Gliadins are major components of the storage proteins in wheat endosperm and are important for the wheat flour quality because their quantity and quality directly affect the wheat gluten property. Gliadins are further separated into three groups, α/3, γ and ω-gliadins, among which the α/3-gliadins are the most abundant. The genes encoding the α/3-gliadins are located at the Gli-2 loci on the short arms of the homoeologous group 6 chromosomes. Complete characterization of the Gli-2 loci is necessary to understand the physical and chemical properties of the α/3-gliadins and to improve the wheat flour quality. However, the Gli-2 loci consist of multiple gene copies with highly similar sequences and include a lot of pseudogenes, which preclude their complete assembling from NGS data and impede a complete characterization of the Gli-2 loci. In fact, the Gli-2 loci is still incomplete even in the first reference genome sequence of wheat, IWGSC RefSeq v1.0 (IWGSC 2018).

In this study, we analyzed the Gli-B2 locus in the wheat chromosome 6B based on the BAC physical map (Kobayashi et al.

2015) using the combined sequence technologies including a long read NGS, Oxford Nanopore. We identified that the GliB2 locus consist of two sub-loci 19 Mb apart from each other on the chromosome 6BS, which were covered with 10 and 3 MTP BAC clones. These MTP BACs were subjected to the sequencing using three NGS platforms, Roche 454 GS FLX+, Illumina short reads and Oxford Nanopore, and the conventional Sanger method, and we finally obtained the highly accurate genome sequences without gaps for the Gli-B2 locus. We also investigated the α/3-gliadin proteins in the mature grains. The α/3-gliadins were prepared from the mature grains of Chinese Spring (CS) and the nullisomic 6B-tetrasomic 6A line followed by Wang et al. (2017). Extracted gliadins were digested by chymotrypsin and then subjected to the MS analysis on LC-MS. The MS peptide data of the gliadin extracts were compared with the Gli-B2 genome sequences mentioned above and identified the population of α/3-gliadins actually expressed in the mature grains. The complete Gli-B2 genome sequences combined with the expressed proteins data (LC-MS) enable us to better understand the α/3-gliadins encoded in the Gli-B2 locus.

Acknowledgements: MS analysis was conducted in the NIMS Molecule & Material Synthesis Platform in "Nanotechnology Platform Project" operated by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.