Volume 26, Issue 6, Pages 782-787 (March 2016) A Causal Gene for Seed Dormancy on Wheat Chromosome 4A Encodes a MAP Kinase Kinase  Atsushi Torada, Michiya Koike, Taiichi Ogawa, Yu Takenouchi, Kazuki Tadamura, Jianzhong Wu, Takashi Matsumoto, Kanako Kawaura, Yasunari Ogihara  Current Biology  Volume 26, Issue 6, Pages 782-787 (March 2016) DOI: 10.1016/j.cub.2016.01.063 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Mapping of Phs1 Region (A) Genetic map of Phs1 region on wheat chromosome arm 4AL developed from 3,535 BC3F2 and BC4F2 plants. (B) Wheat BAC contigs harboring portions of Phs1 region. (C) Regions of rice chromosomes 3 and 6 with co-linearity to wheat Phs1 region. Diamonds in (A) and (B) show mapped markers derived from BAC sequences and wheat expressed sequence tags (ESTs) corresponding to the rice gene on chromosome 3. M1–M11 on the diamonds correspond to Table S2. Squares in (B) show genes annotated in the sequences from the two BACs (WCS1133O06 and WCS0255A18). G1–G4 on the squares correspond to Table S1. Circles in (C) show genes annotated in rice. (D and E) Diagrams of genomic DNA (D) and cDNA (E) showing ORFs (red bars), introns (black lines), untranslated regions (UTRs) (white bars), and the position of the candidate functional SNP within the MKK3 gene on 4AL. (F) The putative structure of deduced protein of TaMKK3-A gene. The predicted TaMKK3-A protein contains two conserved domains, cd06623 (catalytic domain of plant dual-specificity mitogen-activated protein kinase kinases and similar proteins) and cd00780 (nuclear transport factor 2 [NTF2] domain). In putative catalytic domain, two signatures, “catalytic loop” and “phosphorylation site,” are predicted. Two threonine residues that are putative phosphorylation targets are colored with red (T) letters. The blue pin between two signatures is the position of the amino acid substitution between Haruyokoi and Leader or Chinese Spring. See also Table S1 and Figures S1 and S2. Current Biology 2016 26, 782-787DOI: (10.1016/j.cub.2016.01.063) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Transgenic Complementation Tests for TaMKK3-A, a Candidate Gene for Phs1 A genomic fragment containing the Haruyokoi (nondormant) allele was transformed into Leader (dormant) by particle bombardment. Lines MEL10, MEL29, and MEL31 were selected by long-PCR experiments to screen for the intact transgenic Haruyokoi allele at the T0 generation. (A) Expression of transgenic Haruyokoi allele in embryos from T2 seeds. RT-PCR was conducted using TaMKK3-A-specific primers (F-Acl-HW1/MEK.Exo-In.R2-T), followed by restriction enzyme (Hpy166 II) digestion to distinguish between the transgenic (Haruyokoi) allele and the endogenous (Leader) allele. Expression of an actin (primers actin 361-L and actin 361-R) gene was used as the internal control. (B) Germination percentages determined using homozygous T3 seeds of nine lines for the transgenic Haruyokoi allele and 13 null lines; both types were derived from segregating progenies of MEL10, MEL29, and MEL31 T0 plants. Plants were grown in a closed-system greenhouse, harvested at physiological maturity, and dried at 25°C for 7 days. Germination tests were carried out at 20°C for 7 days. (C) Germination percentages determined using homozygous T5 seeds for the transgenic Haruyokoi allele, derived from MEL29 and MEL31 T0 plants. Plants were grown in a closed-system greenhouse and harvested at 30, 40, and 50 days after anthesis (DAA). Germination tests were carried out at 20°C for 7 days as soon as spikes were harvested. Error bars show SD of three replications. (D) Relative expression in embryos from homozygous T5 seeds for the transgenic Haruyokoi allele, derived from MEL29 and MEL31 T0 plants. Plants were grown in a closed-system greenhouse and harvested at 30, 40, and 50 DAA. Real-time PCR was conducted using TaMKK3-A-specific primers (MEKA RT F2/MEKA RT R2-2). Expression of an actin (primers QRT-Actin-F/QRT-Actin-R) gene was used as the internal control. Error bars show SD of three replications. See also Figures S3 and S4 and Table S4. Current Biology 2016 26, 782-787DOI: (10.1016/j.cub.2016.01.063) Copyright © 2016 Elsevier Ltd Terms and Conditions