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

BACKGROUND: 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 through mutagenesis. Other seed quality traits could be improved by altering biosynthetic enzyme activities. Targeting Induced Local Lesions in Genomes (TILLING), a reverse-genetics approach, was used to identify mutations affecting seed traits in peanut. RESULTS: 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 major allergen Ara h 2. Due to the challenge of discriminating between homeologous genes in allotetraploid peanut, nested PCR was employed, in which both gene copies were amplified using unlabeled primers. This was followed by a second PCR using gene-specific labeled primers, heteroduplex formation, CEL1 nuclease digestion, and electrophoretic detection of labeled fragments. Using ethyl methanesulfonate (EMS) as a mutagen, a mutation frequency of 1 SNP/967 kb (3,420 M2 individuals screened) was observed. The most significant mutations identified were 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 Ara h 2.02 protein was confirmed. Several Ara h 1 protein isoforms were eliminated or reduced according to 2D gel analyses. TILLING 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. A frameshift mutation was identified, resulting in truncation and inactivation of AhFAD2B protein. A mutation in AhFAD2A was predicted to restore function to the normally inactive enzyme. CONCLUSIONS: This work represents the first steps toward the goal of creating a peanut cultivar with reduced allergenicity. TILLING in peanut can be extended to virtually any gene, and could be used to modify other traits such as nutritional properties of the seed, as shown in this study.

Identification and characterization of a hypoallergenic ortholog of Ara h 2.01.

Peanut (Arachis hypogaea L.), can elicit type I allergy becoming the most common cause of fatal food-induced anaphylactic reactions. Strict avoidance is the only effective means of dealing with this allergy. Ara h 2, a peanut seed storage protein, has been identified as the most potent peanut allergen and is recognized by approximately 90% of peanut hypersensitive individuals in the US. Because peanut has limited genetic variation, wild relatives are a good source of genetic diversity. After screening 30 Arachis duranensis accessions by EcoTILLing, we characterized five different missense mutations in ara d 2.01. None of these polymorphisms induced major conformational modifications. Nevertheless, a polymorphism in the immunodominant epitope #7 (S73T) showed a 56-99% reduction in IgE-binding activity and did not affect T cell epitopes, which must be retained for effective immunotherapy. The identification of natural hypoallergenic isoforms positively contributes to future immunological and therapeutic studies and peanut cultivar development.

Spontaneous and induced variability of allergens in commodity crops: Ara h 2 in peanut as a case study.

Many commodity crops are grown for human consumption, and the resulting food products usually contain proteins, some of which may be allergenic. The legumes, peanut and soybean, as well as tree nuts and some cereal grains are well recognized sources of food allergens. In peanut, there are 11 documented allergenic proteins, although the major allergens are considered to be Ara h 1 and Ara h 2, both of which are seed storage proteins. Methods to reduce or eliminate these proteins from seeds are available and allow the feasibility of this approach to be tested. Greatly reduced amounts of Ara h 2 can be achieved by RNA silencing in transgenic peanut; however, mutagenesis is a more viable and socially acceptable approach to allergen elimination. Although the techniques for mutagenesis are not new, methods for mutant detection at the molecular level have recently been developed. However, these methods are dependent on genome sequence. These methods will facilitate discovery of spontaneous and induced mutations that may be useful over the long term to eliminate certain allergens from peanut.

Chromosomal and phylogenetic context for conglutin genes in Arachis based on genomic sequence.

Comparative genomic and cDNA sequence analysis of ara h 2, a major peanut allergen, and a related conglutin ara h 6 were performed in Arachis hypogaea L. and its putative progenitors, Arachis duranensis and Arachis ipaensis. The complete identity between sequences encoding Ara h 2 isoforms demonstrated that these are homeologous genes and represent orthologs from diploid ancestors. Three copies of ara h 6 were identified in A. hypogaea, one of them located in the A-genome and the other two in the B-genome. Expression analysis showed higher levels of ara h 2 transcripts compared with ara h 6. Dual-labeled genomic in situ hybridization permitted identification of two subgenomes, each of which contained one pair of ara h 2-ara h 6 signals localized by fluorescence in situ hybridization. Characterization of genomic clones showed close genetic linkage between Ara h 2.02 and one copy of ara h 6 in the B-genome. The physical linkage may have arisen by tandem duplication and divergence of an ancestral gene. A gene duplication event specific to the B-genome progenitor has resulted in ara h 6 paralogs. These data provide further evidence for progenitor relationships and genomic organization of the conglutin gene family in the genus Arachis and could contribute to the development of a hypoallergenic peanut.