A 25 KASP SNP genotyping assay for Humulus identification. Tissue for KASP genotyping were shipped after the addition of silica to each well using the Plant Sample Collection kit (according to the manufacturer’s recommendations) to LGC Biosearch Technologies, where DNA extraction and genotyping was conducted. Raw allele calls for the 25 assays were received from LGC Biosearch Technologies and assessed via their SNPviewer software.
A Humulus 9 SSR multiplex fingerprinting set containing SSRs with trinucleotide or higher core repeats was developed to distingish European and wild North American accessions and confirm identity and pedigree in the NCGR germplasm collection. PCR reactions 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. Primers were optimized to the following concentration in the primer mix HI-AGA7 6-FAM-0.40 μM; K10315842 6-FAM-0.31 μM; K10315910 6-FAM-0.50 μM; hop primer 6 VIC-0.40 μM; K10316221 NED with pigtailed reverse (ptR)- 0.63 μM; K10315931 VIC with ptR-3 μM; K10316016 VIC-0.50 μM; hop primer 1 NED-0.50 μM; ACA 1-K9-3 PET-0.63 μM. PCR reactions for 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 65°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 55 °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 seperated on an Applied Biosystems (ABI) 3500 Genetic Analyzer
CEQ platform: Applied Biosystems (ABI) 3500 Genetic Analyzer