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Molecular Genetic Resources for Development of 1% Linolenic Acid Soybeans

^a USDA-ARS, Plant Genetics Research Unit, 210 Waters Hall, Univ. of Missouri Columbia, MO 65211
^b Dep. of Agronomy, Univ. of Missouri, Columbia, MO 65211

View larger version (14K): [in a new window]   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.

View larger version (25K): [in a new window]   Fig. 2. Identification of a mutation in the A29 allele of GmFAD3C. (A) Amino acid alignment of a portion of the soybean and Arabidopsis FAD3 protein sequences (At2 g29980, GenBank accessions AY204710, AY204711, and AY204712). Identical amino acids are highlighted in black while similar amino acids are highlighted in gray. The wild-type and A29 GmFAD3C alleles are shown from amino acid 280 to 329. A SNP in the coding sequence results in a H304Y mutation (indicated above the alignment with an asterisk) for A29 GmFAD3C. Histidine-rich region II (Shanklin et al., 1994) is underlined. (B) Sequence of genomic DNA from Williams 82 GmFAD3C beginning at base 864 and ending at base 951 of the cDNA sequence. Only exon sequences are in the listed region. The base mutated in A29 is underlined (base 910 of the coding sequence) and the BccI recognition sequence is in parentheses.

View larger version (11K): [in a new window]   Fig. 3. Association of linolenic acid phenotype with FAD3 genotype in segregating BC1F2 seeds derived from a backcross (SS97–6946 x [SS97–6946 x IA3017]). Sixty-one BC1F2 seeds were chipped for fatty acid analysis with the remainder of the seed germinated and then subjected to genotyping for A29 or wild-type alleles of GmFAD3A, GmFAD3B, and GmFAD3C. It was not possible to distinguish Aa from AA genotypes. Samples were organized by increasing linolenic acid concentration. Black diamonds () represent soybean lines containing homozygous GmFAD3A mutant alleles and any combination of wild-type or mutant GmFAD3B and GmFAD3A alleles (aaxxxx). Circle symbols () represent soybean lines either heterozygous or homozygous wild-type for the GmFAD3A allele (Aa or AA, designated A?). The black square () represents the only line with the complete homozygous mutant genotype (aabbcc). The black triangle () represents the only line with a genotype of aaBbcc. No lines were identified which had the alternate genotype, aabbCc. Circles with gray shading indicate those lines which had either Aabbcc or AAbbcc genotypes (A?bbcc). Other selected genotypes are identified with arrows, and the genotype is listed.

View larger version (12K): [in a new window]   Fig. 4. Association of linolenic acid phenotype with genotype in plants segregating for mutant alleles of GmFAD3B and GmFAD3C derived from a backcross (SS97–6946 x (SS97–6946 x IA3017)). Fifty BC1F2:3 seeds produced on a aaBbCc plant (26 g kg–1 of oil) were chipped for fatty acid analysis with the remainder of the seed germinated and then subjected to genotyping for A29 or wild-type alleles of GmFAD3B and GmFAD3C. The samples were pooled by genotype, as listed. The number of lines represented in each pool follows the genotype in parentheses. No lines were recovered with the complete mutant genotype. Histograms represent mean linolenic acid (g kg–1 of oil) plus one standard deviation from the mean.