PCR-RFLP protocols

GRA15, ROP16 and ROP18 PCR-RFLP protocols were followed in accordance with a previous study [29]. ROP5 allele was analyzed as previously described [29, 30]. For each locus, PCR was run on genomic DNA using external primers, followed by PCR amplification with internal primers. All sequences of the primers for each locus are shown in Table 1. A Phusion High-Fidelity PCR Kit (Thermo Fisher Scientific,California, USA) was used for PCR amplification with individual marker primers and a thermocycler program of 5 min at 94 °C, 37 cycles of 30 s at 94 °C, 30 s at 58 °C, and 45 s at 72 °C, and finally, 5 min at 72 °C. PCR products were purified after agarose gel electrophoresis conformation using Axygen PCR clean up kit (Axygen,California, USA). The new primers used in this study are shown in Table 1.

PCR primes for genotyping ROP16, GRA3 and GRA15