Genetic variations of γ-/ω-gliadin and Spa (storage protein activator) in 40 Korean wheat cultivars were evaluated to provide genetic information for improving end-use quality in wheat breeding programs. Korean wheat cultivars were classified into 13 patterns at the Gli-1 locus based on the allelic variation using A-PAGE (acidic-polyacrylamide gel electrophoresis). Seven, five, and six alleles were identified at Gli-A1, Gli-B1, and Gli-D1 loci, respectively. Allele-specific PCR markers for γ-gliadin corresponded to specific allele at Gli-1 loci on A-PAGE, which Gli-A1f, Gli-A1h and Gli-A1l alleles corresponded to GliA1.2, Gli-B1h and Gli-B1f alleles corresponded to GliB1.2 and Gli-D1f, Gli-D1m and Gli-D1o alleles corresponded to GliD1.1. DNA markers for γ-45 and γ-42 also corresponded to the γ-gliadin patterns around 40kDa on A-PAGE, except in Sukang, Ol and Joongmo2003. However, allelic specific PCR markers for ω5-gliadin did not correspond to that of A-PAGE. Three alleles were identified at Spa-A1 locus, whereas there was no variation at Spa-B1 and Spa-D1 loci.

The ω5-gliadins are the major allergens in wheat-dependent excise-induced anaphylaxis (WDEIA). In this study, SDS-PAGE analysis was used to assign the ω5-gliadins (Gli-B1) alleles in thirty two Korean wheat cultivars, compared with eleven standard wheat cultivars for Gli-B1a~m alleles. These results were reconfirmed with their complementary Glu-B3 low-molecular-weight glutenin subunits alleles tightly linked with Gli-B1 locus revealed with 2-DGE in our previous study. As a result, one Gli-B1b, four Gli-B1d, two Gli-B1f, six Gli-B1m and nineteen Gli-B1h varieties were identified. This is the first report on revealing the Gli-B1 alleles in Korean wheat cultivars and represents valuable basic data on wheat allergy, relationship between gliadin and wheat quality, and development of hypo-allergenic wheat.

Celiac disease (CD) is classified as an autoimmune disease of small intestine and occurred with people with the human leucocyte antigen (HLA) DQ2(8) cells. The gluten commonly called for the gliadins and glutenins from wheat and related proteins from barley and rye is significant cause of celiac disease. There are many sequences that recognized by T-cell according to species and different types of gliadins. In ω-gliadin, two sort of epitopes were figured out that consisting of some proline(P) and glutamine(Q) scattered in gliadin sequence. All registered ω-gliadin sequences deposited in NCBI database were downloaded and collected. In order to classify groups depending on sequence difference, sequence similarity and their closeness were analyzed by phylogenetic trees using by MEGA (ver.6.06). Chinese spring genome sequence database offered by URGI (Unité de Recherche Génomique Info) is used for sequence assembly. Primers to validate presence of epitopes were designed by two different type from conserved and specific region. Primer pair from consensus region were designed in conserved domain of ω-gliadin sequences from public database by sequence alignment. And, sequence-specific primers of ω-gliadin were designed from the unique region of each ω-gliadin sequence comparing ω-gliadin sequences from NCBI database with draft sequence of Chinese spring in URGI. The two known epitopes of ω-gliadin were located on same site, approximately from the 315th nucleotide to the 348th nucleotide in CDS. Candidate epitopes present in ω-gliadin were divided into three categories based on analysis of sequence similarity. This categorization shows similar pattern with groups that were previously reported by sequence motifs such as SRLL, AREL, ARQL and KELQ. However, sequence which has AREL motif and sequence ARQL motif were not distinguished obviously in ω-gliadin based on sequence alignment.

Gliadins are the main class of wheat seed storage proteins. Since gliadins show a high level of polymorphism as well as genetically fixed, it can be used as a marker for the genetic identification. Gliadin subunit diversity information can be useful for wheat quality breeding programs. Tunisia is a country in the North Africa bordered with the Mediterranean Sea to the north and the east but with the Sahara desert to the south, which represent extremely different growth climate. Therefore, there may be a numerous variation in Tunisian common wheat and durum wheat. Total 48 lines of wheat consisted of 32 common wheat (16 Korean wheat and 16 Tunisian common wheat) and 16 Tunisian durum wheat were incorporated in this study. Gliadins were extracted with 70% ethanol and fractionated by acid polyacrylamide gel electrophoresis (A-PAGE) at 8% in aluminum lactate buffer (pH 3.1). The gel was stained with 0.25% Coomassie Brilliant Blue (CBB) R-250. The presence of each gliadin subunit band was scored and cluster analysis was carried out. The cluster showed that wheat varieties were classified into some groups and their genetic distance could be identified. The obtained information will be helpful to the future breeding program of tetraploid durum wheat as well as hexaploid common wheats. Acknowledgement: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2012K1A3A1A09028123) and carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project title: Development of high yielding wheat with stress tolerance via molecular breeding strategies, Project No. PJ008031)”, Rural Development Administration, Republic of Korea.

The environment in which a given genotype is grown may influence its grain quality characteristics. When varieties are ~times evaluated over numerous environments, a variety environment interaction usually is observed, but the relative magnitude of environmental(E), genetic(G), and G ~times E effects on quality is unclear. In order to determine relative contribution of genotype, environment, and G ~times E interaction to the variations observed in grain quality characteristics, 18 Korean wheat cultivars and experimental lines were evaluated in two environments in 1998 and 1999. Correlation coefficients between grain quality and agronomic characteristics were also estimated. The analysis of variance for the optical density obtained by reaction bet- ween gliadin and anti-gliadin polyclonal antibody (AGPab) indicated that gliadin content measured by Enzyme-Linked Immunosorbent Assay(ELISA) was significantly in- fluenced by environment and cultivar differences. The significant differences of year and year ~times location were also found. The ratio of the variances associated with environmental effects to the variances associated with genetic effect gave relatively greater influence of environmental factor on gliadin content. The different protein content from same genotype grown in different environment might be associated with degree of storage protein accumulations. Significant relationships between ELISA and protein content, yield, ten spike weight, and ten spike number were detected. Polyphenol oxidase (PPO) activity was significantly influenced by year, location, cultivar and year ~times location. The variance in grain PPO activities among growing years appeared larger than the variation produced by the cultivar examined. This suggested that the growing environment contributed more to variability in grain PPO concentration.

Immunological method has been applied in biochemical genetic analysis of seed storage proteins. We developed and characterized anti-gliadin polyclonal antibody (AGPab) specific to gliadin fractions whose quality and quantity were known to be associated with wheat end-use quality. Reactions of anti-gliadin polyclonal antibody (AGPab) to gliadin were linearly decreased as AGPab and antigen were diluted. Dot-blot and immunoblot assay showed that produced AGPab specifically reacted to gliadin and mainly α -, β -, and ~gamma -gliadin subunits. Enzyme-linked immuno- sorbent assay (ELISA) was applied for quantifi-cation of gliadins in Korean wheat cultivars and breeding lines by using AGPab. High reactions between AGPab and gliadins were found in wheat cultivars Olmil and Olgeurumil. Significant difference of optical densities for alcohol soluble proteins among crop species was found, as wheat showed the highest value (0.697) followed by rye (0.295), and barley (0.066).