Evaluation location: Oregon, United States
The European Cooperative Program for Plant Genetic Resources (ECPGR) developed a 12 dinucleotide SSR fingerprinting set to genotype and compare individuals within and between germplasm collections (Evans et al., 2015). The SSRs in the ECPGR fingerprinting set are amplified in two PCR reactions. They contain dinucleotide motifs and thus exhibit various artifacts such as stutters, split peaks, and binning errors, making the ECPGR fingerprinting set difficult to use (Evans et al., 2015). In this study, an easy-to-use, single reaction, 10-SSR fingerprint set containing mostly high-core repeat SSRs was developed and compared to the ECPGR set and to single nucleotide polymorphism (SNP) markers found on the new 70K pear Axiom™ array (Montanari et al., 2019) in 152 and 237 accessions, respectively, for its ability to assess diversity, population structure, pedigree, and identity. PCR reactions for each of the three multiplexes in the ECPGR set and for this new U.S. Pyrus Genetic Resources (USPGR) fingerprinting set were conducted in 15 μL volumes consisting of 8.3 μL of 2× Type-it Multiplex PCR Master Mix (Qiagen N.V.), 1.5 μL of PCR grade water, 1.7 μL of primer mix, and 3.5 μL of DNA at a concentration of 3 ng/μL. The primers were optimized to the following concentration in the primer mix TXY276-0.25µM; TsuENH046-0.25µM; TsuENH083-0.375µM; TsuENH089-0.12µM; TsuENH076-0.18µM; CH04e03-0.5µM; CH01d08-0.36µM; NAUpy40d-0.18µM; TsuENH080_354-0.375µM; TXY144_347-0.18µM. The ECPGR optimized primer concentrations were as follows for Mpx 1:EMPc117-1µM; CH05c06-1.5µM; GD147-2µM; EMPc11-0.5µM:Mpx 2: CH04e03-1µM; CH01f07a-0.38µM; CH03g07-1.5µM; CH01d08-2µM: and Mpx 3: CH03d12-2µM; CH02b10-0.5µM; CH01d09-0.63µM; GD96-2µM. Reactions for both fingerprinting sets were amplified in an Eppendorf Gradient thermocycler (Eppendorf Inc., Westbury, NY, U.S.A.) using a program consisting of an initial denaturation at 95 °C for 5 min; 10 cycles of denaturation at 95 °C for 30 s, annealing for 1.5 min starting at 62°C and decreasing by 1 °C per cycle, and extension at 72 °C for 30 s; 30 cycles of denaturation at 95 °C for 30 s, annealing at 52 °C for 1.5 min, and extension at 72 °C for 30 s; followed by a 30 min hold at 60 °C. PCR products were pooled for Mpx 1 and Mpx 2 of the ECPGR set and all PCR products were separated via capillary electrophoresis using a Beckman Coulter CEQ 8000 (Beckman Coulter Inc., Brea, CA, U.S.A.).