Experimental evidence supports assembly 9. A. PCR amplification confirms the sizes of the regions surrounding the A2, B8, D1-b, and E2 genes. Amplicons in lanes 1 (~4 kb), 2 (~3.6 kb), 4 (~3.6 kb), and 5 (~4 kb), correspond to the sizes of the A2, B8, D1- b, and E2 genes plus their flanking regions according to sizes predicted in all the candidate assemblies (see Figure 2). A single amplicon of ~4 kb (lane 3) was generated from primers predicted to amplify each D1-g, and D1-y genes plus flanking regions. See Table 2 and Figure 2 for primer information. B. Diagram of a region of assembly 9 showing the D1 genes (B8, orange; see also Figure 2). The subcloned regions of 7096 containing D1 genes (amplified with primers 2F and 1R; see Table 2 and Figure 2) are indicated. The assembled sequence for these subclones contains either one (D1-y) or two (D1-g) Ase I restriction sites (purple lines). One of the Ase I sites in the D1-g gene is mutated by a SNP. Because of the gap in assembly 9, which includes this region (dashed line), one of the Ase I sites is predicted (dashed purple line) based on sequence similarity with the D1-y subclone. C. A SNP obliterates an Ase I restriction site and differentiates D1-y and D1-g genes. PCR amplicons using 2F and 1R primers produce 4 kB fragments. When digested with Ase I the clones containing a D1-y gene could be differentiated from those with a D1-g gene. Lane 1, D1-y gene (4.2 kb, 2.3 kb, and 0.9 kb). Lane 2, D1-g gene (4.2 kb and 3.2 kb). Lane 3, vector without insert has one Ase I site (4 kb).