Lingrang Kong

Expression analysis of defense-related genes in wheat in response to infection by Fusarium graminearum.

Fusarium head blight (FHB), caused by the fungi Fusarium graminearum and Fusarium culmorum, is a worldwide disease of wheat (Triticum aestivum L.). The Chinese cultivar Ning 7840 is one of a few wheat cultivars with resistance to FHB. GeneCalling, an open-architecture mRNA-profiling technology, was used to identify differentially expressed genes induced or suppressed in spikes of Ning 7840 after infection by F. graminearum. One hundred and twenty-five cDNA fragments representing transcripts differentially expressed in wheat spikes were identified. Based on BLASTN and BLASTX analyses, putative functions were assigned to some of the genes: 28 were assigned functions in primary metabolism and photosynthesis, 7 were involved in defense response, 14 were involved in gene expression and regulation, 24 encoded proteins associated with structure and protein synthesis, 42 lacked homology to sequences in the database, and 3 were similar to cloned multidrug resistance or disease resistance proteins. Of particular interest in this study were genes associated with resistance and defense against pathogen infection. Real-time quantitative reverse-transcription PCR indicated that of 51 genes tested, 19 showed 2-fold or greater induction or suppression in infected Ning 7840 in comparison with the water-treated control. The remaining 32 genes were not significantly induced or suppressed in infected Ning 7840 compared with the control. Subsequently, these 19 induced or suppressed genes were examined in the wheat line KS24-1, containing FHB resistance derived from Lophopyrum elongatum, and Len, an FHB-susceptible wheat cultivar. The temporal expression of some of these sequences encoding resistance proteins or defense-related proteins showed FHB (resistance specific) induction, suggesting that these genes play a role in protection against toxic compounds in plant-fungus interactions. On the basis of comprehensive expression profiling of various biotic or abiotic stress response genes revealed by quantitative PCR in this study and other supporting data, we hypothesized that the plant-pathogen interactions may be highly integrated into a network of diverse biosynthetic pathways.

A genetic analysis of segregation distortion revealed by molecular markers in Lophopyrum ponticum chromosome 7E

Segregation distortion can be defined as deviations from normal Mendelian segregation (Sandler et al. 1958). It is a common phenomenon found in most genetic mapping studies in many species (Faris et al. 1998). Lophopyrum ponticum (also syn. Agropyron elongatum (Host) Beau.) has been one of the most important perennial Triticeae germplasm sources for wheat improvement (Oliver et al. 2006). Chromosome 7E of L. ponticum is crucial because it is the carrier of a number of elite agronomic traits (Zhang et al. 2011). However, there is still no report about molecular markers showing segregation distortion region of L. ponticum chromosome 7E. Segregation distortion caused by preferential transmission may be affected by genetic background. Kong et al. (2008) reported that a segment of the Thinopyrum intermedium chromosome 7E present in the translocation line P98134 was preferentially transmitted through male gametes under different genetic background conditions. However, the transmission frequency of T. intermedium 7E segment present in another wheat T. intermedium translocation line, P961341, varied with different genetic backgrounds. Similarly, studies on the translocations involving T. ponticum chromosome 7el1 indicated that Thinopyrum chromosome 7el1 was transmitted normally in Thatcher and China Spring wheat backgrounds (McIntosh et al. 1976). However, high levels of distortion were observed when chromosome 7el1 was transferred in other genetic backgrounds (McIntosh et al. 1995). Both 7el1 and 7el2, are gametocidal chromosomes (KibirigeSebunya and Knott 1983; Prins et al. 1996). Gametocidal chromosomes cause abortion of gametes lacking the gametocidal chromosome, which results in partial sterility and exclusive transmission of these chromosomes (Endo 1990).

Molecular and Cytological Comparisons of Chromosomes 7el1, 7el2, 7Ee, and 7Ei Derived from Thinopyrum

Thinopyrum chromosomes 7el₁, 7el₂, 7E^e, and 7Eⁱ, homoeologous to group 7 chromosomes of common wheat (Triticum aestivum), were determined to have many useful agronomical traits for wheat improvement. To analyze the genetic relationships among the 4 Thinopyrum 7E chromosomes, the conserved orthologous set markers, genomic in situ hybridization (GISH), and meiotic chromosome pairing were used in this study. The unweighted pair-group method with arithmetical averages (UPGMA) analysis indicated that 7el₁, derived from T. ponticum, and 7Eⁱ, derived from T. intermedium, were the most closely related. 7el₂, derived from T. ponticum, was relatively distant from the 7el₁-7Eⁱ complex. While 7E^e, derived from T. elongatum, was more distantly related to 7el₁, 7el₂, and 7Eⁱ. This is the first report showing that 7el₁ and 7Eⁱ may be similar, which could be explained by the similar chromosome signal distribution revealed by GISH as well as UPGMA analysis revealed by both molecular markers and the highest frequency of meiotic pairing. The newly developed genome-specific molecular markers may be useful for marker-assisted selection of Lr19, Bdv3, and Fhblop.

Phylogenetic relationships of Thinopyrum and Triticum species revealed by SCoT and CDDP markers

To evaluate the phylogenetic relationships among Thinopyrum species and Triticum species, 7 accessions of Thinopyrum species (2 Th. bessarabicum, 1 Th. elongatum, 2 Th. intermedium and 2. Th. ponticum), 11 accessions of Triticum species (2 Aeglips tauschii, 1 T. monococcum, 1 T. turgidum, 2 T. timopheevii and 5 T. aestivum) together with Hordeum vulgare cv. Golden were analysed using 17 SCoT and 10 CDDP markers. The mean number of observed alleles was 8.5 and 6.6 among the species for SCoT and CDDP markers, respectively. Based on the genetic data produced by the SCoT and CDDP markers, cluster analysis among Thinopyrum species, Triticum species and Hordeum was performed to generate dendrograms. The genetic relationships among Thinopyrum species, Triticum species and H. vulgare revealed by the SCoT markers were in agreement with the results of the CDDP markers. On both dendrograms, these species formed three clusters. The results indicated that Thinopyrum species and Triticum species were the most closely related, whereas H. vulgare was relatively distant from both genera. In addition, seven markers, i.e. SCoT 9, SCoT 31, SCoT 34, WRKY-R1, WRKY-R2, MADS-1 and MADS-4 were developed for monitoring introgression of Thinopyrum chromosomes or chromosome segments into Triticum species.

Functional analysis of a wheat pleiotropic drug resistance gene involved in Fusarium head blight resistance

The pleiotropic drug resistance (PDR) sub-family of adenosine triphosphate (ATP)-binding cassette (ABC) transporter had been reported to participate in diverse biological processes of plant. In this study, we cloned three novel PDR genes in Fusarium head blight (FHB) resistant wheat cultivar Ning 7840, which were located on wheat chromosomes 6A, 6B and 6D. In phylogeny, these genes were members of cluster I together with AePDR7 and BdPDR7. Subcellular localization analysis showed that TaPDR7 was expressed on the plasmalemma. The quantitative real time PCR (RT-PCR) analysis showed that this gene and its probable orthologues in chromosomes 6B and 6D were both up-regulated sharply at 48 h after infected by Fusarium graminearum and trichothecene deoxynivalenol (DON) in spike. When knocking down the transcripts of all TaPDR7 members by barely stripe mosaic virus-induced gene silencing (BSMV-VIGS) system, it could promote the F. graminearum hyphae growth and made larger pathogen inoculation points in Ning 7840, which suggested that TaPDR7 might play an important role in response to F. graminearum. Although salicylic acid (SA), methyl jasmonate (MeJA) and abscisic acid (ABA) had been reported to possibly regulate wheat FHB resistance, here, we found that the three members of TaPDR7 were negatively regulated by these three hormones but positively regulated by indoleacetic acid (IAA).

Association analysis of grain traits with SSR markers between Aegilops tauschii and hexaploid wheat（Triticum aestivum L.）

Abstract Seven important grain traits, including grain length (GL), grain width (GW), grain perimeter (GP), grain area (GA), grain length/width ratio (GLW), roundness (GR), and thousand-grain weight (TGW), were analyzed using a set of 139 simple sequence repeat (SSR) markers in 130 hexaploid wheat varieties and 193 Aegilops tauschii accessions worldwide. In total, 1 612 alleles in Ae. tauschii and 1 360 alleles in hexaploid wheat ( Triticum aestivum L.) were detected throughout the D genome. 197 marker-trait associations in Ae. tauschii were identified with 58 different SSR loci in 3 environments, and the average phenotypic variation value ( R 2 ) ranged from 0.68 to 15.12%. In contrast, 208 marker-trait associations were identified in wheat with 66 different SSR markers in 4 environments and the average phenotypic R 2 ranged from 0.90 to 19.92%. Further analysis indicated that there are 6 common SSR loci present in both Ae. tauschii and hexaploid wheat, which are significantly associated with the 5 investigated grain traits (i.e., GA, GP, GR, GL, and TGW) and in total, 16 alleles derived from the 6 aforementioned SSR loci were shared by Ae. tauschii and hexaploid wheat. These preliminary data suggest the existence of common alleles may explain the evolutionary process and the selection between Ae. tauschii and hexaploid wheat. Furthermore, the genetic differentiation of grain shape and thousand-grain weight were observed in the evolutionary developmental process from Ae. tauschii to hexaploid wheat.

Cloning and characterization of novel fast ω-gliadin genes in Triticum monococcum

Fast ω-gliadin is the major allergen to wheat-dependent exercise-induced anaphylaxis (WDEIA). It is generally accepted that the proteins were expressed inGli-B1 loci located on the short arm of chromosome 1B in wheat. The knowledge of the orthologous genes in A and D genomes is still limited. In this study, the novel fastω-gliadin genes were cloned from genome A of diploid wheat Triticum monococcum. The deduced amino acid sequences of N-terminal domain showed that they referred to TRQ-type ω-gliadins. This type of ωgliadins have been identified in genome A by using mass spectroscopy and amino acid sequencing in previous studies (DuPont et al. 2004), but their coding sequences were lacking. Further analysis of IgE-binding epitopes suggested that the TRQ-typeω-gliadins have potential to trigger WDEIA. Wheat is one of the most important sources of food in the human diet. In the kernels of wheat, gliadins are the main components of the gluten. Traditionally, gliadins are further classified into α-gliadins, γ-gliadins and ω-gliadins on the basis of their mobility in electrophoresis at low pH (Wan et al. 2014). The ω-gliadins are minor components among wheat prolamins and they have not been studied well due to difficulties in cloning their genes caused by highly repetitive sequences (Anderson et al. 2009). In common wheat, most of the ω-gliadins were encoded by the Gli-A1, Gli-B1 and Gli-D1 on the short arm of chromosomes 1A, 1B and 1D, respectively (Pogna et al. 1990; Shewry et al. 1995; DuPont et al. 2000, 2004). Theω-gliadins have been categorized into ω1,ω2 andω5 types (DuPont et al. 2004). Theω5-gliadin, also referred as fastω-gliadin, are encoded byGli-B1 and the proteins can be distinguished by the amino acid sequences at the N-terminal domain, which are Ser-Arg-Leu (SRL) in ω5-gliadin, Ala-Arg-Gln (ARQ) or Lys-Glu-Leu (KEL)

Multi-functional roles of TaSSI2 involved in Fusarium head blight and powdery mildew resistance and drought tolerance

Abstract The mutation of the gene encoding a stearoyl-acyl carrier protein fatty acid desaturase (ssi2) has been proved to enhance pathogen resistance in several plants, while its potential to regulate biotic and abiotic stresses in wheat is still unclear. In this study, we cloned TaSSI2 gene in wheat and provided several evidences of its involvement in multiple biological functions. By using barley stripe mosaic virus (BSMV)-induced gene silencing (VIGS) in wheat, it was found that TaSSI2 negatively regulated both powdery mildew and Fusarium head blight (FHB) resistance, which was consistent with the phenotype observed in knock-out mutants of Kronos. The expression of TaSSI2 was down-regulated by in vitro treatments of methyl jasmonate (MeJA), but positively regulated by salicylic acid (SA) and abscisic acid (ABA), implying the cross-talk between different hormone signaling pathways involved in wheat to regulate biotic stresses is still to be elucidated. Furthermore, the up-regulated expression of PR4 and PR5 indicated that TaSSI2 probably regulated FHB resistance by depressing the SA signaling pathway in wheat. In addition, the over-expression of TaSSI2 increased the content of linolenic acid (18:3) and subsequently enhanced drought tolerance of transgenic Brachypodium. This phenomenon might be associated with its subcellular localization in the whole cytosol, partly overlapping with Golgi apparatus and the secreted vesicles. As a stearoyl-acyl carrier protein fatty acid desaturase, TaSSI2 was proposed to be involved in cell lipid metabolism and carried targets out of the cell from membrane or wax synthesis, resulting in enhanced drought tolerance in plant.

Modification of a novel x-type high-molecular-weight glutenin subunit gene from Aegilops markgrafii to improve dough strength of wheat flour

Abstract. High-molecular-weight glutenin subunits (HMW-GS) in bread wheat are major determinants of dough viscoelastic properties and the end-use quality of wheat flour. Cysteine residues, which form intermolecular disulphide bonds in HMW-GS, could improve the strength of gluten. To our knowledge, the number and position of cysteine residues in HMW-GS are conserved between wheat (Triticum aestivum) and Aegilops markgrafii. In the present study, we modified a gene (1Cx1.1) from Ae. markgrafii for an HMW-GS that possessed the typical structure and conserved number of cysteines. Site-directed mutagenesis was carried out in 1Cx1.1 to investigate how the position of cysteine residues in HMW-GS affects the mixing properties of dough. Six HMW-GS containing an extra cysteine residue were expressed in Escherichia coli, and the proteins were purified at sufficient scale for incorporation into flour to test dough quality. There were large differences in dough property among samples containing different modified subunits. Cysteine substituting in the N-terminal or repetitive-domain of HMW-GS could significantly improve dough quality. The results showed that the strategy was useful for providing genetic resources for gene engineering, and hence could be valuable for improving the processing quality of wheat.

Molecular characterization of the IgE-binding epitopes in the fast ω-gliadins of Triticeae in relation to wheat-dependent, exercise-induced anaphylaxis.

Fast ω-gliadins were minor components of wheat storage proteins but a major antigen triggering allergy to wheat. Sixty-six novel full-length fast ω-gliadin genes with unique characteristics were cloned and sequenced from wheat and its relative species using a PCR-based strategy. Their coding regions ranged from 177bp to 987bp in length and encoded 4.28kDa to 37.56kDa proteins. On the base of first three deduced amino acids at the N-terminal, these genes could be classified into the six subclasses of SRL-, TRQ-, GRL-, NRL-, SRP- and SRM-type ω-gliadin genes. Compared by multiple alignments, these genes were significantly different from each other, due to the insertion or deletion at the repetitive domain. An analysis of the IgE-binding epitopes of the 66 deduced fast ω-gliadins demonstrated that they contained 0-24 IgE-binding epitopes. The phylogenetic tree demonstrated that the fast ω-gliadins and slow ω-gliadins were separated into two groups and their divergence time was 21.64millionyears ago. Sequence data of the fast ω-gliadin genes assist in the study of the origins and evolutions of the different types of ω-gliadins while also providing a basis for the synthesis of monoclonal antibodies to detect wheat antigen content.