title: TILLING for allergen reduction and improvement of quality traits in peanut (Arachis hypogaea L.).
abstract: 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.
authors.affiliation: Department of Horticulture/NESPAL, University of Georgia-Tifton Campus, Tifton, GA 31793, USA.
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Knoll
authors.forename: M Laura
authors.initials: ML
authors.lastname: Ramos
authors.forename: Yajuan
authors.initials: Y
authors.lastname: Zeng
authors.forename: C Corley
authors.initials: CC
authors.lastname: Holbrook
authors.forename: Marjorie
authors.initials: M
authors.lastname: Chow
authors.forename: Sixue
authors.initials: S
authors.lastname: Chen
authors.forename: Soheila
authors.initials: S
authors.lastname: Maleki
authors.forename: Anjanabha
authors.initials: A
authors.lastname: Bhattacharya
authors.forename: Peggy
authors.initials: P
authors.lastname: Ozias-Akins
mesh_terms: D055516:2S Albumins, Plant / Q000235:genetics*; D000485:Allergens / Q000235:genetics; D052179:Antigens, Plant / Q000235:genetics*; D010367:Arachis / Q000235:genetics*; D015139:Blotting, Southern; D004252:DNA Mutational Analysis / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional; D005020:Ethyl Methanesulfonate; D044943:Fatty Acid Desaturases / Q000235:genetics; D018628:Gene Dosage; D055786:Gene Knockout Techniques; D005838:Genotype; D006023:Glycoproteins / Q000235:genetics*; D008565:Membrane Proteins; D009154:Mutation; D010940:Plant Proteins / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D020033:Protein Isoforms; D012639:Seeds / Q000187:drug effects / Q000235:genetics; D016415:Sequence Alignment
chemical_list: D055516:2S Albumins, Plant; D000485:Allergens; D052179:Antigens, Plant; C500889:Ara h 1 protein, Arachis hypogaea; C500890:Ara h 2 allergen, Arachis hypogaea; D006023:Glycoproteins; D008565:Membrane Proteins; D010940:Plant Proteins; D020033:Protein Isoforms; D005020:Ethyl Methanesulfonate; D044943:Fatty Acid Desaturases; C040448:delta-12 fatty acid desaturase