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Hexaploid wheat ( Triticum aestivum ), a major staple crop, has a remarkably large genome of ~14.4 Gb (containing 106 913 high‐confidence [HC] and 159 840 low‐confidence [LC] genes in the Chinese Spring v2.1 reference genome), which poses a major challenge for functional genomics studies. To overcome this hurdle, we performed whole‐exome sequencing to generate a nearly saturated wheat mutant database containing 18 025 209 mutations induced by ethyl methanesulfonate (EMS), carbon (C)‐ion beams, or γ‐ray mutagenesis. This database contains an average of 47.1 mutations per kb in each gene‐coding sequence: the potential functional mutations were predicted to cover 96.7% of HC genes and 70.5% of LC genes. Comparative analysis of mutations induced by EMS, γ‐rays, or C‐ion beam irradiation revealed that γ‐ray and C‐ion beam mutagenesis induced a more diverse array of variations than EMS, including large‐fragment deletions, small insertions/deletions, and various non‐synonymous single nucleotide polymorphisms. As a test case, we combined mutation analysis with phenotypic screening and rapidly mapped the candidate gene responsible for the phenotype of a yellow‐green leaf mutant to a 2.8‐Mb chromosomal region. Furthermore, a proof‐of‐concept reverse genetics study revealed that mutations in gibberellic acid biosynthesis and signalling genes could be associated with negative impacts on plant height. Finally, we built a publically available database of these mutations with the corresponding germplasm (seed stock) repository to facilitate advanced functional genomics studies in wheat for the broad plant research community.

Salinity stress has become an increasing threat to food security worldwide and elucidation of the mechanism for salinity tolerance is of great significance. Induced mutation, especially spaceflight mutagenesis, is one important method for crop breeding. In this study, we show that a spaceflight-induced wheat mutant, named salinity tolerance 1 ( st1 ), is a salinity-tolerant line. We report the characteristics of transcriptomic sequence variation induced by spaceflight, and show that mutations in genes associated with sodium ion transport may directly contribute to salinity tolerance in st1 . Furthermore, GO and KEGG enrichment analysis of differentially expressed genes (DEGs) between salinity-treated st1 and wild type suggested that the homeostasis of oxidation-reduction process is important for salt tolerance in st1 . Through KEGG pathway analysis, “Butanoate metabolism” was identified as a new pathway for salinity responses. Additionally, key genes for salinity tolerance, such as genes encoding arginine decarboxylase, polyamine oxidase, hormones-related, were not only salt-induced in st1 but also showed higher expression in salt-treated st1 compared with salt-treated WT, indicating that these genes may play important roles in salinity tolerance in st1 . This study presents valuable genetic resources for studies on transcriptome variation caused by induced mutation and the identification of salt tolerance genes in crops.

Background Heading time is one of the most important agronomic traits in wheat, as it largely affects both adaptation to different agro-ecological conditions and yield potential. Identification of genes underlying the regulation of wheat heading and the development of diagnostic markers could facilitate our understanding of genetic control of this process. Results In this study, we developed 400 recombinant inbred lines (RILs) by crossing a γ-ray-induced early heading mutant ( eh1 ) with the late heading cultivar, Lunxuan987. Bulked Segregant Analysis (BSA) of both RNA and DNA pools consisting of various RILs detected a quantitative trait loci (QTL) for heading date located on chromosomes 5B, and further genetic linkage analysis limited the QTL to a 3.31 cM region. We then identified a large deletion in the first intron of the vernalization gene VRN-B1 in eh1, and showed it was associated with the heading phenotype in the RIL population. However, it is not the mutation loci that resulted in early heading phonotype in the mutant compared to that of wildtype. RNA-seq analysis suggested that Vrn-B3 and several newly discovered genes, including beta-amylase 1 ( BMY1 ) and anther-specific protein ( RTS ), were highly expressed in both the mutant and early heading pool with the dominant Vrn-B1 genotype compared to that of Lunxuan987 and late heading pool. Enrichment analysis of differentially expressed genes (DEGs) identified several key pathways previously reported to be associated with flowering, including fatty acid elongation, starch and sucrose metabolism, and flavonoid biosynthesis. Conclusion The development of new markers for Vrn-B1 in this study supplies an alternative solution for marker-assisted breeding to optimize heading time in wheat and the DEGs analysis provides basic information for VRN-B1 regulation study.

Crop productivity is highly dependent on the application of N fertilizers, but ever-increasing N application is causing serious environmental impacts. To facilitate the development of new wheat cultivars that can thrive in low N growth conditions, key loci and genes associated with wheat responses to low N must be identified. In this GWAS and t-test study of 190 M6 mutant wheat lines (Jing 411-derived) based on genotype data from the wheat 660k SNP array, we identified a total of 221 significant SNPs associated four seedling phenotypic traits that have been implicated in resistance to low N: relative root length, relative shoot length, relative root weight, and relative shoot weight. Notably, we detected large numbers of significantly associated SNP in what appear to be genomic 'hotspots' for resistance to low N on chromosomes 2A and 6B, strongly suggesting that these regions are functionally related to the resistance phenotypes that we observed in some of the mutant lines. Moreover, the candidate genes, including genes encoding high-affinity nitrate transporter 2.1, gibberellin responsive protein, were identified for resistance to low N. This study raises plausible mechanistic hypotheses that can be evaluated in future applied or basic efforts by breeders or plant biologists seeking to develop new high-NUE wheat cultivars.

Background Heavy ion beam irradiation is a potent mutagenic technique for developing new germplasm resources and breeding novel plant varieties. However, the biological effects and molecular variations caused by different dosages of heavy ion beam irradiation in crops are still not well understood. Results In this study, we investigated the biological effects and molecular variations in the M 1  generation of wheat, along with extensive phenotype screening in the M 2  generation, to thoroughly assess the mutagenic impact of carbon-ion beam irradiation. Our findings indicate that radiation doses of 60–120 Gy significantly reduced seedling height and root length, with a 50% survival rate occurring at the dose of 87 Gy. We examined a total of 1840 M 2 plants for phenotypic mutations and found a mean mutation frequency of 2.68%. Transcriptome sequencing revealed that irradiation induced single nucleotide polymorphisms (SNPs) and small insertions and deletions (indels), with transition/transversion ratios ranging from 1.96 to 2.03. Mutation hotspots were identified in the Chr1A, 3A, 6D, and 7D regions. Additionally, weighted gene co-expression network analysis (WGCNA) identified genes such as CAT, NPF, and BZIP which showed a high correlation with dosage response and are predominantly involved in starch and sucrose metabolism. Metabolome analysis at a radiation dose of 100 Gy revealed that the differential metabolites were predominantly involved in tryptophan metabolism. These pathways may play a crucial role in the response to radiation. Conclusion This study provides valuable information and resources for mutation breeding in crops, enhancing our understanding of carbon-ion-beam-induced mutations in wheat.

Agronomic traits such as heading date (HD), plant height (PH), thousand grain weight (TGW), and spike length (SL) are important factors affecting wheat yield. In this study, we constructed a high-density genetic linkage map using the Wheat55K SNP Array to map quantitative trait loci (QTLs) for these traits in 207 recombinant inbred lines (RILs). A total of 37 QTLs were identified, including 9 QTLs for HD, 7 QTLs for PH, 12 QTLs for TGW, and 9 QTLs for SL, which explained 3.0–48.8% of the phenotypic variation. Kompetitive Allele Specific PCR (KASP) markers were developed based on sequencing data and used for validation of the stably detected QTLs on chromosomes 3A, 4B and 6A using 400 RILs. A QTL cluster on chromosome 4B for PH and TGW was delimited to a 0.8 Mb physical interval explaining 12.2–22.8% of the phenotypic variation. Gene annotations and analyses of SNP effects suggested that a gene encoding protein Photosynthesis Affected Mutant 68, which is essential for photosystem II assembly, is a candidate gene affecting PH and TGW. In addition, the QTL for HD on chromosome 3A was narrowed down to a 2.5 Mb interval, and a gene encoding an R3H domain-containing protein was speculated to be the causal gene influencing HD. The linked KASP markers developed in this study will be useful for marker-assisted selection in wheat breeding, and the candidate genes provide new insight into genetic study for those traits in wheat.

Plant height is one of the most important agronomic traits that affects yield in wheat, owing to that the utilization of dwarf or semi-dwarf genes is closely associated with lodging resistance. In this study, we identified a semi-dwarf mutant, jg0030 , induced by γ-ray mutagenesis of the wheat variety ‘Jing411’ (wild type). Compared with the ‘Jing411’, plant height of the jg0030 mutant was reduced by 7%-18% in two years’ field experiments, and the plants showed no changes in yield-related traits. Treatment with gibberellic acid (GA) suggested that jg0030 is a GA-sensitive mutant. Analysis of the frequency distribution of plant height in 297 F 3 families derived from crossing jg0030 with the ‘Jing411’ indicated that the semi-dwarf phenotype is controlled by a major gene. Using the wheat 660K SNP array-based Bulked Segregant Analysis (BSA) and the exome capture sequencing-BSA assay, the dwarf gene was mapped on the long arm of chromosome 2B. We developed a set of KASP markers and mapped the dwarf gene to a region between marker PH1 and PH7. This region encompassed a genetic distance of 55.21 cM, corresponding to a physical distance of 98.3 Mb. The results of our study provide a new genetic resource and linked markers for wheat improvement in molecular breeding programs.

Background Transient starch provides carbon and energy for plant growth, and its synthesis is regulated by the joint action of a series of enzymes. Starch synthesis IV (SSIV) is one of the important starch synthase isoforms, but its impact on wheat starch synthesis has not yet been reported due to the lack of mutant lines. Results Using the TILLING approach, we identified 54 mutations in the wheat gene TaSSIVb - D , with a mutation density of 1/165 Kb. Among these, three missense mutations and one nonsense mutation were predicted to have severe impacts on protein function. In the mutants, TaSSIVb - D was significantly down-regulated without compensatory increases in the homoeologous genes TaSSIVb - A and TaSSIVb - B . Altered expression of TaSSIVb - D affected granule number per chloroplast; compared with wild type, the number of chloroplasts containing 0–2 granules was significantly increased, while the number containing 3–4 granules was decreased. Photosynthesis was affected accordingly; the maximum quantum yield and yield of PSII were significantly reduced in the nonsense mutant at the heading stage. Conclusions These results indicate that TaSSIVb - D plays an important role in the formation of transient starch granules in wheat, which in turn impact the efficiency of photosynthesis. The mutagenized population created in this study allows the efficient identification of novel alleles of target genes and could be used as a resource for wheat functional genomics.

Background Plant height (PH) and spike compactness (SC) are important agronomic traits that affect yield improvement in wheat crops. The identification of the loci or genes responsible for these traits is thus of great importance for marker-assisted selection in wheat breeding. Results In this study, we used a recombinant inbred line (RIL) population with 139 lines derived from a cross between the mutant Rht8-2 and the local wheat variety NongDa5181 (ND5181) to construct a high-density genetic linkage map by applying the Wheat 40 K Panel. We identified seven stable QTLs for PH (three) and SC (four) in two environments using the RIL population, and found that Rht8-B1 is the causal gene of qPH2B.1 by further genetic mapping, gene cloning and gene editing analyses. Our results also showed that two natural variants from GC to TT in the coding region of Rht8-B1 resulted in an amino acid change from G (ND5181) to V ( Rht8-2 ) at the 175 th position, reducing PH by 3.6%~6.2% in the RIL population. Moreover, gene editing analysis suggested that the height of T 2 generation in Rht8-B1 edited plants was reduced by 5.6%, and that the impact of Rht8-B1 on PH was significantly lower than Rht8-D1 . Additionally, analysis of the distribution of Rht8-B1 in various wheat resources suggested that the Rht8-B1b allele has not been widely utilized in modern wheat breeding. Conclusions The combination of Rht8-B1b with other favorable Rht genes might be an alternative approach for developing lodging-resistant crops. Our study provides important information for marker-assisted selection in wheat breeding.

Starch is synthesized from a series of reactions catalyzed by enzymes. ADP-glucose pyrophosphorylase (AGPase) initiates the synthesis pathway and synthesizes ADP-glucose, the substrate of starch synthase (SS), of which SSIV is an isoform. Mutations of the AGPase subunit and SSIV-coding genes affect starch content and cause variation in the number of granules. Here, we pyramided the functional mutation alleles of the AGPase subunit gene TaAGP.L-B1 and the SSIV-coding gene TaSSIVb-D to elucidate their synergistic effects on other key starch biosynthesis genes and their impact on starch content. Both the TaAGP.L-B1 and TaSSIVb-D genes were expressed in wheat grain development, and the expression level of TaAGP.L-B1 was higher than that of TaSSIVb-D. The TaAGP.L-B1 gene was downregulated in the agp.L-B1 single and agp.L-B1/ssIV-D double mutants at 12 to 18 days after flowering (DAF). TaSSIVb-D expression was significantly reduced at 6 DAF in both ssIV-D single and double mutants. In the agp.L-B1/ssIV-D double mutant, TaGBSSII was upregulated, while TaAGPSS, TaSSI, and TaSBEII were downregulated. Under the interaction of these genes, the total starch and amylopectin contents were significantly decreased in agp.L-B1 and agp.L-B1/ssIV-D mutants. The results suggested that the mutations of TaAGP.L-B1 and TaSSIVb-D genes resulted in variation in the expression patterns of the other four starch synthetic genes and led to a reduction in starch and amylopectin contents. These mutants could be used further as germplasm for resistant starch analysis.