Mapping and characterization of major QTL for spike traits in common wheat.

The spike traits of wheat can directly affect yield. F2 and F2:3 lines derived from the cross of the multi-spikelet female 10-A and the uni-spikelet male BE89 were used to detect QTLs for spike length (SL), total spikelet number per spike (TSS), kernel number per spike (KNS) and thousand-kernel weight (TKW) in four different environments. A total of 1098 SNP and 5 SSR were used to construct genetic map of 2398.1 cM with the average distance of 2.2 cM between markers. A total of 11 QTLs were identified for spike traits, including three QTLs for SL, five QTLs for TSS, two QTLs for KNS and one QTL for TKW. The QTLs mapped to chromosomes 2D, 4A, 6A, 7A and 7B explained 8.2-37.8% of the phenotypic variation in single environment. The major QTL confidence interval with distance of 0.5 cM was located on chromosome 4A and detected in multiple environments, which can explain more than 30% of the phenotypic variation for SL, TSS and KNS. Combining IWGSC RefSeq v1.0 and RNA-seq data for 10-A and BE89, we identified 16 genes expressed on spike or grain in four QTL regions. These findings provide insights into improving wheat yield through increasing spikletes in wheat, particularly through the use of the multi-spikelet female 10-A for breeding.

Fusarium graminearum FgCWM1 Encodes a Cell Wall Mannoprotein Conferring Sensitivity to Salicylic Acid and Virulence to Wheat.

Fusarium graminearum causes Fusarium head blight (FHB), a devastating disease of wheat. Salicylic acid (SA) is involved in the resistance of wheat to F. graminearum. Cell wall mannoprotein (CWM) is known to trigger defense responses in plants, but its role in the pathogenicity of F. graminearum remains unclear. Here, we characterized FgCWM1 (FG05_11315), encoding a CWM in F. graminearum. FgCWM1 was highly expressed in wheat spikes by 24 h after initial inoculation and was upregulated by SA. Disruption of FgCWM1 (ΔFgCWM1) reduced mannose and protein accumulation in the fungal cell wall, especially under SA treatment, and resulted in defective fungal cell walls, leading to increased fungal sensitivity to SA. The positive role of FgCWM1 in mannose and protein accumulation was confirmed by its expression in Saccharomyces cerevisiae. Compared with wild type (WT), ΔFgCWM1 exhibited reduced pathogenicity toward wheat, but it produced the same amount of deoxynivalenol both in culture and in spikes. Complementation of ΔFgCWM1 with FgCWM1 restored the WT phenotype. Localization analyses revealed that FgCWM1 was distributed on the cell wall, consistent with its structural role. Thus, FgCWM1 encodes a CWM protein that plays an important role in the cell wall integrity and pathogenicity of F. graminearum.

Re-acquisition of the brittle rachis trait via a transposon insertion in domestication gene Q during wheat de-domestication.

De-domestication is a unique evolutionary process during which crops re-acquire wild-like traits to survive and persist in agricultural fields without the need for human cultivation. The re-acquisition of seed dispersal mechanisms is crucial for crop de-domestication. Common wheat is an important cereal crop worldwide. Tibetan semi-wild wheat is a potential de-domesticated common wheat subspecies. However, the crucial genes responsible for its brittle rachis trait have not been identified. Genetic mapping, functional analyses and phylogenetic analyses were completed to identify the gene associated with Qbr.sau-5A, which is a major locus for the brittle rachis trait of Tibetan semi-wild wheat. The cloned Qbr.sau-5A gene is a new Q allele (Qt ) with a 161-bp transposon insertion in exon 5. Although Qt is expressed normally, its encoded peptide lacks some key features of the APETALA2 family. The abnormal functions of Qt in developing wheat spikes result in brittle rachises. Phylogenetic and genotyping analyses confirmed that Qt originated from Q in common wheat and is naturally distributed only in Tibetan semi-wild wheat populations. The identification of Qt provides new evidence regarding the origin of Tibetan semi-wild wheat, and new insights into the re-acquisition of wild traits during crop de-domestication.

Transcriptional reference map of hormone responses in wheat spikes.

BACKGROUND: Phytohormones are key regulators of plant growth, development, and signalling networks involved in responses to diverse biotic and abiotic stresses. Transcriptional reference maps of hormone responses have been reported for several model plant species such as Arabidopsis thaliana, Oryza sativa, and Brachypodium distachyon. However, because of species differences and the complexity of the wheat genome, these transcriptome data are not appropriate reference material for wheat studies. RESULTS: We comprehensively analysed the transcriptomic responses in wheat spikes to seven phytohormones, including indole acetic acid (IAA), gibberellic acid (GA), abscisic acid (ABA), ethylene (ET), cytokinin (CK), salicylic acid (SA), and methyl jasmonic acid (MeJA). A total of 3386 genes were differentially expressed at 24 h after the hormone treatments. Furthermore, 22.7% of these genes exhibited overlapping transcriptional responses for at least two hormones, implying there is crosstalk among phytohormones. We subsequently identified genes with expression levels that were significantly and differentially induced by a specific phytohormone (i.e., hormone-specific responses). The data for these hormone-responsive genes were then compared with the transcriptome data for wheat spikes exposed to biotic (Fusarium head blight) and abiotic (water deficit) stresses. CONCLUSION: Our data were used to develop a transcriptional reference map of hormone responses in wheat spikes.

Functional Analysis of FgNahG Clarifies the Contribution of Salicylic Acid to Wheat (Triticum aestivum) Resistance against Fusarium Head Blight.

Salicylic acid (SA) is a key defense hormone associated with wheat resistance against Fusarium head blight, which is a severe disease mainly caused by Fusarium graminearum. Although F. graminearum can metabolize SA, it remains unclear how this metabolic activity affects the wheat⁻F. graminearum interaction. In this study, we identified a salicylate hydroxylase gene (FG05_08116; FgNahG) in F. graminearum. This gene encodes a protein that catalyzes the conversion of SA to catechol. Additionally, FgNahG was widely distributed within hyphae. Disrupting the FgNahG gene (ΔFgNahG) led to enhanced sensitivity to SA, increased accumulation of SA in wheat spikes during the early infection stage and inhibited development of head blight symptoms. However, FgNahG did not affect mycotoxin production. Re-introducing a functional FgNahG gene into the ΔFgNahG mutant recovered the wild-type phenotype. Moreover, the expression of FgNahG in transgenic Arabidopsis thaliana decreased the SA concentration and the resistance of leaves to F. graminearum. These results indicate that the endogenous SA in wheat influences the resistance against F. graminearum. Furthermore, the capacity to metabolize SA is an important factor affecting the ability of F. graminearum to infect wheat plants.

Mechanisms of wheat (Triticum aestivum) grain storage proteins in response to nitrogen application and its impacts on processing quality.

Basis for the effects of nitrogen (N) on wheat grain storage proteins (GSPs) and on the establishment of processing quality are far from clear. The response of GSPs and processing quality parameters to four N levels of four common wheat cultivars were investigated at two sites over two growing seasons. Except gluten index (GI), processing quality parameters as well as GSPs quantities were remarkably improved by increasing N level. N level explained 4.2~59.2% and 10.4~80.0% variability in GSPs fractions and processing quality parameters, respectively. The amount of N remobilized from vegetative organs except spike was significantly increased when enhancing N application. GSPs fractions and processing quality parameters except GI were only highly and positively correlated with the amount of N remobilized from stem with sheath. N reassimilation in grain was remarkably strengthened by the elevated activity and expression level of glutamine synthetase. Transcriptome analysis showed the molecular mechanism of seeds in response to N levels during 10~35 days post anthesis. Collectively, we provided comprehensive understanding of N-responding mechanisms with respect to wheat processing quality from N source to GSPs biosynthesis at the agronomic, physiological and molecular levels, and screened candidate genes for quality breeding.

Fusarium graminearum ATP-Binding Cassette Transporter Gene FgABCC9 Is Required for Its Transportation of Salicylic Acid, Fungicide Resistance, Mycelial Growth and Pathogenicity towards Wheat.

ATP-binding cassette (ABC) transporters hydrolyze ATP to transport a wide range of substrates. Fusarium graminearum is a major causal agent of Fusarium head blight, which is a severe disease in wheat worldwide. FgABCC9 (FG05_07325) encodes an ABC-C (ABC transporter family C) transporter in F. graminearum, which was highly expressed during the infection in wheat and was up-regulated by the plant defense hormone salicylic acid (SA) and the fungicide tebuconazole. The predicted tertiary structure of the FgABCC9 protein was consistent with the schematic of the ABC exporter. Deletion of FgABCC9 resulted in decreased mycelial growth, increased sensitivity to SA and tebuconazole, reduced accumulation of deoxynivalenol (DON), and less pathogenicity towards wheat. Re-introduction of a functional FgABCC9 gene into ΔFgABCC9 recovered the phenotypes of the wild type strain. Transgenic expression of FgABCC9 in Arabidopsis thaliana increased the accumulation of SA in its leaves without activating SA signaling, which suggests that FgABCC9 functions as an SA exporter. Taken together, FgABCC9 encodes an ABC exporter, which is critical for fungal exportation of SA, response to tebuconazole, mycelial growth, and pathogenicity towards wheat.

Alternative splicing results in a lack of starch synthase IIa-D in Chinese wheat landrace.

We evaluated the SGP-1 protein composition of 368 Chinese wheat landraces using SDS-PAGE. The SGP-D1 null type was identified in three accessions (Xiaoqingmang, Pushanbamai, and P119). An 18-bp deletion and 9-bp variation were found at the junction region of the 7th intron and 8th exon, leading to deletion of the intron-exon junction recognition site AG when aligned the 8261-bp DNA sequence of TaSSIIa-D in Pushanbamai with that of Chinese Spring. Four cDNA types with mis-spliced isoforms were subsequently detected through amplification of TaSSIIa-D cDNAs. Among these, nine type II cDNAs with a 16-bp deletion in the 8th exon were detected, indicating that the major transcriptional pattern of TaSSIIa in Pushanbamai is type II. In the type IV cDNA, a 97-bp sequence remains undeleted in the end of the 5th exon. The amylose content in Pushanbamai was significantly higher than that in all control lines under field conditions, which suggested that deletion of SGP-D1 has an efficient impact on amylose content. As the TaSSIIa gene plays an important role in regulating the content of amylose, it is anticipated that these natural variants of TaSSIIa-D will provide useful resources for quality improvement in wheat.

An Overexpressed Q Allele Leads to Increased Spike Density and Improved Processing Quality in Common Wheat (Triticum aestivum).

Spike density and processing quality are important traits in modern wheat production and are controlled by multiple gene loci. The associated genes have been intensively studied and new discoveries have been constantly reported during the past few decades. However, no gene playing a significant role in the development of these two traits has been identified. In the current study, a common wheat mutant with extremely compact spikes and good processing quality was isolated and characterized. A new allele (Qc1 ) of the Q gene (an important domestication gene) responsible for the mutant phenotype was cloned, and the molecular mechanism for the mutant phenotype was studied. Results revealed that Qc1 originated from a point mutation that interferes with the miRNA172-directed cleavage of Q transcripts, leading to its overexpression. It also reduces the longitudinal cell size of rachises, resulting in an increased spike density. Furthermore, Qc1 increases the number of vascular bundles, which suggests a higher efficiency in the transportation of assimilates in the spikes of the mutant than that of wild type. This accounts for the improved processing quality. The effects of Qc1 on spike density and wheat processing quality were confirmed by analyzing nine common wheat mutants possessing four different Qc alleles. These results deepen our understanding of the key roles of Q gene, and provide new insights for the potential application of Qc alleles in wheat quality breeding.

Inheritance analysis and mapping of quantitative trait loci (QTL) controlling individual anthocyanin compounds in purple barley (Hordeum vulgare L.) grains.

Anthocyanin-rich barley can have great potential in promoting human health and in developing nutraceuticals and functional foods. As different anthocyanin compounds have different antioxidant activities, breeding cultivars with pre-designed anthocyanin compositions could be highly desirable. Working toward this possibility, we assessed and reported for the first time the genetic control of individual anthocyanin compounds in barley. Of the ten anthocyanins assessed, two, peonidin-3-glucoside (P3G) and cyanidin-3-glucoside (C3G), were major components in the purple pericarp barley genotype RUSSIA68. Quantitative trait locus (QTL) mapping showed that both anthocyanin compounds were the interactive products of two loci, one located on chromosome arm 2HL and the other on 7HS. However, the two different anthocyanin components seem to be controlled by different interactions between the two loci. The effects of the 7HS locus on P3G and C3G were difficult to detect without removing the effect of the 2HL locus. At least one copy of the 2HL alleles from the purple pericarp parent was required for the synthesis of P3G. This does not seem to be the case for the production of C3G which was produced in each of all the different allele combinations between the two loci. Typical maternal effect was also observed in the inheritance of purple pericarp grains in barley. The varied values of different compounds, coupled with their different genetic controls, highlight the need for targeting individual anthocyanins in crop breeding and food processing.

Identification of quantitative trait loci for seedling root traits from Tibetan semi-wild wheat (Triticum aestivum subsp. tibetanum).

As a primitive hexaploid wheat resource distributed only in Tibet, Tibetan semi-wild wheat (Triticum aestivum subsp. tibetanum Shao) possesses unique characteristics that could be exploited in wheat breeding programs. Its good root system could offer a stable platform for above-ground components. To detect possible excellent locus for root traits from Tibetan semi-wild wheat, we identified QTLs for root traits using a recombinant inbred line population derived from a cross between Tibetan semi-wild wheat Q1028 and Zhengmai 9023. A total of 15 QTLs on eight chromosomes were detected, including four major QTLs, QMrl.sau-7B, QTrl.sau-4B, QAd.sau-7A, and QSa.sau-4B. The phenotypic variation explained by each of these QTLs ranges from 5.67% to 16.68%. Positive alleles of six QTLs were derived from Q1028. Several novel QTLs for root traits were identified. In addition, significant correlations were detected amongst root traits and agronomic traits. Taken together, these results suggest that Tibetan semi-wild wheat and the newly identified novel QTLs could be useful in future breeding programs.

Linoleic acid isomerase gene FgLAI12 affects sensitivity to salicylic acid, mycelial growth and virulence of Fusarium graminearum.

Fusarium graminearum is the major causal agent of fusarium head blight in wheat, a serious disease worldwide. Linoleic acid isomerase (LAI) catalyses the transformation of linoleic acid (LA) to conjugated linoleic acid (CLA), which is beneficial for human health. We characterised a cis-12 LAI gene of F. graminearum (FGSG_02668; FgLAI12), which was downregulated by salicylic acid (SA), a plant defence hormone. Disruption of FgLAI12 in F. graminearum resulted in decreased accumulation of cis-9,trans-11 CLA, enhanced sensitivity to SA, and increased accumulation of LA and SA in wheat spikes during infection. In addition, mycelial growth, accumulation of deoxynivalenol, and pathogenicity in wheat spikes were reduced. Re-introduction of a functional FgLAI12 gene into ΔFgLAI12 recovered the wild-type phenotype. Fluorescent microscopic analysis showed that FgLAI12 protein was usually expressed in the septa zone of conidia and the vacuole of hyphae, but was expressed in the cell membrane of hyphae in response to exogenous LA, which may be an element of LA metabolism during infection by F. graminearum. The cis-12 LAI enzyme encoded by FgLAI12 is critical for fungal response to SA, mycelial growth and virulence in wheat. The gene FgLAI12 is potentially valuable for biotechnological synthesis of cis-9,trans-11 CLA.

Genome-Wide Association Study for Pre-harvest Sprouting Resistance in a Large Germplasm Collection of Chinese Wheat Landraces.

Pre-harvest sprouting (PHS) is mainly caused by the breaking of seed dormancy in high rainfall regions, which leads to huge economic losses in wheat. In this study, we evaluated 717 Chinese wheat landraces for PHS resistance and carried out genome-wide association studies (GWAS) using to 9,740 DArT-seq and 178,803 SNP markers. Landraces were grown across six environments in China and germination testing of harvest-ripe grain was used to calculate the germination rate (GR) for each accession at each site. GR was highly correlated across all environments. A large number of landraces (194) displayed high levels of PHS resistance (i.e., mean GR < 0.20), which included nine white-grained accessions. Overall, white-grained accessions displayed a significantly higher mean GR (42.7-79.6%) compared to red-grained accessions (19.1-56.0%) across the six environments. Landraces from mesic growing zones in southern China showed higher levels of PHS resistance than those sourced from xeric areas in northern and north-western China. Three main quantitative trait loci (QTL) were detected by GWAS: one on 5D that appeared to be novel and two co-located with the grain color transcription factor Tamyb10 on 3A and 3D. An additional 32 grain color related QTL (GCR-QTL) were detected when the set of red-grained landraces were analyzed separately. GCR-QTL occurred at high frequencies in the red-grained accessions and a strong correlation was observed between the number of GCR-QTL and GR (R2 = 0.62). These additional factors could be critical for maintaining high levels of PHS resistance and represent targets for introgression into white-grained wheat cultivars. Further, investigation of the origin of haplotypes associated with the three main QTL revealed that favorable haplotypes for PHS resistance were more common in accessions from higher rainfall zones in China. Thus, a combination of natural and artificial selection likely resulted in landraces incorporating PHS resistance in China.

Transposon insertion resulted in the silencing of Wx-B1n in Chinese wheat landraces.

KEY MESSAGE: A novel Wx-B1 allele was characterized; a transposon insertion resulted in the loss of its function, which is different from the previously reported gene silencing mechanisms at the Wx-B1 locus. The waxy protein composition of 53 Chinese wheat landraces was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis; of these, 10 did not show the expression of Wx-A1 (four accession) or Wx-B1 (six accessions) protein. The results of molecular marker detection revealed that the Wx-B1 allele (Wx-B1n) showed normal expression, inconsistent with the findings of SDS-PAGE for the Xiaobaipi accession. Further cloning of the 9160-bp region covering the Wx-B1 coding region and 3'-downstream region revealed that a 2178-bp transposon fragment had been inserted at 2462 bp within the tenth exon of Wx-B1n ORF, leading to the absence of Wx-B1 protein. Sequence analysis indicated that the insertion possessed the structural features of invert repeat and target repeat elements, we deduced that it was a transposon. Further PCR analysis revealed that this fragment had moved, but not copied itself, from 3B chromosome to the current location in Wx-B1n. Therefore, the reason for the inactivation of Wx-B1n was considerably different from those for the inactivation of Wx-B1b, Wx-B1k, and Wx-B1m; to our knowledge, this kind of structural mutation has never been reported in Wx-B1 alleles. This novel allele is interesting, because it was not associated with the deletion of other quality-related genes included in the 67 kb region lost with the common null allele Wx-B1b. The null Wx-B1n might be useful for investigating gene inactivation and expression as well as for enriching the genetic resource pool for the modification of the amylose/amylopectin ratio, thereby improving wheat quality.

Identification and characterization of genes on a single subgenome in the hexaploid wheat (Triticum aestivum L.) genotype 'Chinese Spring'.

Gene loss during the formation of hexaploid bread wheat has been repeatedly reported. However, our knowledge on genome-wide analysis of the genes present on a single subgenome (SSG) in bread wheat is still limited. In this study, by analysing the 'Chinese Spring' chromosome arm shotgun sequences together with high-confidence gene models, we detected 433 genes on a SSG. Greater gene loss was observed in A and D subgenomes compared with B subgenome. More than 79% of the orthologs for these SSG genes were detected in diploid and tetraploid relatives of hexaploid wheat. Unexpectedly, no bias in expression breadth or in the distribution patterns of GO (gene ontology) terms for these genes was detected among the high-confidence genes. Further, network and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses indicated that most of these genes were not functionally related to each other. Interestingly, 30.7% of these SSG genes were most highly expressed in root, showing biased distribution given the distribution of the whole high-confidence genes. Collectively, these results facilitate our understanding of the loss of the genes that were retained in a SSG during the formation of hexaploid wheat.

Identification of QTLs associated with tissue culture response of mature wheat embryos.

Mature embryo is an excellent explant for tissue culture as it is convenient to be obtained without limitation of growing seasons and development stages. However, regeneration ability of the calli from wheat mature embryos is limited, thus hindering its application. To identify genes associated with the tissue culture response (TCR) of wheat, QTLs for callus induction from mature embryos and callus regeneration were detected using a recombinant inbred lines (RILs) population derived from the cross between a synthetic hexaploid wheat genotype, SHW-L1 and a commercial cultivar Chuanmai 32. Three QTLs for callus rate were identified and they were located on chromosomes 1D, 5A, and 6D, respectively, with explained phenotypic variation ranging from 10.16 to 11.82 %. One QTL for differentiation rate was detected only with 10.96 % of the phenotypic variation explained. Two QTLs for emergence rate were identified and they were located on 3B and 4A, respectively, with 9.88 and 10.30 % of phenotypic variation. The results presented in this study with those reported previously indicated that group 1, 3, and 5 chromosomes are likely to play important roles in TCR of wheat.

A super twin T-DNA vector that allows independent gene expression during Agrobacterium-mediated transformation.

In this study, we designed and constructed a super twin T-DNA vector (pTRIDT313-g) containing two independent T-DNA cassettes-one for the selection gene Hyg and the other for the target gene Gus-to produce marker-free transgenic lines. The resulting vector was transformed into tobacco, and polymerase chain reaction (PCR) analysis showed four types of gene combinations in the T1 and T2 generations: Gus only, Hyg only, Gus+Hyg, and untransformed lines. The intermediate region from the T-DNA of the right border of Hyg to the left border of Gus in the Hyg and Gus lines was not amplified. Genome walking confirmed that the Hyg and Gus T-DNA cassettes were independently inserted in different regions of the tobacco genome. Thus, the two T-DNA cassettes were integrated randomly as independent loci into the tobacco genome. The results of reverse transcription-PCR indicated that Hyg could normally be expressed in the roots, stems, and leaves of transgenic lines, and the resistance test showed that all Hyg transgenic lines could grow in the presence of 50mg/L hygromycin. All Gus transgenic lines showed obvious blue coloration in enzyme activity tests, indicating that the Gus gene could be normally expressed in all the lines. Therefore, the super twin T-DNA vector (pTRIDT313-g) exhibits independent integration, heredity, and normal gene function from two T-DNA cassettes. This vector could be a useful and valuable tool in the production of marker-free transgenic lines.

Characterization and expression analyses of the H⁺-pyrophosphatase gene in rye.

The H⁺-pyrophosphatase (H⁺-PPase) gene plays an important role in maintaining intracellular proton gradients. Here, we characterized the full-length complementary DNA (cDNA) and DNA of the H⁺-PPase gene ScHP1 in rye (Secale cereale L. 'Qinling'). We determined the subcellular localization of this gene and predicted the corresponding protein structure. We analysed the evolutionary relationship between ScHP1 and H⁺-PPase genes in other species, and did real-time quantitative polymerase chain reaction to explore the expression patterns of ScHP1 in rye plants subjected to N, P and K deprivation and to cold, high-salt and drought stresses. ScHP1 cDNA included a 2289 bp open reading frame (ORF) encoding 762 amino acid residues with 14 transmembrane domains. The genomic ScHP1 DNA was 4354 bp and contained eight exons and seven introns. ScHP1 was highly homologous with other members of the H⁺-PPase gene family. When the full-length ORF was inserted into the expression vector pA7-YFP, the fluorescent microscopy revealed that ScHP1-YFP fusion protein was located in the plasma membrane. Rye plants that were subjected to N deprivation, cold and high-salt stresses, ScHP1 expression was higher in the leaves than roots. Conversely, plants subjected to P and K deprivation and drought stress, ScHP1 expression was higher in the roots than leaves. Under all the investigated stress conditions, expression of ScHP1 was lower in the stem than in the leaves and roots. Our results imply that ScHP1 functions under abiotic stress response.

Structure and expression analysis of genes encoding ADP-glucose pyrophosphorylase large subunit in wheat and its relatives.

ADP-glucose pyrophosphorylase (AGP), which consists of two large subunits (AGP-L) and two small subunits (AGP-S), controls the rate-limiting step in the starch biosynthetic pathway. In this study, a full-length open reading frame (ORF) of AGP-L gene (named as Agp2) in wheat and a series of Agp2 gene sequences in wheat relatives were isolated. The coding region of Agp2 contained 15 exons and 14 introns including a full-length ORF of 1566 nucleotides, and the deduced protein contained 522 amino acids (57.8 kDa). Generally, the phylogenetic tree of Agp2 indicated that sequences from A- and D-genome donor species were most similar to each other and sequences from B-genome donor species contained more variation. Starch accumulation and Agp2 expression in wheat grains reached their peak at 21 and 15 days post anthesis (DPA), respectively.