR, 48 NAU, 41 MUCS, and 254 SWU, were surveyed for their polymorphisms in 132 genotypes belonging to seven cotton species. Then, 111 EST and genomic SSR (based on D-genome) polymorphic primers from the Cotton Marker Database (CMD; http://www.cottonmarker.org/) were used in the SSR analysis. The reaction contained 5 µL 2× Taq Master Mix (containing buffer, dNTPs, and Taq DNA Polymerase), 2 µL primers, 1 µL DNA, and 2 µL H2O. The PCR reaction was performed using a together TP 600 thermal cycler (TAKARA Bio Inc., Kusatsu, Japan) and then followed by silver staining according to a previous method described by Zhang et al. [75]. The PCR temperature program was two cycles of 95 ◦C for 3 min pre-denaturing followed by 30 cycles of 94 ◦C for 45 s denaturing, 57 ◦C for 36 s annealing, 72 ◦C for 1 min extension, with a final step of 1 cycle at 72 ◦C for 5 min extension. To confirm that the observed amplicons were amplified from genomic DNA and not a primer artifact, genome DNA was omitted from the control reaction. No amplification products were detected without genomic DNA in any PCR.
