*2.2.6. Comparison of DNA samples prepared from hop pellets and cones*

Comparison was made between the results obtained in the two cases where DNA samples were extracted from either the hop pellets or the dried hop cones, as described in Section 2.2. It was confirmed that DNA extraction and sequencing were possible with both types of DNA samples. Also, analyses using DNA samples of both types yielded the same results. This result further demonstrates that inspection at a processing step (e.g., inspection for contamination at a pelletization step) is technically possible.

#### *2.2.7. Comparison between three Saaz clones*

About 1 g each of dried cones of three Saaz clones (Osvald's clones 31, 72, and 114) was ground in a mortar in the presence of liquid nitrogen, and the DNA was extracted from about 50 mg each of the ground materials by the CTAB method described in Section 2.2. Each of the extracted DNA sample was subjected to amplification of the DNA fragments from regions A1, B1, and C1 and followed by sequencing of the amplified fragments, according to the proce‐ dures as described above. For each region, the nucleotide sequences of the three clones were aligned for comparison with each other.

The results obtained confirmed that there was no difference among the three Saaz clones in terms of the nucleotide sequences in the analyzed regions. In other words, it was found that any of Saaz clones could be identified through the above-described analysis using the inventive variety identification regions. These results demonstrated that the analysis using regions A1, B1, and C1 as the variety identification regions required no determination by clone.

#### *2.2.8. Identification of variety mixtures using the newly identified SNP markers*

Because it is important to detect contamination of other varieties, we developed a method for detecting such contamination by using the SNPs markers. The mixed samples of Saaz and Premiant hops were analyzed (for region A1). To prepare different samples, pellets of these varieties were ground as described above followed by mixing of the ground materials in the relative proportions by weight as mentioned in Table 7.

Origin Variety Diplotype

336 Next Generation Sequencing - Advances, Applications and Challenges

*2.2.6. Comparison of DNA samples prepared from hop pellets and cones*

Galena

Super Galena

**Table 6.** Successful identification of 21 hop varieties

a pelletization step) is technically possible.

*2.2.7. Comparison between three Saaz clones*

aligned for comparison with each other.

Zeus 2 c i β2 P Summit 2 c i β3 Q

11 c

Nugget 10 c i T Columbus/Tomahawk 12 c i γ U

h

i

2

Comparison was made between the results obtained in the two cases where DNA samples were extracted from either the hop pellets or the dried hop cones, as described in Section 2.2. It was confirmed that DNA extraction and sequencing were possible with both types of DNA samples. Also, analyses using DNA samples of both types yielded the same results. This result further demonstrates that inspection at a processing step (e.g., inspection for contamination at

About 1 g each of dried cones of three Saaz clones (Osvald's clones 31, 72, and 114) was ground in a mortar in the presence of liquid nitrogen, and the DNA was extracted from about 50 mg each of the ground materials by the CTAB method described in Section 2.2. Each of the extracted DNA sample was subjected to amplification of the DNA fragments from regions A1, B1, and C1 and followed by sequencing of the amplified fragments, according to the proce‐ dures as described above. For each region, the nucleotide sequences of the three clones were

The results obtained confirmed that there was no difference among the three Saaz clones in terms of the nucleotide sequences in the analyzed regions. In other words, it was found that any of Saaz clones could be identified through the above-described analysis using the inventive variety identification regions. These results demonstrated that the analysis using regions A1,

Because it is important to detect contamination of other varieties, we developed a method for detecting such contamination by using the SNPs markers. The mixed samples of Saaz and

B1, and C1 as the variety identification regions required no determination by clone.

*2.2.8. Identification of variety mixtures using the newly identified SNP markers*

A1 B1 C1 A1-2 A1+B1+C1+ (A1-2)

i R2

i O2

i S1 v S2

R1

A representative electropherogram, containing the SNP position 77 in the A1 region (A1\_#77), is shown in Figure 7. Peaks in the electropherogram represent fluorescence intensity of 4 different nucleotides at each DNA positions, which are depicted in different colors; A, C, G, and T, are shown in green, blue, black, and red, respectively. Variety I is homozygous (TT) at position 77, and variety II is homozygous (CC) at the same position. In the case of mixing of varieties in 50% proportion, overlap of 2 peaks of T and C was observed at this position, reflecting contamination. Figure 7 also shows peaks in the same region when variety II was mixed at 5 and 10%. Peaks in blue at this position, derived from variety II, were detected even at 5% contamination. It was the same for A1\_#199 and A1\_#204, in which variety I was heterozygous (GC) and variety II was homozygous (CC), so it was not easy to recognize the contamination by peak height of electropherogram.

**Figure 7.** Representative electropherogram containing the SNP positions 77, 199, and 204 in the A1 region (A1\_#77, A1\_#199, and A1\_#204): "T" is represented in red color, and "C" is in blue color.

We estimated more SNPs, with a focus on homozygous SNPs, and calculated an average of the mixing rate in variety II. As a result, we obtained a closer estimate of the mixing rate. We postulate that contamination at levels lower than 5% could be estimated; however, the


acceptable contamination level for us might be 5% with reference to the genetically modified organism contamination in Japanese food. Thus, the values below 5% have a slight importance.

**Table 7.** Preparation of the 2-variety mixtures and results obtained for each sample.

Each sample was prepared by mixing pellets of two varieties in specified proportions by weight.
