Effects of Aegilops longissima chromosome 1Sl on wheat bread-making quality in two types of translocation lines.

KEY MESSAGE: Two wheat-Ae. longissima translocation chromosomes (1BS·1SlL and 1SlS·1BL) were transferred into three commercial wheat varieties, and the new advanced lines showed improved bread-making quality compared to their recurrent parents. Aegilops longissima chromosome 1Sl encodes specific types of gluten subunits that may positively affect wheat bread-making quality. The most effective method of introducing 1Sl chromosomal fragments containing the target genes into wheat is chromosome translocation. Here, a wheat-Ae. longissima 1BS·1SlL translocation line was developed using molecular marker-assisted chromosome engineering. Two types of translocation chromosomes developed in a previous study, 1BS·1SlL and 1SlS·1BL, were introduced into three commercial wheat varieties (Ningchun4, Ningchun50, and Westonia) via backcrossing with marker-assisted selection. Advanced translocation lines were confirmed through chromosome in situ hybridization and genotyping by target sequencing using the wheat 40 K system. Bread-making quality was found to be improved in the two types of advanced translocation lines compared to the corresponding recurrent parents. Furthermore, 1SlS·1BL translocation lines displayed better bread-making quality than 1BS·1SlL translocation lines in each genetic background. Further analysis revealed that high molecular weight glutenin subunit (HMW-GS) contents and expression levels of genes encoding low molecular weight glutenin subunits (LMW-GSs) were increased in 1SlS·1BL translocation lines. Gliadin and gluten-related transcription factors were also upregulated in the grains of the two types of advanced translocation lines compared to the recurrent parents. This study clarifies the impacts of specific glutenin subunits on bread-making quality and provides novel germplasm resources for further improvement of wheat quality through molecular breeding.

Genetic analysis of iron, zinc and grain yield in wheat-Aegilops derivatives using multi-locus GWAS.

BACKGROUND: Wheat is a major staple crop and helps to reduce worldwide micronutrient deficiency. Investigating the genetics that control the concentrations of iron (Fe) and zinc (Zn) in wheat is crucial. Hence, we undertook a comprehensive study aimed at elucidating the genomic regions linked to the contents of Fe and Zn in the grain. METHODS AND RESULTS: We performed the multi-locus genome-wide association (ML-GWAS) using a panel of 161 wheat-Aegilops substitution and addition lines to dissect the genomic regions controlling grain iron (GFeC), and grain zinc (GZnC) contents. The wheat panel was genotyped using 10,825 high-quality SNPs and phenotyped in three different environments (E1-E3) during 2017-2019. A total of 111 marker-trait associations (MTAs) (at p-value < 0.001) were detected that belong to all three sub-genomes of wheat. The highest number of MTAs were identified for GFeC (58), followed by GZnC (44) and yield (9). Further, six stable MTAs were identified for these three traits and also two pleiotropic MTAs were identified for GFeC and GZnC. A total of 1291 putative candidate genes (CGs) were also identified for all three traits. These CGs encode a diverse set of proteins, including heavy metal-associated (HMA), bZIP family protein, AP2/ERF, and protein previously associated with GFeC, GZnC, and grain yield. CONCLUSIONS: The significant MTAs and CGs pinpointed in this current study are poised to play a pivotal role in enhancing both the nutritional quality and yield of wheat, utilizing marker-assisted selection (MAS) techniques.

Disomic 1M (1B) Triticum aestivum-Aegilops comosa Substitution Line with Favorable Protein Properties.

Aegilops comosa (2n = 2x = 14, MM) contains many excellent genes/traits for wheat breeding. Wheat-Ae. comosa introgression lines have potential value in the genetic improvement of wheat quality. A disomic 1M (1B) Triticum aestivum-Ae. comosa substitution line NAL-35 was identified by fluorescence in situ hybridization and genomic in situ hybridization analysis from a hybridization cross between a disomic 1M (1D) substitution line NB 4-8-5-9 with CS N1BT1D. The observation of pollen mother cells showed that NAL-35 had normal chromosome pairing, suggesting that NAL-35 could be used for the quality test. NAL-35 with alien Mx and My subunits showed positive effects on some protein-related parameters including high protein content and high ratios of high-molecular-weight glutenin subunits (HMW-GSs)/glutenin and HMW-GS/low-molecular-weight glutenin subunits. The changes in gluten composition improved the rheological properties of the dough of NAL-35, resulting in a tighter and more uniform microstructure. NAL-35 is a potential material for wheat quality improvement that transferred quality-related genes from Ae. comosa.

Wheat Ym2 originated from Aegilops sharonensis and confers resistance to soil-borne Wheat yellow mosaic virus infection to the roots.

Wheat yellow mosaic virus (WYMV) is a pathogen transmitted into its host's roots by the soil-borne vector Polymyxa graminis. Ym1 and Ym2 genes protect the host from the significant yield losses caused by the virus, but the mechanistic basis of these resistance genes remains poorly understood. Here, it has been shown that Ym1 and Ym2 act within the root either by hindering the initial movement of WYMV from the vector into the root and/or by suppressing viral multiplication. A mechanical inoculation experiment on the leaf revealed that the presence of Ym1 reduced viral infection incidence, rather than viral titer, while that of Ym2 was ineffective in the leaf. To understand the basis of the root specificity of the Ym2 product, the gene was isolated from bread wheat using a positional cloning approach. The candidate gene encodes a CC-NBS-LRR protein and it correlated allelic variation with respect to its sequence with the host's disease response. Ym2 (B37500) and its paralog (B35800) are found in the near-relatives, respectively, Aegilops sharonensis and Aegilops speltoides (a close relative of the donor of bread wheat's B genome), while both sequences, in a concatenated state, are present in several accessions of the latter species. Structural diversity in Ym2 has been generated via translocation and recombination between the two genes and enhanced by the formation of a chimeric gene resulting from an intralocus recombination event. The analysis has revealed how the Ym2 region has evolved during the polyploidization events leading to the creation of cultivated wheat.

The heat stress transcription factor family in Aegilops tauschii: genome-wide identification and expression analysis under various abiotic stresses and light conditions.

Heat stress transcription factors (Hsfs) are known to play a vital role in protecting plants against various abiotic stresses. Among the wild wheat relatives, Aegilops tauschii offers an excellent source of abiotic stress tolerance genes for improvement of bread wheat. However, little is known about its stress tolerance mechanisms. In this study, 22 AetHsf genes were identified in the genome of Aegilops tauschii and their chromosomal location, exon-intron structures, sub-cellular localization, phylogenetic and syntenic relationship were analyzed. Based on the conserved motif analysis, these Hsfs were further divided into group A, B and C. The interaction network analysis and expression profile of AetHsfs in different tissues predicted their interaction with diverse types of proteins and suggested their involvement in different developmental processes of the plant. The promoter analysis of AetHsfs showed the presence of abiotic stress-responsive, phytohormone-responsive, plant development-related and light-related cis-elements. Thus, we investigated the expression of Hsfs in Aegilops tauchii seedlings under various abiotic stress conditions and irradiated with different monochromatic lights. Most of the AetHsfs were found to be upregulated by heat stress, while some showed expression in drought, salinity and high light stress as well. Notably, the expression pattern of various AetHsfs showed their responsiveness toward dark and various light conditions (blue red and far-red) as well. Thus, this study provides novel insights into the potential role of AetHsfs in stress and light signaling pathways, which can further facilitate understanding of the stress tolerance mechanisms in Aegilops tauschii.

Identification and validation of TCONS_00093333 for regulating fertility conversion of thermo-sensitive cytoplasmic male-sterility wheat with Aegilops kotschyi cytoplasm.

An increasing number of studies have shown that long non-coding RNAs (lncRNAs) play an important role in regulating plant fertility, however, they are less studied in wheat. Here, we analyzed lncRNA sequencing data and showed that the fixation carbon sequestration pathway was closely associated with pollen development and fertility conversion in KTM3315A, and eight differentially expressed genes under different fertility conditions were significantly regulated by TCONS_00093333 (designed as TaHTMAR) and transcription factors TaBBX25 and TaOBF1. Among them, TaBBX25 and TaOBF1 were located in the nucleus and expressed in the early stage of fertile anther development. Gene silencing experiments of TaHTMAR showed that TaHTMAR positively regulated the expression of TaBBX25 and TaOBF1 under fertile conditions, thereby reducing male fertility of KTM3315A. This study confirms the effective roles of TaHTMAR, TaBBX25, and TaOBF1 in the regulation of male fertility in wheat and provides a valuable molecular basis for hybrid wheat breeding.

Comparative transcriptome analysis of two contrasting resistant and susceptible Aegilops tauschii accessions to wheat leaf rust (Puccinia triticina) using RNA-sequencing.

Leaf rust, caused by Puccinia triticina Eriks., is the most common rust disease of wheat (Triticum aestivum L.) worldwide. Owing to the rapid evolution of virulent pathotypes, new and effective leaf rust resistance sources must be found. Aegilops tauschii, an excellent source of resistance genes to a wide range of diseases and pests, may provide novel routes for resistance to this disease. In this study, we aimed to elucidate the transcriptome of leaf rust resistance in two contrasting resistant and susceptible Ae. tauschii accessions using RNA-sequencing. Gene ontology, analysis of pathway enrichment and transcription factors provided an apprehensible review of differentially expressed genes and highlighted biological mechanisms behind the Aegilops-P. triticina interaction. The results showed the resistant accession could uniquely recognize pathogen invasion and respond precisely via reducing galactosyltransferase and overexpressing chromatin remodeling, signaling pathways, cellular homeostasis regulation, alkaloid biosynthesis pathway and alpha-linolenic acid metabolism. However, the suppression of photosynthetic pathway and external stimulus responses were observed upon rust infection in the susceptible genotype. In particular, this first report of comparative transcriptome analysis offers an insight into the strength and weakness of Aegilops against leaf rust and exhibits a pipeline for future wheat breeding programs.

Identification of QTLs and a Candidate Gene for Reducing Pre-Harvest Sprouting in Aegilops tauschii-Triticum aestivum Chromosome Segment Substitution Lines.

Wheat pre-harvest sprouting (PHS) causes serious losses in wheat yield. In this study, precise mapping was carried out in the chromosome segment substitution lines (CSSL) F2 population generated by a direct cross of Zhoumai 18 (PHS-sensitive) and Aegilops tauschii accession T093 (highly PHS-resistant). Three Ae. tauschii-derived quantitative trait loci (QTLs), QDor.3D.1, QDor.3D.2, and QDor.3D.3, were detected on chromosome 3DL using four simple sequence repeats (SSR) markers and 10 developed Kompetitive allele-specific PCR (KASP) markers. Alongside these QTL results, the RNA-Seq and qRT-PCR analysis revealed expression levels of TraesCS3D01G466100 in the QDor.3D.2 region that were significantly higher in CSSLs 495 than in Zhoumai 18 during the seed imbibition treatment. The cDNA sequencing results of TraesCS3D01G466100 showed two single nucleotide polymorphisms (SNPs), resulting in two changed amino acid substitutions between Zhoumai 18 and line 495, and the 148 nt amino acid substitution of TraesCS3D01G466100, derived from Ae. tauschii T093, which may play an important role in the functioning of ubiquitin ligase enzymes 3 (E3) according to the homology protein analysis, which could lead to differential PHS-resistance phenotypes. Taken together, our results may foster a better understanding of the mechanism of PHS resistance and are potentially valuable for marker-assisted selection in practical wheat breeding efforts.

Exploring the alpha-gliadin locus: the 33-mer peptide with six overlapping coeliac disease epitopes in Triticum aestivum is derived from a subgroup of Aegilops tauschii.

Most alpha-gliadin genes of the Gli-D2 locus on the D genome of hexaploid bread wheat (Triticum aestivum) encode for proteins with epitopes that can trigger coeliac disease (CD), and several contain a 33-mer peptide with six partly overlapping copies of three epitopes, which is regarded as a remarkably potent T-cell stimulator. To increase genetic diversity in the D genome, synthetic hexaploid wheat lines are being made by hybridising accessions of Triticum turgidum (AB genome) and Aegilops tauschii (the progenitor of the D genome). The diversity of alpha-gliadins in A. tauschii has not been studied extensively. We analysed the alpha-gliadin transcriptome of 51 A. tauschii accessions representative of the diversity in A. tauschii. We extracted RNA from developing seeds and performed 454 amplicon sequencing of the first part of the alpha-gliadin genes. The expression profile of allelic variants of the alpha-gliadins was different between accessions, and also between accessions of the Western and Eastern clades of A. tauschii. Generally, both clades expressed many allelic variants not found in bread wheat. In contrast to earlier studies, we detected the 33-mer peptide in some A. tauschii accessions, indicating that it was introduced along with the D genome into bread wheat. In these accessions, transcripts with the 33-mer peptide were present at lower frequencies than in bread wheat varieties. In most A. tauschii accessions, however, the alpha-gliadins do not contain the epitope, and this may be exploited, through synthetic hexaploid wheats, to breed bread wheat varieties with fewer or no coeliac disease epitopes.

Transcriptome Analysis Reveals Important Candidate Genes Related to Nutrient Reservoir, Carbohydrate Metabolism, and Defence Proteins during Grain Development of Hexaploid Bread Wheat and Its Diploid Progenitors.

Wheat grain development after anthesis is an important biological process, in which major components of seeds are synthesised, and these components are further required for germination and seed vigour. We have made a comparative RNA-Seq analysis between hexaploid wheat and its individual diploid progenitors to know the major differentially expressed genes (DEGs) involved during grain development. Two libraries from each species were generated with an average of 55.63, 55.23, 68.13, and 103.81 million reads, resulting in 79.3K, 113.7K, 90.6K, and 121.3K numbers of transcripts in AA, BB, DD, and AABBDD genome species respectively. Number of expressed genes in hexaploid wheat was not proportional to its genome size, but marginally higher than that of its diploid progenitors. However, to capture all the transcripts in hexaploid wheat, sufficiently higher number of reads was required. Functional analysis of DEGs, in all the three comparisons, showed their predominance in three major classes of genes during grain development, i.e., nutrient reservoirs, carbohydrate metabolism, and defence proteins; some of them were subsequently validated through real time quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR). Further, developmental stage-specific gene expression showed most of the defence protein genes expressed during initial developmental stages in hexaploid contrary to the diploids at later stages. Genes related to carbohydrates anabolism expressed during early stages, whereas catabolism genes expressed at later stages in all the species. However, no trend was observed in case of different nutrient reservoirs gene expression. This data could be used to study the comparative gene expression among the three diploid species and homeologue-specific expression in hexaploid.

Reducing the size of an alien segment carrying leaf rust and stripe rust resistance in wheat.

BACKGROUND: Leaf and stripe rusts are two major wheat diseases, causing significant yield losses. The preferred way for protecting wheat from rust pathogens is by introgression of rust resistance traits from wheat-related wild species. To avoid genetic drag due to replacement of large wheat chromosomal segments by the alien chromatin, it is necessary to shorten the alien chromosome segment in primary recombinants. RESULTS: Here we report on shortening of an alien chromosome segment in wheat that carries leaf and stripe rust resistance from Sharon goatgrass (Aegilops sharonensis). Rust resistant wheat introgression lines were selected and the alien region was mapped using genotyping by sequencing. Single polymorphic nucleotides (SNP) were identified and used to generate diagnostic PCR markers. Shortening of the alien fragment was achieved by induced homoeologous pairing and lines with shortened alien chromosome were identified using the PCR markers. Further reduction of the segment was achieved in tertiary recombinants without losing the rust resistance. CONCLUSIONS: Alien chromatin in wheat with novel rust resistance genes was characterized by SNP markers and shortened by homoeologous recombination to avoid deleterious traits. The resulting wheat lines are resistant to highly virulent races of leaf and stripe rust pathogens and can be used as both resistant wheat in the field and source for gene transfer to other wheat lines/species.

Yield reduction historically associated with the Aegilops ventricosa 7DV introgression is genetically and physically distinct from the eyespot resistance gene Pch1.

KEY MESSAGE: Yield penalty and increased grain protein content traits associated with Aegilops ventricosa 7D introgression have been mapped for the first time, and they are physically distinct from the eyespot resistance locus Pch1. Wheat wild relatives represent an important source of genetic variation, but introgression of agronomically relevant genes, such as for disease resistance, may lead to the simultaneous introduction of genetically linked deleterious traits. Pch1 is a dominant gene, conferring resistance to eyespot and was introgressed to wheat from Aegilops ventricosa as part of a large segment of the 7DV chromosome. This introgression has been associated with a significant yield reduction and a concomitant increase in grain protein content. In this study, we evaluated both traits and their relationship to the location of the Pch1 gene. We found that both QTLs were clearly distinct from the Pch1 gene, being located on a different linkage group to Pch1. In addition, we found that the QTL for increased grain protein content was strong and consistent across field trials, whereas the yield penalty QTL was unstable and environmentally dependent. The yield and grain protein content QTLs were genetically linked and located in the same linkage group. This finding is due in part to the small size of the population, and to the restricted recombination between wheat 7D and Ae. ventricosa 7Dv chromosomes. Although recombination in this interval is rare, it does occur. A recombinant line containing Pch1 and 7D_KASP6, the marker associated with increase in grain protein content, but not Xwmc221, the marker associated with the yield penalty effect, was identified.

The production of wheat - Aegilops sharonensis 1Ssh chromosome substitution lines harboring alien novel high-molecular-weight glutenin subunits.

In our previous work, a novel high-molecular-weight glutenin subunit (HMW-GS) with an extremely large molecular weight from Aegilops sharonensis was identified that may contribute to excellent wheat (Triticum aestivum) processing quality and increased dough strength, and we further generated HMW-GS homozygous lines by crossing. In this study, we crossed the HMW-GS homozygous line 66-17-52 with 'Chinese Spring' Ph1 mutant CS ph1b to induce chromosome recombination between wheat and Ae. sharonensis. SDS-PAGE was used to identify 19 derived F2 lines with the HMW-GSs of Ae sharonensis. The results of non-denaturing fluorescence in situ hybridization (ND-FISH) indicated that lines 6-1 and 6-7 possessed a substitution of both 5D chromosomes by a pair of 1Ssh chromosomes. Further verification by newly developed 1Ssh-specific chromosome markers showed that these two lines amplified the expected fragment. Thus, it was concluded that lines 6-1 and 6-7 are 1Ssh(5D) chromosome substitution lines. The 1Ssh(5D) chromosome substitution lines, possessing alien subunits with satisfactory quality-associated structural features of large repetitive domains and increased number of subunits, may have great potential in strengthening the viscosity and elasticity of dough made from wheat flour. Therefore, these substitution lines can be used for wheat quality improvement and further production of 1Ssh translocation lines.

The basis of resistance mechanism to mesosulfuron-methyl in Tausch's goatgrass (Aegilops tauschii Coss.).

Tausch's goatgrass (Aegilops tauschii Coss.) is one of the most troublesome weeds in winter wheat-growing regions of China. In recent years, the recommended field rate of mesosulfuron-methyl failed to control the Tausch's goatgrass population in Shanxi province (SX), China. Experiments were conducted to characterize the herbicide resistance level and investigate the basis of mesosulfuron-methyl resistance in Tausch's goatgrass. Whole-plant dose-response tests showed that the SX population exhibited 11.42-fold resistance to mesosulfuron-methyl than the susceptible HN population, and the resistance level in the SX population could be significantly reduced by malathion, a cytochrome P450 inhibitor. The SX population also exhibited cross-resistance to imazethapyr, pyroxsulam and bispyribac­sodium. Acetohydroxyacid synthase (AHAS) sequencing and enzyme activity assays demonstrated that the mesosulfuron-methyl resistance was not conferred by target-site substitution. A sensitive AHAS, together with the malathion revisable resistance, suggested that herbicide metabolism likely plays a main role in the mechanism of mesosulfuron-methyl resistance in the SX population. To our knowledge, this is the first report elucidating the mesosulfuron-methyl resistance in Tausch's goatgrass.

Transcriptomic analysis of Aegilops tauschii during long-term salinity stress.

Aegilops tauschii is the diploid progenitor of the bread wheat D-genome. It originated from Iran and is a source of abiotic stress tolerance genes. However, little is known about the molecular events of salinity tolerance in Ae. tauschii. This study investigates the leaf transcriptional changes associated with long-term salt stress. Total RNA extracted from leaf tissues of control and salt-treated samples was sequenced using the Illumina technology, and more than 98 million high-quality reads were assembled into 255,446 unigenes with an average length of 1398 bp and an N50 of 2269 bp. Functional annotation of the unigenes showed that 93,742 (36.69%) had at least a significant BLAST hit in the SwissProt database, while 174,079 (68.14%) showed significant similarity to proteins in the NCBI nr database. Differential expression analysis identified 4506 salt stress-responsive unigenes. Bioinformatic analysis of the differentially expressed unigenes (DEUs) revealed a number of biological processes and pathways involved in the establishment of ion homeostasis, signaling processes, carbohydrate metabolism, and post-translational modifications. Fine regulation of starch and sucrose content may be important features involved in salt tolerance in Ae. tauschii. Moreover, 82% of DEUs mapped to the D-subgenome, including known QTL for salt tolerance, and these DEUs showed similar salt stress responses in other accessions of Ae. tauschii. These results could provide fundamental insight into the regulatory process underlying salt tolerance in Ae. tauschii and wheat and facilitate identification of genes involved in their salt tolerance mechanisms.

Cloning and characterization of a novel low-molecular-weight glutenin subunit gene with an unusual molecular structure of Aegilops uniaristata.

Low-molecular-weight glutenin subunits (LMW-GSs) are one of the important factors for the dough processing quality. In this study, a novel LMW-GS, designated LMW-N13, from the wheat relative species Aegilops uniaristata PI 554421 was cloned and characterized. Unlike previously published LMW-GSs, LMW-N13 has a large molecular weight and is the largest LMW-GS published thus far. Sequence alignments demonstrated that LMW-N13 is a LMW-i-type subunit but contains nine cysteine residues which is one more than typical LMW-i-type subunits. In addition, four insertions are present in the repetitive domain that resulted in the large molecular weight. In vitro analysis showed that LMW-N13 could improve the dough quality of different base flours.

Genetic diversity of submergence stress response in cytoplasms of the Triticum-Aegilops complex.

Genetic diversity in cytoplasmic and nuclear genomes and their interaction affecting adaptive traits is an attractive research subject in plants. We addressed submergence stress response of wheat that has become increasingly important but remained largely uninvestigated. Our primary aim was to disclose cytoplasmic diversity using nucleus-cytoplasm (NC) hybrids possessing a series of heterologous cytoplasms in a common nuclear background. Effects of submergence on seedling emergence and growth from imbibed seeds were studied and compared with euplasmic lines. Marked phenotypic variabilities were observed among both lines, demonstrating divergent cytoplasmic and nuclear effects on submergence response. NC hybrids with cytoplasm of Aegilops mutica showed a less inhibition, indicative of their positive contribution to submergence tolerance, whereas cytoplasms of Aegilops umbellulata and related species caused a greater inhibition. Superoxide dismutase (SOD) activity showed a marked increase accompanied by retardation of seedling growth in a susceptible NC hybrid. The observation suggested that the elevated SOD activity was resulted from a high level of reactive oxygen species accumulated and remained in susceptible seedlings. Taken together, our results point to the usefulness of NC hybrids in further studies needed to clarify molecular mechanisms underlying the nucleus-cytoplasm interaction regulating submergence stress response in wheat.