We sequenced the two lower bands, Band-A and Band-B, derived from different cultivars showing different genotypes for each of the five markers. Two representative cultivars, Chunpoong and Yunpoong, were sequenced for all five markers and other cultivars were also sequenced, including Sunpoong for the gm47n marker, Sunun for the gm129 marker, Sunone for the gm175 marker, and Sunpoong, Sunone, and Gopoong for the gm184 marker. A total of 34 high-quality sequences derived from individual bands was obtained. Multiple sequence comparison allowed us to classify the multiple bands as representing different loci in the same cultivar (paralogs) or allelic forms of the same locus in different

cultivars (alleles; Fig. 2). The bands close to the expected size (Band-B of gm45n, gm47n, and gm175 and Band-A of gm129 and gm184) were derived Selleck Nivolumab from same locus as the reported EST. The other bands (Band-B of gm45n, gm47n, and gm175 and Band-A of gm129 and gm184) find more amplified from a paralogous locus showed relatively different sizes from those expected. The paralogous sequences

were characterized by SNP or InDel variations as well as much larger variations in SSR unit number. For example, the gm175 marker showed polymorphism for both loci among cultivars. Each of the two bands showed one or two copy differences of the AGG SSR motif among cultivars. There was a maximum copy number difference of four for the AGG SSR motif as well as a 21 bp InDel variation between Band-A and Band-B (Table 1). The Band-B sequence of Chunpoong corresponded to the EST, indicating that the EST is derived from the locus of Band-B (Fig. 2A). The gm45n marker showed a maximum copy number difference of five for the TGG SSR motif, (TGG)5 and (TGG)10, as well as two SNPs between Band-A and Band-B. The allelic form of Band-B showed only a two-copy difference for the TGG SSR motif, (TGG)8 and (TGG)10, in Chunpoong and Yunpoong

cultivars, respectively (Table 1). By contrast, Band-A showed no variation Hydroxychloroquine among the different cultivars. Similarly, only one of the two bands, Band-B, was polymorphic among cultivars, except for the gm175 marker. Among the five markers, four had SNPs and the other had an InDel between Band-A and Band-B that served as a signature to distinguish paralogous sequences (Fig. 2, Table 1). We next tried to develop locus-unique markers to amplify selectively single bands derived from one of two paralogous regions. We focused on the SNP regions between paralogous sequences. The gm47n marker showed a more than four SSR unit difference as well as one SNP between Band-A and Band-B (Fig. 2B). The SNP was identified at the position 51 bp as “C” and “T” for Band-A and Band-B, respectively (Table 1). For the polymorphic Band-B-specific primer, we designed an additional left primer, 5′-CTCTGTTTTCTTCCCTTTTCTCTGT-3′, which has the Band-B specific nucleotide “T” at the end and an additional modified nucleotide “G” ( Fig. 2B).