TILLING for allergen reduction and improvement of quality traits in peanut (Arachis hypogaea L.)

Authors: Knoll, Joseph - Joe; RAMOS, M. LAURA - University Of Georgia; ZENG, YAJUAN - University Of Georgia; Holbrook, Carl - Corley; CHOW, MARJORIE - University Of Florida; CHEN, SIXUE - University Of Florida; Maleki, Soheila; BHATTACHARYA, ANJANABHA - University Of Georgia; OZIAS-AKINS, PEGGY - University Of Georgia

Submitted to: BMC Plant Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/13/2011
Publication Date: 5/12/2011
Citation: Knoll, J.E., Ramos, M., Zeng, Y., Holbrook Jr, C.C., Chow, M., Chen, S., Maleki, S.J., Bhattacharya, A., Ozias-Akins, P. 2011. TILLING for allergen reduction and improvement of quality traits in peanut (Arachis hypogaea L.). Biomed Central (BMC) Plant Biology. 11:81.

Interpretive Summary: Allergic reactions to peanuts (Arachis hypogaea L.) can cause severe symptoms and in some cases can be fatal. Avoidance of peanuts is the best strategy to prevent allergic reactions, but this can be difficult because peanut-derived products are common in processed foods. One strategy of reducing the allergenicity of peanuts is to alter or eliminate the allergenic proteins in the seed through mutagenesis. Other seed quality traits such as oil composition also could be improved through this approach. We subjected peanut seeds to mutagenic chemicals ethylmethane sulfonate (EMS) or diethyl sulfate (DES), planted the seeds, and then screened the plants for mutations in specific genes. A treatment of 0.4% EMS for 12 hr yielded the highest mutation frequency, while we did not detect any mutations in the DES treatment. To identify mutations we used a technique called TILLING (Targeting Induced Local Lesions in Genomes), which identifies mismatches between mutant and wild-type DNA sequences. We showed that peanut possesses two copies of the gene Ara h 1, a major allergen. Using TILLING, we identified a premature stop codon mutation in one copy (Ara h 1.02), which should result in elimination of an allergen protein. Though there are only two genes, many Ara h 1 protein products are produced, so we used two-dimensional protein analysis to show that several of these products were reduced or eliminated in homozygous mutant offspring. We also have shown that another major allergen Ara h 2 has two gene copies in peanut. Through TILLING, we identified several mutations in these genes. Two Ara h 2.01 proteins with altered amino acid sequence were tested for allergenicity using patient serum, but no changes were observed. We also found a mutation in Ara h 2.02 that disrupts the start codon, eliminating this protein altogether. TILLIING was also used to identify mutations in AhFAD2, a gene which controls the ratio of oleic to linoleic acid in peanut oil. This work represents the first steps toward the eventual goal of creating a peanut cultivar with reduced allergenicity. Alterations in seed oil composition will also improve the nutritional quality of peanuts.

Technical Abstract: Allergic reactions to peanuts (Arachis hypogaea L.) can cause severe symptoms and in some cases can be fatal, but avoidance is difficult due to the prevalence of peanut-derived products in processed foods. One strategy of reducing the allergenicity of peanuts is to alter or eliminate the allergenic proteins in the seed through mutagenesis. Other seed quality traits such as oil composition also could be improved by altering biosynthetic enzyme activities. Targeting Induced Local Lesions in Genomes (TILLING), a reverse-genetics approach developed to screen mutagenized populations for individuals carrying mutations in specific genes, was used to identify mutants affecting seed traits in peanut. Two similar copies of a major allergen gene, Ara h 1, have been identified in tetraploid peanut, one in each subgenome. The same situation has been shown for Ara h 2, also a major allergen. Thus, TILLING presents challenges for discriminating between homeologous genes in an allotetraploid such as peanut. Nested PCR was employed, in which both gene copies were amplified in the first PCR, followed by a second PCR using gene-specific labelled primers. Mutations were detected upon heteroduplex formation, CEL1 nuclease digestion, and separation of fragments on a Li-Cor DNA Analyzer. The highest mutation frequency (1 SNP/931 kb) was achieved by treating seeds in 0.4% ethyl methanesulfonate (EMS) for 12 hr. The two most significant mutations identified to date are a disrupted start codon in Ara h 2.02 and a premature stop codon in Ara h 1.02. Homozygous individuals were recovered in succeeding generations for each of these mutations, and elimination of the Ara h 2.02 protein was confirmed. Several Ara h 1 protein isoforms were eliminated or reduced in 2D analyses, suggesting elimination of Ara h 1.02 as well. The TILLING approach also was used to identify mutations in fatty acid desaturase AhFAD2 (also present in two copies), a gene which controls the ratio of oleic to linoleic acid in the seed. This work represents the first steps toward the eventual goal of creating a peanut cultivar with reduced allergenicity. Alterations in seed oil composition will also improve the nutritional quality of peanuts.