*2.2.1. DNA extraction from hop varieties*

DNA was extracted from the pellets or dried cones of the three hop varieties (A, B, and C), used in the transcriptome analysis described above. DNA extraction by the cetyltrimethylam‐ monium bromide (CTAB; Sigma-Aldrich Co. LLC., St. Louis, USA) method was carried out as follows. The CTAB solution comprised of 2% (w/v) CTAB, 100 mM Tris–HCl (pH 8.0), 20 mM EDTA (pH 8.0), 1.4 M NaCl, and 1% (w/v) polyvinylpyrrolidone (PVP). About 10–50 g of hop pellets or 1 g of dried cones were ground in a mortar with or without liquid nitrogen. Next, 650 μL of the CTAB solution and 2 μL 1 mg/mL RNaseA solution were added to the ground material in a 1.5 mL microtube and stirred well. This microtube containing the sample and the CTAB solution was submerged in a constant temperature water bath held at 65°C such that the content of the tube was completely underwater; the mixture was incubated for 1 h to disrupt hop cells. An equal volume (650 μL) of chloroform/isoamyl alcohol (24:1; Sigma-Aldrich Co. LLC.) was subsequently added, and the mixture was manually mixed by inversion for 3 min. The mixture was centrifuged in a Beckman's Allegra 21R centrifuge (with F2402H rotor) at 15,000 rpm (ca. 15,000×*g*) for 1–5 min and thereby fractionated into an organic solvent layer (lower) and an aqueous layer (upper). The aqueous layer (ca. 400 μL) was removed to a new tube. After the addition of an equal volume of isopropyl alcohol (Wako Pure Chemical Industries, Ltd.), the mixture was mixed by inversion and then centrifuged in the same way as described above. The supernatant was discarded and the remaining sediment was rinsed with about 500 μL of 70% ethanol (Wako Pure Chemical Industries, Ltd.) and dried in an MV-100 MicroVac Mini Vacuum-Centrifugal Evaporator (TOMY Seiko Co., Ltd.) for about 5 min. The residue was dissolved in 20–50 μL of TE buffer (pH 8.0, Nippon Gene Co., Ltd.) to obtain the DNA sample.

The extracted DNAs were subjected to amplification of the respective regions A1, B1, C1, and A1-2 using the primer sets shown in Table 4. The primers were designed using the DNASIS Pro software (Hitachi Software Engineering Co., Ltd.).


**Table 4.** PCR primers used in the study.

**Reference/ variety supplied** **Total number of contigs**

328 Next Generation Sequencing - Advances, Applications and Challenges

**Table 3.** Results for SNP analytical regions.

*2.2.1. DNA extraction from hop varieties*

obtain the DNA sample.

Pro software (Hitachi Software Engineering Co., Ltd.).

for further confirmation of the analysis regions.

Variety A 43638 10472 6

**Total number of SNPs detected**

Variety B 42395 14408 9 1.0k B1 Variety C 45432 19330 24 2.8k C1

Primers were designed for each region using DNASIS Pro software (Hitachi Software Engi‐ neering Co., Ltd.; Tokyo, Japan), and PCR amplifications of the four regions were performed

**2.2. Application of NGS-SNP genotypes to identify hop varieties by Sanger sequencing**

DNA was extracted from the pellets or dried cones of the three hop varieties (A, B, and C), used in the transcriptome analysis described above. DNA extraction by the cetyltrimethylam‐ monium bromide (CTAB; Sigma-Aldrich Co. LLC., St. Louis, USA) method was carried out as follows. The CTAB solution comprised of 2% (w/v) CTAB, 100 mM Tris–HCl (pH 8.0), 20 mM EDTA (pH 8.0), 1.4 M NaCl, and 1% (w/v) polyvinylpyrrolidone (PVP). About 10–50 g of hop pellets or 1 g of dried cones were ground in a mortar with or without liquid nitrogen. Next, 650 μL of the CTAB solution and 2 μL 1 mg/mL RNaseA solution were added to the ground material in a 1.5 mL microtube and stirred well. This microtube containing the sample and the CTAB solution was submerged in a constant temperature water bath held at 65°C such that the content of the tube was completely underwater; the mixture was incubated for 1 h to disrupt hop cells. An equal volume (650 μL) of chloroform/isoamyl alcohol (24:1; Sigma-Aldrich Co. LLC.) was subsequently added, and the mixture was manually mixed by inversion for 3 min. The mixture was centrifuged in a Beckman's Allegra 21R centrifuge (with F2402H rotor) at 15,000 rpm (ca. 15,000×*g*) for 1–5 min and thereby fractionated into an organic solvent layer (lower) and an aqueous layer (upper). The aqueous layer (ca. 400 μL) was removed to a new tube. After the addition of an equal volume of isopropyl alcohol (Wako Pure Chemical Industries, Ltd.), the mixture was mixed by inversion and then centrifuged in the same way as described above. The supernatant was discarded and the remaining sediment was rinsed with about 500 μL of 70% ethanol (Wako Pure Chemical Industries, Ltd.) and dried in an MV-100 MicroVac Mini Vacuum-Centrifugal Evaporator (TOMY Seiko Co., Ltd.) for about 5 min. The residue was dissolved in 20–50 μL of TE buffer (pH 8.0, Nippon Gene Co., Ltd.) to

The extracted DNAs were subjected to amplification of the respective regions A1, B1, C1, and A1-2 using the primer sets shown in Table 4. The primers were designed using the DNASIS

**Number of SNPs homozygous in 2 varieties other than reference (per contig)**

**Contig size (bp)**

> 1.4k A1 719 A1-2

**Analysis region**

> PCR was performed in a Veriti 96 Well Thermal Cycler (Life Technologies Japan Ltd.) with PerfectShot Ex Taq (Loading dye mix, Takara Bio Inc.), as described in its manual. The temperature during the 30 PCR cycles was 98°C for 10 s, 60°C or 50°C for 30 s, and 72°C for 60 s.
