Fig. 1. Identification of a splice-site mutation in the CX1512-44 GmFAD3A allele. A. schematic diagram of GmFAD3A exons (5–7, black rectangles) connected by introns (horizontal lines). Normal splicing and intron removal is represented by dashed lines above the introns. Abnormal splicing (black and gray lines below the introns) characterized CX1512-44 mRNA with either exclusion of exon 6 or activation of a cryptic splice site within an intron leading to improper expansion of exon 6 coupled to proper splicing of exons 5 and 7. A mutation in the splice site was identified at the first base following exon 6, (position denoted by an asterisk). B. Sequence comparison of Williams 82 and CX1512-44 GmFAD3A splice site following exon 6. PCR products generated from amplification of genomic DNA were cloned and sequenced. Sense sequence is listed 5' to 3', with the vertical line indicating the separation of exon 6 and intron sequence. The first base of the intron in the CX1512-44 allele is changed from G to A (underlined), mutating a consensus splice site (G^XXXGT, bold, Brown et al., 1996). C. Sequence of genomic DNA from Williams 82 GmFAD3A beginning with base 763 of the cDNA sequence and including the exon 6/intron 6 boundary. Exon sequences are listed in capitals while intron sequences are lowercase. The sequences corresponding to the PCR primers used for the GmFAD3A MaeIII assay are shown in bold (see Materials and Methods). The base mutated in CX1512-44 is underlined, and the MaeIII recognition sequence (GTNAC) is in parentheses.