MODIFICATION OF SOYBEAN SEED COMPOSITION FOR FOOD, FEED, AND OTHER INDUSTRIAL USES
Location: Plant Genetics Research
Title: Soybean Seed Lipoxygenase Genes: Molecular Characterization and Development of Molecular Marker Assays
Submitted to: Theoretical and Applied Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 8, 2009
Publication Date: January 10, 2010
Citation: Lenis, J., Gillman, J.D., Lee, J., Shannon, J., Bilyeu, K.D. 2010. Soybean Seed Lipoxygenase Genes: Molecular Characterization and Development of Molecular Marker Assays. Theoretical and Applied Genetics. 120(6):1139-1149.
Interpretive Summary: Grassy and beany flavors present in soyfood products are the result of the activity of lipoxygenase enzymes in developing seeds. Soybeans have been identified and developed that do not produce one, two, or all three of the seed lipoxygenase activities. However, advanced breeding for lipoxygenase-free soybeans has been hampered by the complicated genetics underlying the trait. The objective of our work was to determine the molecular genetic basis for all of the known sources for two of the mutant lipoxygenases and then to develop molecular marker assays that could be used by plant breeders to hasten the development of lipoxygenase-free soybeans for use in the soyfood industry. Along with the previously determined mutation in the lipoxygenase 2 gene, we discovered mutations in the other lipoxygenase genes for all available sources of the trait. We developed molecular marker assays and validated the efficiency of their use in a population segregating for the lipoxygenase-free trait. The impact of this research is the ability to rapidly develop food grade soybeans with an important seed trait.
Background - Soybean seeds contain three lipoxygenase enzymes that are controlled by three separate genes, Lox1, Lox2 and Lox3. Lipoxygenases play a role in the development of unpleasant flavors in foods containing soybean by oxidation of polyunsaturated fatty acids. Null alleles for all three enzymes have been identified, lox1, lox2 and lox3, and are known to be inherited as simple recessive alleles. Previous studies determined that a T2849A mutation, relative to start codon, resulted in a H532Q substitution, rendering the lipoxygenase 2 enzyme inactive. The molecular genetic basis underlying the lox1 and lox3 mutations was not known. The objectives of this study were to determine the molecular genetic basis of known sources of lox1 and lox3 mutant alleles and to develop molecular marker assays to accelerate the development of lipoxygenase-free soybean varieties.
Results - Two independent mutations, a 74 bp deletion and a nonsense C2880A mutation, were identified as causative of the lack of Lox1 in seeds of independent lox1 mutants. Similarly, a single base deletion of a guanine within a run of five guanine nucleotides from position 97 to 101, relative to start codon, results in a frame shift that truncates the Lox3 protein in lox3 mutants. Molecular marker assays were designed to distinguish mutant from wild type alleles for Lox1, Lox2 and Lox3 genes. Genotype and lipoxygenase activity phenotype of two segregating populations derived from a cross between M23 (Lox1, Lox2, Lox3) and Jinpumkong 2 (lox1, lox2, lox3) were examined. Results indicated a complete association between the inheritance of homozygous lox mutant alleles and the lack of lipoxygenase activity. It was also observed that recombination within Lox1 locus led to the generation of the triple lox mutant phenotype in the cultivar Jinpumkong 2.
Conclusions - The genetic basis of mutations at the Lox1 and Lox3 soybean genes were discovered, and molecular marker assays perfectly associated with the causative mutations were developed for the three soybean seed Lox genes. Molecular markers herein reported represent a rapid, simple and accurate alternative to evaluate soybean genotypes for seed lipoxygenase activity, allowing for efficient introgression of the lipoxygenase-free trait in soybean genotypes.