*2.2.4. Identification markers for 21 hop varieties*

*2.2.3. Confirmation of SNP markers by Sanger DNA sequencing*

330 Next Generation Sequencing - Advances, Applications and Challenges

a popular multiple sequence alignment program for DNA.

of an intron.

3' end (primer R).

a

Including indel (insertion/deletion) sequence.

**Table 5.** Properties of SNP-rich analytical regions.

C1, and A1-2, were 651, 729, 640/646, and ca. 1500 bp, respectively.

the size with or without the 6-nucleotide insertion in this region.

Name No. of SNPsa Size (bp) A1 24 541 B1 14 645 C1 43 559a A1-2-L 21 538 A1-2-R 67 516 A1-2-R-2 59 542

Cycle sequencing was performed using the BigDye terminator v1.1 cycle sequencing kit (Life Technologies Japan Ltd.), according to the protocol prescribed by the vendor. The purification of the product as the template DNA for sequencing was performed by the Centri-Sep Spin Column (Life Technologies Japan Ltd.) according to the manufacturer's protocol. DNA sequencing was performed on an ABI PRISM 310 genetic analyzer (Life Technologies Japan Ltd.). Sequence data obtained from the 5' and 3' ends were checked, and the correct base sequence was determined. The nucleotide sequences determined from the tested varieties were aligned in each analysis region by using ClustalW (DDBJ; DNA Data Bank of Japan), which is

Thus, the amplified regions were confirmed. Comparison was also made with the data obtained using the next-generation sequencer, to confirm that the identification of the three varieties A, B, and C was possible. The size of the amplified products from the regions, A1, B1,

The PCR product from the B1 region was bigger in size than expected, and on close scrutiny, it was found that this DNA region included a 111-bp insertion sequence, which might be that

For reference, the two different sizes of the amplicons obtained from the C1 region represented

Since the fragment of region A1-2 had a length of about 1,500 bp, the sequencing data obtained for this region consisted of sequences from two regions: an analytical region, A1-2- L of 538 bp from the 5' end (primer L), and an analytical region A1-2-R of 516 bp from the

It was determined that the analysis of regions A1, B1, C1, and A1-2 (and the SNPs con‐ tained, therein) was also applicable to the identification of other varieties in addition to the

three mentioned above. Thus, analysis of a number of varieties was carried out.

Twenty-one varieties of hops were selected for identification based on the nucleotide sequence in region A1.

Nucleotide sequences were aligned to determine the consensus sequence in region A1. The nucleotide sequences determined in the varieties were evaluated for 24 SNPs in region A1 corresponding to the nucleotide positions 74, 77, 87, 103, 116, 118, 121, 125, 134, 135, 148, 192, 195, 197, 199, 203, 204, 226, 230, 235, 306, 316, 330, and 532. These 21 hop varieties showed 12 types (types 1 to 12) of combinations of 24 SNPs in region A1. The SNP positions are depicted in red in Figure 1.


**Figure 1.** Nucleotide sequence of region A1. Each nucleotide is shown as per the IUPAC definition. Gaps are inserted after every 10 letters to increase clarity. Number at the right in every line denotes the number of nucleotides up to that position. Red letters represent the SNP positions.

Nucleotide sequences determined in the 21 varieties were aligned to determine the consensus sequence in region B1. These sequences were evaluated for 14 SNPs in region B1, correspond‐ ing to the nucleotide positions 178, 204, 227, 234, 245, 246, 247, 248, 370, 426, 439, 547, 562, and 624. The 21 hop varieties to be identified showed 9 types (types a to i) of combinations of 14 SNPs in region B1. The SNP positions are depicted in red in Figure 2.

Similarly, 43 SNPs were found in region C1 (6 of these 43 SNPs, at positions 129–134, consti‐ tuted an indel portion) corresponding to the nucleotide positions 3, 13, 17, 76, 77, 87, 88, 93, 129, 130, 131, 132, 133, 134, 136, 138, 163, 165, 245, 254, 313, 321, 331, 356, 373, 375, 376, 380, 396, 398, 399, 421, 435, 438, 460, 474, 475, 476, 477, 480, 481, 500, and 547. The 21 hop varieties to be identified showed 5 types (types i to v) of combinations of 43 SNPs or an indel portion in region C1. The SNP positions are depicted in red in Figure 3.

The analyses of 24 SNPs in region A1, 14 SNPs in region B1, and 43 SNPs or an indel portion in region C1 made it possible to classify 16 of the 21 varieties, excluding Perle, Northern Brewer, Premiant, Zeus, and Summit, into distinct types, respectively. For identifying Perle, Northern


**Figure 2.** Nucleotide sequence of region B1. Please consult the legend of Figure 1 for details.


**Figure 3.** Nucleotide sequence of region C1. Please consult the legend of Figure 1 for details.

Brewer, Premiant, Zeus, and Summit, the nucleotide sequences in region A1-2 of these varieties were further analyzed. These nucleotide sequences were aligned to determine the consensus sequence in region A1-2-L. The nucleotide sequences were evaluated for 21 SNPs in region A1-2-L (10 of these 21 SNPs constitute indel portions) corresponding to the nucleotide positions 34, 101, 118, 124, 164, 168, 171, 186, 187, 188, 189, 190, 191, 192, 193, 194, 393, 398, 399, 459, and 502 (positions 186–194 and position 399 each constituted an indel portion). The SNP positions are highlighted in red in Figure 4.

These nucleotide sequences were aligned to determine the consensus sequence in region A1-2- R. The nucleotide sequences determined in the varieties were evaluated for 67 SNPs in region A1-2-R (4 of these 67 SNPs, at positions 57–59 and 65, constituted an indel portion) corre‐ sponding to the nucleotide positions 1, 2, 3, 5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 21, 25, 26, 27, 28, 29, 30, 31, 33, 35, 36, 37, 38, 41, 42, 43, 44, 46, 47, 48, 50, 51, 56, 57, 58, 59, 63, 65, 68, 72, 78, 79, 84, 86, 88, 90, 92, 118, 153, 154, 191, 205, 206, 226, 228, 233, 254, 289, 315, 350, 392, and 405. The SNP positions are highlighted in red in Figure 5.

Zeus, Summit, and Premiant were subjected to further analysis for region A1-2-R2. In region A1-2-R2 (34 of these 59 SNPs constituted an indel portion), the SNPs corresponded to the nucleotide positions 3, 8, 19, 20, 27, 28, 40, 41, 46, 47, 57, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 90, 112, 116, 118, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 140, 156, 178, 191, 192, 244, 266, 271, 377, and 430 (positions 64–85, and 120–131 constituted the indel portions). The SNP positions are depicted in red in Figure 6.

Using nucleotides at the SNP positions in the fragments of A1-2-L (sequenced from 5' primer) and A1-2-R/R2 (sequenced from 3' primer), Perle and Northern Brewer as well as Premiant, Zeus, and Summit were successfully identified, when 21 and 67 SNPs were found between Perle and Northern Brewer, and 59 SNPs were found among Premiant, Zeus, and Summit.


**Figure 4.** Nucleotide sequence of region A1-2-L. Please consult the legend of Figure 1 for details.

#### *2.2.5. Characterization of SNP-rich regions and identification of hop varieties*

Brewer, Premiant, Zeus, and Summit, the nucleotide sequences in region A1-2 of these varieties were further analyzed. These nucleotide sequences were aligned to determine the consensus sequence in region A1-2-L. The nucleotide sequences were evaluated for 21 SNPs in region A1-2-L (10 of these 21 SNPs constitute indel portions) corresponding to the nucleotide positions 34, 101, 118, 124, 164, 168, 171, 186, 187, 188, 189, 190, 191, 192, 193, 194, 393, 398, 399, 459, and 502 (positions 186–194 and position 399 each constituted an indel portion). The SNP positions

**Figure 2.** Nucleotide sequence of region B1. Please consult the legend of Figure 1 for details.

332 Next Generation Sequencing - Advances, Applications and Challenges

**Figure 3.** Nucleotide sequence of region C1. Please consult the legend of Figure 1 for details.

are highlighted in red in Figure 4.

In every SNP-rich region, consensus sequence and SNP positions were detected by the alignment analysis. Nucleotide polymorphisms in each variety were evaluated at the SNPs positions in all the regions. Within the 21 studied hop varieties, 24 SNPs were identified in the A1 region and 14 SNPs (including indel) were observed in the B1 region. *H. lupulus*, with normally a diploid chromosome, had heterozygous or homozygous SNPs in each DNA region. For example, in the A1 region, European varieties had homozygous SNPs at 77th (C/T) and


**Figure 5.** Nucleotide sequence of region A1-2-R. Please consult the legend of Figure 1 for details.


**Figure 6.** Nucleotide sequence of region A1-2-R2. Please consult the legend of Figure 1 for details.

103th (A/G) positions. Such homozygous SNPs can be analyzed more easily than the hetero‐ zygous SNPs when mixed with another variety, having different nucleotides at the SNP positions. In a case, where the variety Saaz was mixed with the variety Sládek, position 77 SNPs were T and C, respectively, in the A1 region (data not shown), and these nucleotides could be easily distinguished on their electropherograms. On the other hand, position 74 SNPs were W (A and T) and A, respectively, in the same region. In this case, it was difficult to recognize whether the variety Saaz was mixed with the variety Sládek or not. Each variety had a specific combination of SNPs as markers, and 12 and 9 DNA types in the A1 and B1 regions, respectively, were identified among the 21 varieties. Indel was also found in the C1 region. Nucleotides at the SNP positions including indel were observed. Forty-four SNPs were found in this region, and 5 DNA types were identified among the 21 varieties. It was revealed that Galena had 2 DNA types in the B1 region and Cascade and Super Galena had 2 DNA types in the C1 region.

Additionally, the A1-2 region was searched because there was no difference in the combination of the SNPs in A1, B1, and C1 regions between Perle and Northern Brewer, among Premiant, Zeus, and Summit. Nucleotides at the SNP positions in the fragments of A1-2-L (sequenced from 5' primer) and A1-2-R/R2 (sequenced from 3' primer) were shown, when 21 and 67 SNPs were found between Perle and Northern Brewer, and 59 SNPs were found among Premiant, Zeus, and Summit. Perle and Northern Brewer as well as Premiant, Zeus, and Summit were successfully identified.

For confirmation, we tried to distinguish between Columbus and Tomahawk, considered genetically identical [22–24], by analyzing the 4 regions and, additionally, the middle area of the A1-2 region, with newly designed primers, A1-2-M\_F and A1-2-M\_R (Table 4). No differences in the nucleotide sequences were detected between them. Therefore, we confirmed that these 2 varieties are indeed identical and could be considered a single variety.

Consequently, 21 hop varieties were successfully identified by a combination of these geno‐ types of SNPs, with differences in the 4 SNP-rich regions. The summary results are shown in Table 6.


103th (A/G) positions. Such homozygous SNPs can be analyzed more easily than the hetero‐ zygous SNPs when mixed with another variety, having different nucleotides at the SNP positions. In a case, where the variety Saaz was mixed with the variety Sládek, position 77 SNPs were T and C, respectively, in the A1 region (data not shown), and these nucleotides could be easily distinguished on their electropherograms. On the other hand, position 74 SNPs were W (A and T) and A, respectively, in the same region. In this case, it was difficult to recognize whether the variety Saaz was mixed with the variety Sládek or not. Each variety had a specific combination of SNPs as markers, and 12 and 9 DNA types in the A1 and B1 regions, respectively, were identified among the 21 varieties. Indel was also found in the C1 region. Nucleotides at the SNP positions including indel were observed. Forty-four SNPs were found

**Figure 6.** Nucleotide sequence of region A1-2-R2. Please consult the legend of Figure 1 for details.

**Figure 5.** Nucleotide sequence of region A1-2-R. Please consult the legend of Figure 1 for details.

334 Next Generation Sequencing - Advances, Applications and Challenges


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