Differences between diploid donors are the main contributing factor for subgenome asymmetry measured in either gene ratio or relative diversity in allopolyploids.

Subgenome asymmetry (SA) has routinely been attributed to different responses between the subgenomes of a polyploid to various stimuli during evolution. Here, we compared subgenome differences in gene ratio and relative diversity between artificial and natural genotypes of several allopolyploid species. Surprisingly, consistent differences were not detected between these two types of polyploid genotypes, although they differ in times exposed to evolutionary selection. The estimated ratio of shared genes between a subgenome and its diploid donor was invariably higher for the artificial allopolyploid genotypes than those for the natural genotypes, which is expected as it is now well-known that many genes in a species are not shared among all individuals. As the exact diploid parent for a given subgenome is unknown, the estimated ratios of shared genes for the natural genotypes would also include difference among individual genotypes of the diploid donor species. Further, we detected the presence of SA in genotypes before the completion of the polyploidization events as well as in those which were not formed via polyploidization. These results indicate that SA may, to a large degree, reflect differences between its diploid donors or that changes occurred during polyploid evolution are defined by their donor genomes.

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.

A Novel QTL Conferring Fusarium Crown Rot Resistance Located on Chromosome Arm 6HL in Barley.

Fusarium crown rot (FCR), caused primarily by Fusarium pseudograminearum, is a devastating disease for cereal production in semi-arid regions worldwide. To identify and characterize loci conferring FCR resistance, we assessed a landrace AWCS799 which is among the best lines identified from a systematic screening of more than 1,000 genotypes. Genetic control of its resistance was investigated by generating and analyzing two populations of recombinant inbred lines with AWCS799 as the common parent. One of the populations was used for QTL detection and the other for validation. A novel QTL, located on the long arm of chromosome 6H (designated as Qcrs.caf-6H), was consistently detected in each of the four FCR severity tests conducted against the mapping population. The QTL explained up to 28.3% of the phenotypic variance, and its effect was confirmed in the validation population. Significant interaction between this resistance locus and either plant height or heading date was not detected, further facilitating its manipulation in breeding programs.

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.

Development of tightly linked markers and identification of candidate genes for Fusarium crown rot resistance in barley by exploiting a near-isogenic line-derived population.

KEY MESSAGE: This study demonstrates the feasibility of developing co-segregating markers and identifying candidate genes for Fusarium crown rot resistance in barley based on the generation and exploitation of a near-isogenic line-derived large population. Fusarium crown rot (FCR) is a chronic and severe disease in cereals in semi-arid regions worldwide. Previous studies showed that FCR assessment could be affected by many factors including plant height, growth rate as well as drought stress. Thus, accurate assessment, which is essential for detailed mapping of any locus conferring FCR resistance, is difficult. Targeting one of the resistance loci reported earlier, we developed a near-isogenic line-derived population consisting of 1820 F9 lines. By analysing this population, the Qcrs.cpi-4H locus was mapped to an interval of 0.09 cM covering a physical distance of about 637 kb and 13 markers co-segregating with the targeted locus were developed. Candidate genes underlying the resistance locus were identified by analysing the expression and sequence variation of genes in the targeted interval. The accurate localization and the development of co-segregating markers should facilitate the incorporation of this large-effect QTL into breeding programmes as well as the cloning of gene(s) underlying the locus.

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.

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.

Contrasting evolutionary patterns of multiple loci uncover new aspects in the genome origin and evolutionary history of Leymus (Triticeae; Poaceae).

Leymus Hochst. (Triticeae: Poaceae), a group of allopolyploid species with the NsXm genomes, is a perennial genus with diversity in morphology, cytology, ecology, and distribution in the Triticeae. To investigate the genome origin and evolutionary history of Leymus, three unlinked low-copy nuclear genes (Acc1, Pgk1, and GBSSI) and three chloroplast regions (trnL-F, matK, and rbcL) of 32 Leymus species were analyzed with those of 36 diploid species representing 18 basic genomes in the Triticeae. The phylogenetic relationships were reconstructed using Bayesian inference, Maximum parsimony, and NeighborNet methods. A time-calibrated phylogeny was generated to estimate the evolutionary history of Leymus. The results suggest that reticulate evolution has occurred in Leymus species, with several distinct progenitors contributing to the Leymus. The molecular data in resolution of the Xm-genome lineage resulted in two apparently contradictory results, with one placing the Xm-genome lineage as closely related to the P/F genome and the other splitting the Xm-genome lineage as sister to the Ns-genome donor. Our results suggested that (1) the Ns genome of Leymus was donated by Psathyrostachys, and additional Ns-containing alleles may be introgressed into some Leymus polyploids by recurrent hybridization; (2) The phylogenetic incongruence regarding the resolution of the Xm-genome lineage suggested that the Xm genome of Leymus was closely related to the P genome of Agropyron; (3) Both Ns- and Xm-genome lineages served as the maternal donor during the speciation of Leymus species; (4) The Pseudoroegneria, Lophopyrum and Australopyrum genomes contributed to some Leymus species.

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.

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.

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.