Fig. 1. Identification of a splice-site mutation in the A29 GmFAD3B allele. (A) Schematic diagram of GmFAD3B exons 3–4 (gray rectangles) connected by an intron (horizontal lines). Normal splicing and intron removal is represented by dashed lines above the introns. Abnormal splicing (solid lines below the introns) characterized A29 mRNA with activation of a cryptic splice site within exon 4 leading to exclusion of the first seven bases of exon 4. A mutation in the splice site was identified at the first base before exon 4 (position denoted by an asterisk). (B) Sequence comparison of Williams 82 and A29 GmFAD3B splice site junction at the intron 3–exon 4 boundary. 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 intron 3 and exon 4. The final base of the intron in the A29 allele is changed from G to A (underlined), mutating a consensus splice site (AG-splice, bold [Brown et al., 1996]). (C) Sequence of genomic DNA from Williams 82 GmFAD3B beginning within the 3' part of intron 3 and ending at base 533 of the cDNA sequence (exon 4 begins at base 466 of the cDNA sequence). Exon sequences are listed in capitals while intron sequences are lowercase. The base mutated in A29 is underlined, the HpaI recognition sequence (GTTAAC) is in parentheses, and the consensus splice sequence is in bold.