Mutations in Soybean Microsomal Omega-3 Fatty Acid Desaturase Genes Reduce Linolenic Acid Concentration in Soybean Seeds

three fatty acid desaturase gene FAD3 (Bilyeu et al., 2003). GmFAD3A was also characterized as the most One major locus (Fan) and several minor loci have been shown highly expressed of the three homologs in developing to contribute to the linolenic acid level in soybean [Glycine max (L.) Merr.] seeds. The Fan gene encodes a microsomal omega-3 fatty acid seeds. The relative importance of GmFAD3B and desaturase (Arabidopsis FAD3 homolog), and soybeans contain three GmFAD3C to seed linolenic acid levels has not yet FAD3 genes. The objective of this work was to characterize candidate been described, although they have been shown to be soybean FAD3 genes from low linolenic acid soybean lines and associexpressed at low levels in developing seeds (Bilyeu et ate those alleles with the trait. Mutations in two of the three soybean al., 2003). Nuclear-encoded, chloroplast-targeted omegaFAD3 genes were identified, and genotypes with the mutant alleles three fatty acid desaturases may also contribute to seed conferred a reduction of over two thirds of the linolenic acid present linolenic acid levels (Yadav et al., 1993). Omega-3 fatty in the seed. The two mutant genes contributed unequally but additively to the phenotype. The results demonstrated that the mutant genotype acid desaturases are members of an enzyme family charcan be identified with mutation-specific molecular markers in the F2 acterized by the presence of a diiron cofactor which generation, and the low linolenic acid trait will be stably inherited in interacts with three regions of conserved histidine motifs subsequent generations. in the protein (Shanklin et al., 1994). The low linolenic acid soybean line CX1512-44 was previously developed from a mutagenesis breeding proT production of soybeans containing low linolenic gram (James Wilcox, pers. comm.). Although dependent acid levels in the oil fraction is desirable for the on the environmental growth conditions, CX1512-44 seeds demands of modern markets. Through mutagenesis breedtypically contain about 3% (30 g kg 1 oil) linolenic acid, ing, a set of soybean lines has been developed with while wild-type lines contain about 7 to 10% (70–100 g lowered seed linolenic acid levels, and these lines have kg 1 oil) linolenic acid. On the basis of segregation analymutations at the fan locus (Wilcox and Cavins, 1985; sis of the fatty acid composition phenotype of F2 progeny Rennie and Tanner, 1991; Rahman et al., 1996). Other from crosses between CX1512-44 and lines with wildlines have been described in which lower linolenic acid type linolenic acid levels, the low linolenic acid trait was phenotypes were generated, and the inheritance was presumed to be multigenic (data not shown). A genetic characterized as multigenic (Fehr et al., 1992; Rahman characterization of CX1512-44 has not been described in et al., 1997; Rahman et al., 1998; Takagi et al., 1999; the literature. The objective of this work was to characRoss et al., 2000). Successful incorporation of multiple terize candidate soybean FAD3 genes from low linolegenes at independent loci into elite lines would be ennic acid soybean lines and associate those alleles with hanced by the use of molecular markers specific for the the trait. We previously determined that soybean conmutated genes. Mutation-specific allele markers would also enable multigenic traits to be efficiently selected in tained three microsomal omega-3 fatty acid desaturase lines used for backcrossing into elite lines, decreasing the genes. We confirmed by sequence analysis that the three dependence on segregation of poor agronomic traits inherCX1512-44 FAD3 genes were candidates for genetic ited from the mutated parent line with the desired trait. lesions associated with the low linolenic acid trait. In Omega-3 fatty acid desaturase enzymes introduce the addition, we were able to associate the linolenic acid third double bond into linoleic acid precursors to prolevel phenotype and the FAD3 genotype for progeny duce linolenic acid precursors. We previously confirmed of crosses derived from CX1512-44. that the Fan locus was one of the three soybean homologs (GmFAD3A) of the Arabidopsis microsomal omegaMATERIALS AND METHODS K.D. Bilyeu and P.R. Beuselinck, USDA-ARS, Plant Genetics ReCloning and Sequence Analysis search Unit, Columbia, MO 65211; L. Palavalli and D.A. Sleper, Primers were designed on the basis of the sequences deposited Dep. of Agronomy, University of Missouri, Columbia, MO 65211. in the GenBank to amplify soybean GmFAD3A (AY204711), Contribution of the Missouri Agric. Exp. Stn. Mention of a trademark, vendor, or proprietary product does not constitute a guarantee or GmFAD3B (AY204712), and GmFAD3C (AY204713) cDNAs warranty of the product by the USDA or the University of Missouri from line CX1512-44. Reverse transcriptase reactions, PCR and does not imply its approval to the exclusion of other products or amplification, isolation of products from agarose gels, and vendors that may also be suitable. Received 28 Oct. 2004. *Correcloning were as previously described (Bilyeu et al., 2003). sponding author ([email protected]). Published in Crop Sci. 45:1830–1836 (2005). Genomics, Molecular Genetics & Biotechnology Abbreviations: bp, base pairs; CAPS, cleaved amplified polymorphic sequence; dNTP, deoxyribonucleotide triphosphate mix; PCR, polydoi:10.2135/cropsci2004.0632 © Crop Science Society of America merase chain reaction; RT, reverse transcriptase; SNP, single nucleotide polymorphism. 677 S. Segoe Rd., Madison, WI 53711 USA 1830 Published online August 1, 2005