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

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 a popular multiple sequence alignment program for DNA.

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

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 of an intron.

For reference, the two different sizes of the amplicons obtained from the C1 region represented the size with or without the 6-nucleotide insertion in this region.

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 3' end (primer R).

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.


a Including indel (insertion/deletion) sequence.

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