Genome resources for the elite bread wheat cultivar Aikang 58 and mining of elite homeologous haplotypes for accelerating wheat improvement.

Despite recent progress in crop genomics studies, the genomic changes brought about by modern breeding selection are still poorly understood, thus hampering genomics-assisted breeding, especially in polyploid crops with compound genomes such as common wheat (Triticum aestivum). In this work, we constructed genome resources for the modern elite common wheat variety Aikang 58 (AK58). Comparative genomics between AK58 and the landrace cultivar Chinese Spring (CS) shed light on genomic changes that occurred through recent varietal improvement. We also explored subgenome diploidization and divergence in common wheat and developed a homoeologous locus-based genome-wide association study (HGWAS) approach, which was more effective than single homoeolog-based GWAS in unraveling agronomic trait-associated loci. A total of 123 major HGWAS loci were detected using a genetic population derived from AK58 and CS. Elite homoeologous haplotypes (HHs), formed by combinations of subgenomic homoeologs of the associated loci, were found in both parents and progeny, and many could substantially improve wheat yield and related traits. We built a website where users can download genome assembly sequence and annotation data for AK58, perform blast analysis, and run JBrowse. Our work enriches genome resources for wheat, provides new insights into genomic changes during modern wheat improvement, and suggests that efficient mining of elite HHs can make a substantial contribution to genomics-assisted breeding in common wheat and other polyploid crops.

Deciphering genetic basis of developmental and agronomic traits by integrating high-throughput optical phenotyping and genome-wide association studies in wheat.

Dissecting the genetic basis of complex traits such as dynamic growth and yield potential is a major challenge in crops. Monitoring the growth throughout growing season in a large wheat population to uncover the temporal genetic controls for plant growth and yield-related traits has so far not been explored. In this study, a diverse wheat panel composed of 288 lines was monitored by a non-invasive and high-throughput phenotyping platform to collect growth traits from seedling to grain filling stage and their relationship with yield-related traits was further explored. Whole genome re-sequencing of the panel provided 12.64 million markers for a high-resolution genome-wide association analysis using 190 image-based traits and 17 agronomic traits. A total of 8327 marker-trait associations were detected and clustered into 1605 quantitative trait loci (QTLs) including a number of known genes or QTLs. We identified 277 pleiotropic QTLs controlling multiple traits at different growth stages which revealed temporal dynamics of QTLs action on plant development and yield production in wheat. A candidate gene related to plant growth that was detected by image traits was further validated. Particularly, our study demonstrated that the yield-related traits are largely predictable using models developed based on i-traits and provide possibility for high-throughput early selection, thus to accelerate breeding process. Our study explored the genetic architecture of growth and yield-related traits by combining high-throughput phenotyping and genotyping, which further unravelled the complex and stage-specific contributions of genetic loci to optimize growth and yield in wheat.

Genome wide association and haplotype analyses for the crease depth trait in bread wheat (Triticum aestivum L.).

Wheat grain has a complex structure that includes a crease on one side, and tissues within the crease region play an important role in nutrient transportation during wheat grain development. However, the genetic architecture of the crease region is still unclear. In this study, 413 global wheat accessions were resequenced and a method was developed for evaluating the phenotypic data of crease depth (CD). The CD values exhibited continuous and considerable large variation in the population, and the broad-sense heritability was 84.09%. CD was found to be positively correlated with grain-related traits and negatively with quality-related traits. Analysis of differentiation of traits between landraces and cultivars revealed that grain-related traits and CD were simultaneously improved during breeding improvement. Moreover, 2,150.8-Mb genetic segments were identified to fall within the selective sweeps between the landraces and cultivars; they contained some known functional genes for quality- and grain-related traits. Genome-wide association study (GWAS) was performed using around 10 million SNPs generated by genome resequencing and 551 significant SNPs and 18 QTLs were detected significantly associated with CD. Combined with cluster analysis of gene expression, haplotype analysis, and annotated information of candidate genes, two promising genes TraesCS3D02G197700 and TraesCS5A02G292900 were identified to potentially regulate CD. To the best of our knowledge, this is the first study to provide the genetic basis of CD, and the genetic loci identified in this study may ultimately assist in wheat breeding programs.

Epigenetic Landscape Is Largely Shaped by Diversiform Transposons in Aegilops tauschii.

Transposons (TEs) account for more than 80% of the wheat genome, the highest among all known crop species. They play an important role in shaping the elaborate genomic landscape, which is the key to the speciation of wheat. In this study, we analyzed the association between TEs, chromatin states, and chromatin accessibility in Aegilops tauschii, the D genome donor of bread wheat. We found that TEs contributed to the complex but orderly epigenetic landscape as chromatin states showed diverse distributions on TEs of different orders or superfamilies. TEs also contributed to the chromatin state and openness of potential regulatory elements, affecting the expression of TE-related genes. Some TE superfamilies, such as hAT-Ac, carry active/open chromatin regions. In addition, the histone mark H3K9ac was found to be associated with the accessibility shaped by TEs. These results suggest the role of diversiform TEs in shaping the epigenetic landscape and in gene expression regulation in Aegilops tauschii. This has positive implications for understanding the transposon roles in Aegilops tauschii or the wheat D genome.

Intra-Varietal Diversity and Its Contribution to Wheat Evolution, Domestication, and Improvement in Wheat.

Crop genetic diversity is essential for adaptation and productivity in agriculture. A previous study revealed that poor allele diversity in wheat commercial cultivars is a major barrier to its further improvement. Homologs within a variety, including paralogs and orthologs in polyploid, account for a large part of the total genes of a species. Homolog diversity, intra-varietal diversity (IVD), and their functions have not been elucidated. Common wheat, an important food crop, is a hexaploid species with three subgenomes. This study analyzed the sequence, expression, and functional diversity of homologous genes in common wheat based on high-quality reference genomes of two representative varieties, a modern commercial variety Aikang 58 (AK58) and a landrace Chinese Spring (CS). A total of 85,908 homologous genes, accounting for 71.9% of all wheat genes, including inparalogs (IPs), outparalogs (OPs), and single-copy orthologs (SORs), were identified, suggesting that homologs are an important part of the wheat genome. The levels of sequence, expression, and functional variation in OPs and SORs were higher than that of IPs, which indicates that polyploids have more homologous diversity than diploids. Expansion genes, a specific type of OPs, made a great contribution to crop evolution and adaptation and endowed crop with special characteristics. Almost all agronomically important genes were from OPs and SORs, demonstrating their essential functions for polyploid evolution, domestication, and improvement. Our results suggest that IVD analysis is a novel approach for evaluating intra-genomic variations, and exploitation of IVD might be a new road for plant breeding, especially for polyploid crops, such as wheat.

DIW1 encoding a clade I PP2C phosphatase negatively regulates drought tolerance by de-phosphorylating TaSnRK1.1 in wheat.

Drought seriously impacts wheat production (Triticum aestivum L.), while the exploitation and utilization of genes for drought tolerance are insufficient. Leaf wilting is a direct reflection of drought tolerance in plants. Clade A PP2Cs are abscisic acid (ABA) co-receptors playing vital roles in the ABA signaling pathway, regulating drought response. However, the roles of other clade PP2Cs in drought tolerance, especially in wheat, remain largely unknown. Here, we identified a gain-of-function drought-induced wilting 1 (DIW1) gene from the wheat Aikang 58 mutant library by map-based cloning, which encodes a clade I protein phosphatase 2C (TaPP2C158) with enhanced protein phosphatase activity. Phenotypic analysis of overexpression and CRISPR/Cas9 mutant lines demonstrated that DIW1/TaPP2C158 is a negative regulator responsible for drought resistance. We found that TaPP2C158 directly interacts with TaSnRK1.1 and de-phosphorylates it, thus inactivating the TaSnRK1.1-TaAREB3 pathway. TaPP2C158 protein phosphatase activity is negatively correlated with ABA signaling. Association analysis suggested that C-terminal variation of TaPP2C158 changing protein phosphatase activity is highly correlated with the canopy temperature, and seedling survival rate under drought stress. Our data suggest that the favorable allele with lower phosphatase activity of TaPP2C158 has been positively selected in Chinese breeding history. This work benefits us in understanding the molecular mechanism of wheat drought tolerance, and provides elite genetic resources and molecular markers for improving wheat drought tolerance.

Genome sequencing of Sitopsis species provides insights into their contribution to the B subgenome of bread wheat.

Wheat (Triticum aestivum, BBAADD) is an allohexaploid species that originated from two polyploidization events. The progenitors of the A and D subgenomes have been identified as Triticum urartu and Aegilops tauschii, respectively. Current research suggests that Aegilops speltoides is the closest but not the direct ancestor of the B subgenome. However, whether Ae. speltoides has contributed genomically to the wheat B subgenome and which chromosome regions are conserved between Ae. speltoides and the B subgenome remain unclear. Here, we assembled a high-quality reference genome for Ae. speltoides, resequenced 53 accessions from seven species (Aegilops bicornis, Aegilops longissima, Aegilops searsii, Aegilops sharonensis, Ae. speltoides, Aegilops mutica [syn. Amblyopyrum muticum], and Triticum dicoccoides) and revealed their genomic contributions to the wheat B subgenome. Our results showed that centromeric regions were particularly conserved between Aegilops and Triticum and revealed 0.17 Gb of conserved blocks between Ae. speltoides and the B subgenome. We classified five groups of conserved and non-conserved genes between Aegilops and Triticum, revealing their biological characteristics, differentiation in gene expression patterns, and collinear relationships between Ae. speltoides and the wheat B subgenome. We also identified gene families that expanded in Ae. speltoides during its evolution and 789 genes specific to Ae. speltoides. These genes can serve as genetic resources for improvement of adaptability to biotic and abiotic stress. The newly constructed reference genome and large-scale resequencing data for Sitopsis species will provide a valuable genomic resource for wheat genetic improvement and genomic studies.

Centromere repositioning and shifts in wheat evolution.

The centromere is the region of a chromosome that directs its separation and plays an important role in cell division and reproduction of organisms. Elucidating the dynamics of centromeres is an alternative strategy for exploring the evolution of wheat. Here, we comprehensively analyzed centromeres from the de novo-assembled common wheat cultivar Aikang58 (AK58), Chinese Spring (CS), and all sequenced diploid and tetraploid ancestors by chromatin immunoprecipitation sequencing, whole-genome bisulfite sequencing, RNA sequencing, assay for transposase-accessible chromatin using sequencing, and comparative genomics. We found that centromere-associated sequences were concentrated during tetraploidization and hexaploidization. Centromeric repeats of wheat (CRWs) have undergone expansion during wheat evolution, with strong interweaving between the A and B subgenomes post tetraploidization. We found that CENH3 prefers to bind with younger CRWs, as directly supported by immunocolocalization on two chromosomes (1A and 2A) of wild emmer wheat with dicentromeric regions, only one of which bound with CENH3. In a comparison of AK58 with CS, obvious centromere repositioning was detected on chromosomes 1B, 3D, and 4D. The active centromeres showed a unique combination of lower CG but higher CHH and CHG methylation levels. We also found that centromeric chromatin was more open than pericentromeric chromatin, with higher levels of gene expression but lower gene density. Frequent introgression between tetraploid and hexaploid wheat also had a strong influence on centromere position on the same chromosome. This study also showed that active wheat centromeres were genetically and epigenetically determined.

Tiller Number1 encodes an ankyrin repeat protein that controls tillering in bread wheat.

Wheat (Triticum aestivum L.) is a major staple food for more than one-third of the world's population. Tiller number is an important agronomic trait in wheat, but only few related genes have been cloned. Here, we isolate a wheat mutant, tiller number1 (tn1), with much fewer tillers. We clone the TN1 gene via map-based cloning: TN1 encodes an ankyrin repeat protein with a transmembrane domain (ANK-TM). We show that a single amino acid substitution in the third conserved ankyrin repeat domain causes the decreased tiller number of tn1 mutant plants. Resequencing and haplotype analysis indicate that TN1 is conserved in wheat landraces and modern cultivars. Further, we reveal that the expression level of the abscisic acid (ABA) biosynthetic gene TaNCED3 and ABA content are significantly increased in the shoot base and tiller bud of the tn1 mutants; TN1 but not tn1 could inhibit the binding of TaPYL to TaPP2C via direct interaction with TaPYL. Taken together, we clone a key wheat tiller number regulatory gene TN1, which promotes tiller bud outgrowth probably through inhibiting ABA biosynthesis and signaling.

Low-affinity SPL binding sites contribute to subgenome expression divergence in allohexaploid wheat.

Expression divergence caused by genetic variation and crosstalks among subgenomes of the allohexaploid bread wheat (Triticum aestivum. L., BBAADD) is hypothesized to increase its adaptability and/or plasticity. However, the molecular basis of expression divergence remains unclear. Squamosa promoter-binding protein-like (SPL) transcription factors are critical for a wide array of biological processes. In this study, we constructed expression regulatory networks by combining DAP-seq for 40 SPLs, ATAC-seq, and RNA-seq. Our findings indicate that a group of low-affinity SPL binding regions (SBRs) were targeted by diverse SPLs and caused different sequence preferences around the core GTAC motif. The SBRs including the low-affinity ones are evolutionarily conserved, enriched GWAS signals related to important agricultural traits. However, those SBRs are highly diversified among the cis-regulatory regions (CREs) of syntenic genes, with less than 8% SBRs coexisting in triad genes, suggesting that CRE variations are critical for subgenome differentiations. Knocking out of TaSPL7A/B/D and TaSPL15A/B/D subfamily further proved that both high- and low-affinity SBRs played critical roles in the differential expression of genes regulating tiller number and spike sizes. Our results have provided baseline data for downstream networks of SPLs and wheat improvements and revealed that CRE variations are critical sources for subgenome divergence in the allohexaploid wheat.

Mapping Quantitative Trait Loci for Type II Fusarium Head Blight Resistance in Two Wheat Recombinant Inbred Line Populations Derived from Yangmai 4 and Yangmai 5.

Fusarium head blight (FHB) is a destructive wheat disease worldwide and significantly affects grain yield and quality in wheat. To understand the genetic basis underlying type II FHB resistance in two elite wheat cultivars-Yangmai 4 (YM4) and Yangmai 5 (YM5)-quantitative trait loci (QTL) mapping was conducted in two recombinant inbred line (RIL) populations derived from the crosses of YM4 and YM5 with susceptible cultivar Yanzhan 1 (YZ1), respectively. A survey with markers linked to Fhb1, Fhb2, Fhb4, and Fhb5 in landrace Wangshuibai indicated the nonexistence of these known FHB resistance genes or QTL in YM4, YM5, and YZ1. One overlapped resistance QTL was identified in both RIL populations (namely, QFhb.Y4.2D/QFhb.Y5.2D) with a large effect on FHB resistance. One novel resistance QTL (QFhb.Y4.5A) mapped on chromosome 5A was detected only in the YM4/YZ1 population. The resistance alleles of both QFhb.Y4.2D/QFhb.Y5.2D and QFhb.Y4.5A did not increase the plant height and did not significantly affect the heading date and flowering date. Kompetitive allele-specific PCR markers for QFhb.Y4.2D/QFhb.Y5.2D and QFhb.Y4.5A had been developed to verify in an additional set of 244 geographically diverse cultivars or lines. Pyramiding of the two resistance alleles decreased the percentage of symptomatic spikelets by 51.77% relative to the cultivars or lines without these two resistance alleles. QFhb.Y4.2D/QFhb.Y5.2D and QFhb.Y4.5A were shown to be useful alternatives in FHB resistance breeding programs. The results will facilitate marker-assisted selection for introgression of the favorable alleles for improving FHB resistance in breeding programs.

Genome-wide association study of agronomic traits related to nitrogen use efficiency in wheat.

KEY MESSAGE: GWAS identified 347 QTLs associated with eight traits related to nitrogen use efficiency in a 389-count wheat panel. Four novel candidate transcription factor genes were verified using qRT-PCR. Nitrogen is an essential nutrient for plants that determines crop yield. Improving nitrogen use efficiency (NUE) should considerably increase wheat yield and reduce the use of nitrogen fertilisers. However, knowledge on the genetic basis of NUE during wheat maturity is limited. In this study, a diversity panel incorporating 389 wheat accessions was phenotyped for eight NUE-related agronomic traits across five different environments. A total of 347 quantitative trait loci (QTLs) for low nitrogen tolerance indices (ratio of agronomic characters under low and high nitrogen conditions) were identified through a genome-wide association study utilising 397,384 single nucleotide polymorphisms (SNPs) within the MLM (Q + K) model, including 11 stable QTLs. Furthermore, 69 candidate genes were predicted for low nitrogen tolerance indices of best linear unbiased predictions values of the eight studied agronomic traits, and four novel candidate transcription factors (TraesCS5A02G237500 for qFsnR5A.2, TraesCS5B02G384500 and TraesCS5B02G384600 for qSLR5B.1, and TraesCS3B02G068800 for qTKWR3B.1) showed differing expression patterns in contrasting low-nitrogen-tolerant wheat genotypes. Moreover, the number of favourable marker alleles calculated using NUE that were significantly related to SNP in accessions decreased over the decades, indicating a decline in the NUE of the 389 wheat varieties. These findings denote promising NUE markers that could be useful in breeding high-NUE wheat varieties, and the candidate genes could further detail the NUE-related regulation network in wheat.

Wheat genomic study for genetic improvement of traits in China.

Bread wheat (Triticum aestivum L.) is a major crop that feeds 40% of the world's population. Over the past several decades, advances in genomics have led to tremendous achievements in understanding the origin and domestication of wheat, and the genetic basis of agronomically important traits, which promote the breeding of elite varieties. In this review, we focus on progress that has been made in genomic research and genetic improvement of traits such as grain yield, end-use traits, flowering regulation, nutrient use efficiency, and biotic and abiotic stress responses, and various breeding strategies that contributed mainly by Chinese scientists. Functional genomic research in wheat is entering a new era with the availability of multiple reference wheat genome assemblies and the development of cutting-edge technologies such as precise genome editing tools, high-throughput phenotyping platforms, sequencing-based cloning strategies, high-efficiency genetic transformation systems, and speed-breeding facilities. These insights will further extend our understanding of the molecular mechanisms and regulatory networks underlying agronomic traits and facilitate the breeding process, ultimately contributing to more sustainable agriculture in China and throughout the world.

Wheat male-sterile 2 reduces ROS levels to inhibit anther development by deactivating ROS modulator 1.

Ms2 is an important dominant male-sterile gene in wheat, but the biochemical function of Ms2 and the mechanism by which it causes male sterility remain elusive. Here, we report the molecular basis underlying Ms2-induced male sterility in wheat. We found that activated Ms2 specifically reduces the reactive oxygen species (ROS) signals in anthers and thereby induces termination of wheat anther development at an early stage. Furthermore, our results indicate that Ms2 is localized in mitochondria, where it physically interacts with a wheat homolog of ROS modulator 1 (TaRomo1). Romo1 positively regulates the ROS levels in humans but has never been studied in plants. We found that single amino acid substitutions in the Ms2 protein that rescue the ms2 male-sterile phenotype abolish the interaction between Ms2 and TaRomo1. Significantly, Ms2 promotes the transition of TaRomo1 proteins from active monomers to inactive oligomers. Taken together, our findings unravel the molecular basis of Ms2-induced male sterility and reveal a regulatory mechanism in which ROS act as essential signals guiding the anther development program in wheat.

Reference genome assemblies reveal the origin and evolution of allohexaploid oat.

Common oat (Avena sativa) is an important cereal crop serving as a valuable source of forage and human food. Although reference genomes of many important crops have been generated, such work in oat has lagged behind, primarily owing to its large, repeat-rich polyploid genome. Here, using Oxford Nanopore ultralong sequencing and Hi-C technologies, we have generated a reference-quality genome assembly of hulless common oat, comprising 21 pseudomolecules with a total length of 10.76 Gb and contig N50 of 75.27 Mb. We also produced genome assemblies for diploid and tetraploid Avena ancestors, which enabled the identification of oat subgenomes and provided insights into oat chromosomal evolution. The origin of hexaploid oat is inferred from whole-genome sequencing, chloroplast genomes and transcriptome assemblies of different Avena species. These findings and the high-quality reference genomes presented here will facilitate the full use of crop genetic resources to accelerate oat improvement.

A new major QTL for flag leaf thickness in barley (Hordeum vulgare L.).

BACKGROUND: Carbohydrate accumulation of photosynthetic organs, mainly leaves, are the primary sources of grain yield in cereals. The flag leaf plays a vital role in seed development, which is probably the most neglected morphological characteristic during traditional selection processes. RESULTS: In this experiment, four flag leaf morphological traits and seven yield-related traits were investigated in a DH population derived from a cross between a wild barley and an Australian malting barley cultivar. Flag leaf thickness (FLT) showed significantly positive correlations with grain size. Four QTL, located on chromosomes 1H, 2H, 3H, and 5H, respectively, were identified for FLT. Among them, a major QTL was located on chromosome 3H with a LOD value of 18.4 and determined 32% of the phenotypic variation. This QTL showed close links but not pleiotropism to the previously reported semi-dwarf gene sdw1 from the cultivated barley. This QTL was not reported before and the thick leaf allele from the wild barley could provide a useful source for improving grain yield through breeding. CONCLUSIONS: Our results also provided valuable evidence that source traits and sink traits in barley are tightly connected and suggest further improvement of barley yield potential with enhanced and balanced source and sink relationships by exploiting potentialities of the wild barley resources. Moreover, this study will provide a novel sight on understanding the evolution and development of leaf morphology in barley and improving barley production by rewilding for lost superior traits during plant evolution.

A recent burst of gene duplications in Triticeae.

Gene duplication provides raw genetic materials for evolution and potentially novel genes for crop improvement. The two seminal genomic studies of Aegilops tauschii both mentioned the large number of genes independently duplicated in recent years, but the duplication mechanism and the evolutionary significance of these gene duplicates have not yet been investigated. Here, we found that a recent burst of gene duplications (hereafter abbreviated as the RBGD) has probably occurred in all sequenced Triticeae species. Further investigations of the characteristics of the gene duplicates and their flanking sequences suggested that transposable element (TE) activity may have been involved in generating the RBGD. We also characterized the duplication timing, retention pattern, diversification, and expression of the duplicates following the evolution of Triticeae. Multiple subgenome-specific comparisons of the duplicated gene pairs clearly supported extensive differential regulation and related functional diversity among such pairs in the three subgenomes of bread wheat. Moreover, several duplicated genes from the RBGD have evolved into key factors that influence important agronomic traits of wheat. Our results provide insights into a unique source of gene duplicates in Triticeae species, which has increased the gene dosage together with the two polyploidization events in the evolutionary history of wheat.

The chromosome-level genome assembly of Astragalus sinicus and comparative genomic analyses provide new resources and insights for understanding legume-rhizobial interactions.

The legume species Astragalus sinicus (Chinese milk vetch [CMV]) has been widely cultivated for centuries in southern China as one of the most important green manures/cover crops for improving rice productivity and preventing soil degeneration. In this study, we generated the first chromosome-scale reference genome of CMV by combining PacBio and Illumina sequencing with high-throughput chromatin conformation capture (Hi-C) technology. The CMV genome was 595.52 Mb in length, with a contig N50 size of 1.50 Mb. Long terminal repeats (LTRs) had been amplified and contributed to genome size expansion in CMV. CMV has undergone two whole-genome duplication (WGD) events, and the genes retained after the WGD shared by Papilionoideae species shaped the rhizobial symbiosis and the hormonal regulation of nodulation. The chalcone synthase (CHS) gene family was expanded and was expressed primarily in the roots of CMV. Intriguingly, we found that resistance genes were more highly expressed in roots than in nodules of legume species, suggesting that their expression may be increased to bolster plant immunity in roots to cope with pathogen infection in legumes. Our work sheds light on the genetic basis of nodulation and symbiosis in CMV and provides a benchmark for accelerating genetic research and molecular breeding in the future.