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Grains of 12 accessions of Triticum timopheevii (Zhuk.) Zhuk. ssp. timopheevii (AAGG, 2n = 4x = 28) and one bread wheat cultivar Chinese Spring (CS) and one durum wheat cultivar Langdon (LDN) grown across two years were analyzed for grain iron (Fe) and zinc (Zn) concentrations. All the 12 tested T. timopheevii ssp. timopheevii genotypes showed significantly higher concentration of grain Fe and Zn than CS and LDN. Aboundant genetic variability of both the Fe and Zn concentrations was observed among the T. timopheevii ssp. timopheevii accessions, averagely varied from 47.06 to 90.26 mg kg−1 and from 30.05 to 65.91 mg kg−1, respectively. Their grain Fe and Zn concentrations between years exhibited a significantly positive correlation with the correlation coefficients r = 0.895 and r = 0.891, respectively, indicating the highly genetic stability. Flag leaf possessed twice or three times higher concentrations for both Fe and Zn than grain, and a significantly high positive correlation appeared between the two organs with r = 0.648 for Fe and r = 0.957 for Zn concentrations, respectively, suggesting flag leaves might be indirectly used for evaluating grain Zn and Fe contents. Significant correlations occurred between grain Fe and Zn concentrations, and between grain Zn concentration and the two agronomic traits of plant height and number of spikelets per spike. Both the concentrations were not related to seed size or weight as well as NAM-G1 gene, implying the higher grain Fe and Zn concentrations of T. timopheevii ssp. timopheevii species are not ascribed to concentration effects of seed and the genetic control of NAM-G1 gene. There might be some other biological factors impacting the grain’s Zn and Fe concentrations. These results indicated T. timopheevii ssp. timopheevii species might be a promising genetic resource with high Fe and Zn concentrations for the biofortification of current wheat cultivars.

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In this study, the cDNA of homocysteine S-methyltransferase was isolated from Aegilops tauschii Coss., with the gene accordingly designated as AetHMT1. Similar to other methyltransferases, AetHMT1 contains a GGCCR consensus sequence for a possible zinc-binding motif near the C-terminal and a conserved cysteine residue upstream of the zinc-binding motif. Analysis of AetHMT1 uncovered no obvious chloroplast or mitochondrial targeting sequences. We functionally expressed AetHMT1 in Escherichia coli and confirmed its biological activity, as evidenced by a positive HMT enzyme activity of 164.516 ± 17.378 nmol min−1 mg−1 protein when catalyzing the transformation of L-homocysteine. Compared with the bacterium containing the empty vector, E. coli harboring the recombinant AetHMT1 plasmid showed much higher tolerance to selenate and selenite. AetHMT1 transcript amounts in different organs were increased by Na2SeO4 treatment, with roots accumulating higher amounts than stems, old leaves and new leaves. We have therefore successfully isolated HMT1 from Ae. tauschii and characterized the biochemical and physiological functions of the corresponding protein.

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Seed germination is a new beginning for the crop life cycle, which is closely related to seed sprouting and subsequent plant growth and development, and ultimately affects grain yield and quality. Salt stress is one of the most important abiotic stress factors that restrict crop production. Therefore, it is highly important to improve crop salt tolerance and sufficient utilization of saline-alkali land. In this study, we identified the phosphorylated proteins involved in salt stress response by combining SEM, 2-DE, Pro-Q Diamond staining and tandem mass spectrometry. The results showed that salt stress significantly inhibited seed germination and starch degradation. In total, 14 phosphorylated protein spots (11 unique proteins) in the embryo and 6 phosphorylated protein spots (4 unique proteins) in the endosperm were identified, which mainly involved in stress/defense, protein metabolism and energy metabolism. The phosphorylation of some proteins such as cold regulated proteins, 27K protein, EF-1β and superoxide dismutase could play important roles in salt stress tolerance.

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Cereal Research Communications
Authors: Z.L. Li, H.Y. Li, G. Chen, X.J. Liu, C.L. Kou, S.Z. Ning, Z.W. Yuan, M. Hao, D.C. Liu, and L.Q. Zhang

Seven Glu-A1 m allelic variants of the Glu-A1 m x genes in Triticum monococcum ssp. monococcum, designated as 1Ax2.1 a, 1Ax2.1 b, 1Ax2.1 c, 1Ax2.1 d, 1Ax2.1 e, 1Ax2.1 f, and 1Ax2.1 g were characterized. Their authenticity was confirmed by successful expression of the coding regions in E. coli, and except for the 1Ax2.1 a with the presence of internal stop codons at position of 313 aa, all correspond to the subunit in seeds. However, all the active six genes had a same DNA size although their encoding subunits showed different molecular weight. Our study indicated that amino acid residue substitutions rather than previously frequently reported insertions/deletions played an important role on the subunit evolution of these Glu-A1 m x alleles. Since variation in the Glu-A1x locus in common wheat is rare, these novel genes at the Glu-A1 m x can be used as candidate genes for further wheat quality improvement.

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Cereal Research Communications
Authors: Z. L. Li, D. D. Wu, H. Y. Li, G. Chen, W. G. Cao, S. Z. Ning, D. C. Liu, and L. Q. Zhang

Gliadin is a main component of gluten proteins that affect functional properties of bread making and contributes to the viscous nature of doughs. In this study, thirteen novel ω-gliadin genes were identified in several Triticum species, which encode the ARH-, ATDand ATN-type proteins. Two novel types of ω-gliadins: ATD- and ATN- have not yet been reported. The lengths of 13 sequences were ranged from 927 to 1269 bp and the deduced mature proteins were varied from 309 to 414 residues. All 13 genes were pseudogenes because of the presence of internal stop codons. The primary structure of these ω-gliadin genes included a signal peptide, a conserved N-terminal domain, a repetitive domain and a conserved C-terminus. In this paper, we first characterize ω-gliadin genes from T. timopheevi ssp. timopheevi and T. timopheevi ssp. araraticum. The ω-gliadin gene variation and the evolutionary relationship of ω-gliadin family genes were also discussed.

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As one of the world’s earliest domesticated crops, barley is a model species for the study of evolution and domestication. Domestication is an evolutionary process whereby a population adapts, through selection; to new environments created by human cultivation. We describe the genome-scanning of molecular diversity to assess the evolution of barley in the Tibetan Plateau. We used 667 Diversity Arrays Technology (DArT) markers to genotype 185 barley landraces and wild barley accessions from the Tibetan Plateau. Genetic diversity in wild barley was greater than in landraces at both genome and chromosome levels, except for chromosome 3H. Landraces and wild barley accessions were clearly differentiated genetically, but a limited degree of introgression was still evident. Significant differences in diversity between barley subspecies at the chromosome level were observed for genes known to be related to physiological and phenotypical traits, disease resistance, abiotic stress tolerance, malting quality and agronomic traits. Selection on the genome of six-rowed naked barley has shown clear multiple targets related to both its specific end-use and the extreme environment in Tibet. Our data provide a platform to identify the genes and genetic mechanisms that underlie phenotypic changes, and provide lists of candidate domestication genes for modified breeding strategies.

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Red coleoptile is an easily observed agronomic trait of wheat and has been extensively studied. However, the molecular mechanism of this trait has not yet been revealed. In this study, the MYB gene TaMYB-D1 was isolated from the wheat cultivar ‘Gy115’, which possesses red coleoptiles. This gene resided at the short arm of the homoelogous group 7 chromosomes. TaMYB-D1 was the only gene expressed in the coleoptiles of ‘Gy115’ and was not expressed in ‘Opata’ and ‘CS’, which have uncoloured coleoptiles. Phylogenetic analysis placed TaMYB-D1 very close to ZmC1 and other MYB proteins regulating anthocyanin biosynthesis. The encoded protein of TaMYB-D1 had an integrated DNA binding domain of 102 amino acids and a transcription domain with 42 amino acids, similar to the structure of ZmC1. Transient expression analysis in onion epidermal cells showed that TaMYB-D1 was located at the plant nucleus, which suggested its role as a transcription factor. The expression of TaMYB-D1 was accompanied with the expression of TaDFR and anthocyanin biosynthesis in the development of the coleoptile of ‘Gy115’. Transient expression analysis showed that only TaMYB-D1 induced a few ‘Opata’ coleoptile cells to synthesize anthocyanin in light, and the gene also induced a colour change to red in many cells with the help of ZmR. All of these results suggested TaMYB-D1 as the candidate gene for the red coleoptile trait of ‘Gy115’.

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Iron deficiency is the most common nutritional disorder, affecting over 30% of the world’s human population. The primary method used to alleviate this problem is nutrient biofortification of crops so as to improve the iron content and its availability in food sources. The over-expression of ferritin is an effective method to increase iron concentration in transgenic crops. For the research reported herein, sickle alfalfa (Medicago falcata L.) ferritin was transformed into wheat driven by the seed-storage protein glutelin GluB-1 gene promoter. The integration of ferritin into the wheat was assessed by PCR, RT-PCR and Western blotting. The concentration of certain minerals in the transgenic wheat grain was determined by inductively coupled plasma-atomic emission spectrometry, the results showed that grain Fe and Zn concentration of transgenic wheat increased by 73% and 44% compared to nontransformed wheat, respectively. However, grain Cu and Cd concentration of transgenic wheat grain decreased significantly in comparison with non-transformed wheat. The results suggest that the over-expression of sickle alfalfa ferritin, controlled by the seed-storage protein glutelin GluB-1 gene promoter, increases the grain Fe and Zn concentration, but also affects the homeostasis of other minerals in transgenic wheat grain.

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Cereal Research Communications
Authors: L. Wei, S.G. Bai, X.J. Hou, J.M. Li, B. Zhang, W.J. Chen, D.C. Liu, B.L. Liu, and H.G. Zhang

Among 20 awnless Tibetan wheat cultivars analyzed by SDS-PAGE, the migration rate of an HMW-GS in XM001584 and XM001593, named 1BX23*. was shown to be slightly faster than 1Bx6. and slower than Bx7. Its nucleotide sequence was isolated based on homology clones. In a phylogenetic tree of 1Bx genes, 1Bx23* was apparently clustered with 1Bx23. Compared with 1Bx23. eight single nucleotide replacements caused four single amino acid replacements in 1Bx23*. The deletion of “G” at base pair 1463 and insertion of “A” at 1509 bps induced a 42-nucleotide frame shift. “GQRQQAGQWQRPGQ” was replaced by “DKGNRQDNGNDRDK”. The new segment cannot be found in other HMW-GSs, and it is very similar to a segment found in collagen. Moreover, an 18-nucleotide deletion made 1Bx23* six amino acids shorter than 1Bx23. The cultivar XM001593 had 28 chromosomes, which signifies that it was tetraploid wheat, and that the new HMW-GS 1Bx23* cannot be used directly for breeding in common wheat.

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Cereal Research Communications
Authors: N. Niu, Y.X. Bai, S. Liu, Q.D. Zhu, Y.L. Song, S.C. Ma, L.J. Ma, X.L. Wang, G.S. Zhang, and J.W. Wang

Studies of the pollen abortion mechanism in thermo-sensitive male sterile lines may provide a strong foundation for breeding hybrid wheat and establishing a theoretical basis for marker-assisted selection. To investigate the cause of pollen abortion in Bainong thermo – sensitive male sterile (BNS) lines, we analyzed the properties of pollen grains, changes in the tapetum and microspores in different anther developmental stages, and the distribution and deposition of nutrient substances in microspores. We found that tapetum degraded in the early uninucleate stage in sterile BNS (S-BNS), which was earlier than that of fertile BNS (F-BNS) tapetum. Large amounts of insoluble polysaccharides, lipids, and proteins were deposited until the trinucleate pollen stage in the nutritive cells in F-BNS. At the binucleate stage, the vacuoles disappeared and pollen inclusion increased gradually. At the trinucleate stage, these nutrients would help pollen grains mature and participate in fertilization normally. Therefore, early degradation of the tapetum, which inhibits normal microspore development, and the limited content of nutrient substances in pollen may be the main factors responsible for male sterility in BNS lines.

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