Figure 4

Confirmation of SNP detected by the RESCAN type I approach. A. RESCAN type I SNP can be identified in sites that are found for the target restriction site in the query (in this case IR64) but are absent in the reference. In most cases, examination of the reference sequence reveals the presence of a proto sequence, i.e. a sequence that diverges by one base from the expected sequence TTAA: VTAA, TVAA, TTBA, TTAB, where V and B are, respectively, not T and not A. For a proto such as GTAA, a T > G SNP is inferred. A SNP cannot be inferred for a proto site such as TTTAG since either T3 > A or G5 > A could have produced the MseI site. B. We chose 20 type I sites that allowed inference and were detected through 1 or 2 RESCAN reads. The products amplified using flanking PCR primers from Nipponbare and IR64 are shown. C. The amplified products were subjected to digestion with MseI and analyzed by agarose gel electrophoresis. The presence of an extra restriction site in the amplified IR64 DNA and not in the control Nipponbare is evident in 17 of the 19 amplified products, confirming the presence of a SNP producing a restriction site in IR64.