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Lesion mimic often exhibits leaf disease-like symptoms even in the absence of pathogen infection, and is characterized by a hypersensitive-response (HR) that closely linked to plant disease resistance. Despite this, only a few lesion mimic genes have been identified in wheat. In this investigation, a lesion mimic wheat mutant named je0297 was discovered, showing no alteration in yield components when compared to the wild type (WT). Segregation ratio analysis of the F2 individuals resulting from the cross between the WT and the mutant revealed that the lesion mimic was governed by a single recessive gene in je0297. Using Bulked segregant analysis (BSA) and exome capture sequencing, we mapped the lesion mimic gene designated as lm6 to chromosome 6BL. Further gene fine mapping using 3315 F2 individuals delimited the lm6 within a 1.18 Mb region. Within this region, we identified 16 high-confidence genes, with only two displaying mutations in je0297. Notably, one of the two genes, responsible for encoding flavonol synthase, exhibited altered expression levels. Subsequent phenotype analysis of TILLING mutants confirmed that the gene encoding flavonol synthase was indeed the causal gene for lm6. Transcriptome sequencing analysis revealed that the DEGs between the WT and mutant were significantly enriched in KEGG pathways related to flavonoid biosynthesis, including flavone and flavonol biosynthesis, isoflavonoid biosynthesis, and flavonoid biosynthesis pathways. Furthermore, more than 30 pathogen infection-related (PR) genes exhibited upregulation in the mutant. Corresponding to this expression pattern, the flavonoid content in je0297 showed a significant decrease in the 4th leaf, accompanied by a notable accumulation of reactive oxygen, which likely contributed to the development of lesion mimic in the mutant. This investigation enhances our comprehension of cell death signaling pathways and provides a valuable gene resource for the breeding of disease-resistant wheat.

Abstract Background Induction of mutation through chemical mutagenesis is a novel approach for preparing diverse germplasm. Introduction of functional alleles in the starch biosynthetic genes help in the improvement of the quality and yield of cereals. Results In the present study, a set of 350 stable mutant lines were used to evaluate dynamic variation of the total starch contents. A megazyme kits were used for measuring the total starch content, resistant starch, amylose, and amylopectin content. Analysis of variance showed significant variation (p

Abstract 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 175th position, reducing PH by 3.6%~6.2% in the RIL population. Moreover, gene editing analysis suggested that the height of T2 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.

The erect leaf plays a crucial role in determining plant architecture, with its growth and development regulated by genetic factors. However, there has been a lack of comprehensive studies on the regulatory mechanisms governing wheat lamina joint development, thus failing to meet current breeding demands. In this study, a wheat erect leaf mutant, mths29, induced via fast neutron mutagenesis, was utilized for QTL fine mapping and investigation of lamina joint development. Genetic analysis of segregating populations derived from mths29 and Jimai22 revealed that the erect leaf trait was controlled by a dominant single gene. Using BSR sequencing and map-based cloning techniques, the QTL responsible for the erect leaf trait was mapped to a 1.03 Mb physical region on chromosome 5A. Transcriptome analysis highlighted differential expression of genes associated with cell division and proliferation, as well as several crucial transcription factors and kinases implicated in lamina joint development, particularly in the boundary cells of the preligule zone in mths29. These findings establish a solid foundation for understanding lamina joint development and hold promise for potential improvements in wheat plant architecture.

Cannabis fructus (Cannabis sativa L.) belongs to the mulberry family, which is one of the earliest cultivated crops by humans and has industrial, agricultural, and medicinal values. In this study, the effect of carbon ion beam irradiation on the physiological and biochemical pathways of cannabis fructus was analyzed by multi-omics. Compared to the unirradiated cannabis fructus stem, 2,891 differentially-expressed genes, 1,145 differentially-expressed proteins, and 954 altered ubiquitination modification sites were identified in cannabis fructus irradiated by carbon ion beam irradiation (60 Gy dosage). Correlation analyses between proteomic, transcriptomic, and ubiquitin modification groups were carried out, and we found that the carbon fixation pathway, fatty acid synthesis pathway, JA synthesis, and glutathione metabolism pathway were enriched in relevant protein levels, and activates the MAPK signaling pathway regulated by gene expression. Further analysis found that the content of some proteins (LOX, AOC, and MDR) was correlated with their respective transcriptional levels, and the content of some proteins (APX, G-6-PD, GST, GAPHD, FBA, AOS, PR1, and ChiB) was correlated with their ubiquitination levels. In addition, M2 generation cannabis fructus seeds exhibited a larger volume, higher thousand-grain weight, and increased linolenic acid content compared to the control group, which was consistent with the trends of our multi-omics analyses. In summary, we demonstrated that 60 Gy carbon ion beam irradiation can enhance the carbon fixation pathway in cannabis fructus and enhance linolenic acid synthesis. Taken together, our multi-omics analysis of the response of cannabis fructus to carbon ion beam irradiation provides a theoretical foundation for readily producing cannabis fructus varieties with advantageous traits in the future.

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 F3 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.

Novel genetic variations can be obtained by inducing mutations in the plant which help to achieve novel traits. The useful mutant can be obtained through radiation mutation in a short period which can be used as a new material to produce new varieties with high yield and good quality wheat. In this paper, the proteomic analysis of wheat treated with different doses of 12C and 7Li ion beam radiation at the seedling stage was carried out through a Tandem Mass Tag (TMT) tagging quantitative proteomic analysis platform based on high-resolution liquid chromatography-mass spectrometry, and the traditional 60Co-γ-ray radiation treatment for reference. A total of 4,764 up-regulated and 5,542 down-regulated differentially expressed proteins were identified. These proteins were mainly enriched in the KEGG pathway associated with amino acid metabolism, fatty acid metabolism, carbon metabolism, photosynthesis, signal transduction, protein synthesis, and DNA replication. Functional analysis of the differentially expressed proteins showed that the oxidative defense system in the plant defense system was fully involved in the defense response after 12C ion beam and 7Li ion beam radiation treatments. Photosynthesis and photorespiration were inhibited after 12C ion beam and 60Co-γ-ray irradiation treatments, while there was no effect on the plant with 7Li ion beam treatment. In addition, the synthesis of biomolecules such as proteins, as well as multiple signal transduction pathways also respond to radiations. Some selected differentially expressed proteins were verified by Parallel Reaction Monitoring (PRM) and qPCR, and the experimental results were consistent with the quantitative results of TMT. The present study shows that the physiological effect of 12C ion beam radiation treatment is different as compared to the 7Li ion beam, but its similar to the 60Co-γ ray depicting a significant effect on the plant by using the same dose. The results of this study will provide a theoretical basis for the application of 12C and 7Li ion beam radiation in the mutation breeding of wheat and other major crops and promote the development of heavy ion beam radiation mutation breeding technology.

Abstract 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.

The wheat AP2 family gene Q controls domestication traits, including spike morphology and threshability, which are critical for the widespread cultivation and yield improvement of wheat. Although many studies have investigated the molecular mechanisms of the Q gene, its direct target genes, especially those controlling spike morphology, are not clear, and its regulatory pathways are not well established. In this study, we conducted gene mapping of a wheat speltoid spike mutant and found that a new allele of the Q gene with protein truncation played a role in spike morphology variation in the mutant. Dynamic expression levels of the Q gene throughout the spike development process suggested that the transcript abundances of the mutant were decreased at the W6 and W7 scales compared to those of the WT. We identified several mutation sites on the Q gene and showed that mutations in different domains resulted in distinct phenotypes. In addition, we found that the Q gene produced three transcripts via alternative splicing and that they exhibited differential expression patterns in nodes, internodes, flag leaves, and spikes. Finally, we identified several target genes directly downstream of Q, including TaGRF1-2D and TaMGD-6B, and proposed a possible regulatory network. This study uncovered the target genes of Q, and the results can help to clarify the mechanism of wheat spike morphology and thereby improve wheat grain yield.

Induced mutagenesis is a powerful approach for the creation of novel germplasm and the improvement of agronomic traits. The evaluation of mutagenic effects and functional variations in crops is needed for breeding mutant strains. To investigate the mutagenic effects of gamma-ray irradiation in wheat, this study characterized genomic variations of wheat early heading mutant (eh1) as compared to wild-type (WT) Zhongyuan 9 (ZY9). Whole-genome resequencing of eh1 and ZY9 produced 737.7 Gb sequencing data and identified a total of 23,537,117 homozygous single nucleotide polymorphism (SNP) and 1,608,468 Indel. Analysis of SNP distribution across the chromosome suggests that mutation hotspots existed in certain chromosomal regions. Among the three subgenomes, the variation frequency in subgenome D was significantly lower than in subgenomes A and B. A total of 27.8 Gb data were obtained by exome-capturing sequencing, while 217,948 SNP and 13,554 Indel were identified. Variation annotation in the gene-coding sequences demonstrated that 5.0% of the SNP and 5.3% of the Indel were functionally important. Characterization of exomic variations in 12 additional gamma-ray-induced mutant lines further provided additional insights into the mutagenic effects of this approach. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) analysis suggested that genes with functional variations were enriched in several metabolic pathways, including plant–pathogen interactions and ADP binding. Kompetitive allele-specific PCR (KASP) genotyping with selected SNP within functional genes indicated that 85.7% of the SNPs were polymorphic between the eh1 and wild type. This study provides a basic understanding of the mechanism behind gamma-ray irradiation in hexaploid wheat.

Stem elongation is a critical phase for yield determination and, as a major trait, is targeted for manipulation for improvement in bread wheat (Triticum aestivum L.). In a previous study, we characterized a mutant showing rapid stem elongation but with no effect on plant height at maturity. The present study aimed to finely map the underlying mutated gene, qd1, in this mutant. By analyzing an F2 segregating population consisting of 606 individuals, we found that the qd1 gene behaved in a dominant manner. Moreover, by using the bulked segregant RNA sequencing (BSR-seq)-based linkage analysis method, we initially mapped the qd1 gene to a 13.55 Mb region on chromosome 4B (from 15.41 to 28.96 Mb). This result was further confirmed in F2 and BC3F2 segregating populations. Furthermore, by using transcriptome sequencing data, we developed 14 Kompetitive Allele-Specific PCR (KASP) markers and then mapped the qd1 gene to a smaller and more precise 5.08 Mb interval from 26.80 to 31.88 Mb. To develop additional markers to finely map the qd1 gene, a total of 4,481 single-nucleotide polymorphisms (SNPs) within the 5.08 Mb interval were screened, and 25 KASP markers were developed based on 10x-depth genome resequencing data from both wild-type (WT) and mutant plants. The qd1 gene was finally mapped to a 1.33 Mb interval from 28.86 to 30.19 Mb on chromosome 4B. Four candidate genes were identified in this region. Among them, the expression pattern of only TraesCS4B02G042300 in the stems was concurrent with the stem development of the mutant and WT. The qd1 gene could be used in conjunction with molecular markers to manipulate stem development in the future.

Manipulation of genes involved in starch synthesis could significantly affect wheat grain weight and yield. The starch-branching enzyme (SBE) catalyzes the formation of branch points by cleaving the α-1,4 linkage in polyglucans and reattaching the chain via an α-1,6 linkage. Three types of SBE isoforms (SBEI, SBEII, and SBEIII) exist in higher plants, with the number of SBE isoforms being species-specific. In this study, the coding sequence of the wheat TaSBEIII gene was amplified. After the multiple sequence alignment of TaSBEIII genome from 20 accessions in a wheat diversity panel, one SNP was observed in TaSBEIII-A, which formed the allelic marker allele-T. Based on this SNP at 294 bp (C/T), a KASP molecular marker was developed to distinguish allelic variation among the wheat genotypes for thousand grain weight (TGW). The results were validated using 262 accessions of mini core collection (MCC) from China, 153 from Pakistan, 53 from CIMMYT, and 17 diploid and 18 tetraploid genotypes. Association analysis between TaSBEIII-A allelic variation and agronomic traits found that TaSBEIII-A was associated with TGW in mini core collection of China (MCC). The accessions possessing Allele-T had higher TGW than those possessing Allele-C; thus, Allele-T was a favorable allelic variation. By analyzing the frequency of the favorable allelic variation Allele-T in MCC, it increased from pre-1950 (25%) to the 1960s (45%) and increased continuously from 1960 to 1990 (80%). The results suggested that the KASP markers can be utilized in grain weight improvement, which ultimately improves wheat yield by marker-assisted selection in wheat breeding. The favorable allelic variation allele-T should be valuable in enhancing grain yield by improving the source and sink simultaneously. Furthermore, the newly developed KASP marker validated in different genetic backgrounds could be integrated into a breeding kit for screening high TGW wheat.

Abstract Background Wheat mutant resources with phenotypic variation have been developed in recent years. These mutants might carry favorable mutation alleles, which have the potential to be utilized in the breeding process. Plant architecture and yield-related features are important agronomic traits for wheat breeders and mining favorable alleles of these traits will improve wheat characteristics. Results Here we used 190 wheat phenotypic mutants as material and by analyzing their SNP variation and phenotypic data, mutation alleles for plant architecture and yield-related traits were identified, and the genetic effects of these alleles were evaluated. In total, 32 mutation alleles, including three pleiotropic alleles, significantly associated with agronomic traits were identified from the 190 wheat mutant lines. The SNPs were distributed on 12 chromosomes and were associated with plant height (PH), tiller number, flag leaf angle (FLA), thousand grain weight (TGW), and other yield-related traits. Further phenotypic analysis of multiple lines carrying the same mutant allele was performed to determine the effect of the allele on the traits of interest. PH-associated SNPs on chromosomes 2BL, 3BS, 3DL, and 5DL might show additive effects, reducing PH by 10.0 cm to 31.3 cm compared with wild type, which means that these alleles may be favorable for wheat improvement. Only unfavorable mutation alleles that reduced TGW and tiller number were identified. A region on chromosome 5DL with mutation alleles for PH and TGW contained several long ncRNAs, and their sequences shared more than 90% identity with cytokinin oxidase/dehydrogenase genes. Some of the mutation alleles we mined were colocalized with previously reported QTLs or genes while others were novel; these novel alleles could also result in phenotypic variation. Conclusion Our results demonstrate that favorable mutation alleles are present in mutant resources, and the region between 409.5 to 419.8 Mb on chromosome 5DL affects wheat plant height and thousand grain weight.

Blast is caused by the host-specific lineages of the fungus Magnaporthe oryzae and is the most important destructive disease in major crop plants, including rice and wheat. The first wheat blast outbreak that occurred in Bangladesh in 2016 and the recent epidemic in Zambia were caused by the M. oryzae Triticum (MoT) pathotype, a fungal lineage belonging to M. oryzae. Although a few reported wheat cultivars show modest resistance to MoT, the patterns of genetic variation and diversity of this pathotype make it crucial to identify additional lines of resistant wheat germplasm. Nearly 40 rice blast resistant and susceptible genes have so far been cloned. Here, we used BLAST analysis to locate two rice blast susceptible genes in the wheat reference genome, bsr-d1 and bsr-k1, and identified six identical homologous genes located on subgenomes A, B, and D. We uncovered a total of 171 single nucleotide polymorphisms (SNPs) in an ethyl methanesulfonate (EMS)-induced population, with mutation densities ranging from 1/1107.1 to 1/230.7 kb through Targeting Induced Local Lesions IN Genomes (TILLING) by sequencing. These included 81 SNPs located in exonic and promoter regions, and 13 coding alleles that are predicted to have severe effects on protein function, including two pre-mature mutants that might affect wheat blast resistance. The loss-of-function alleles identified in this study provide insights into new wheat blast resistant lines, which represent a valuable breeding resource.

The stems of cereal crops provide both mechanical support for lodging resistance and a nutrient supply for reproductive organs. Elongation, which is considered a critical phase for yield determination in winter wheat (Triticum aestivum L.), begins from the first node detectable to anthesis. Previously, we characterized a heavy ion beam triggered wheat mutant qd, which exhibited an altered stem elongation pattern without affecting mature plant height. In this study, we further analyzed mutant stem developmental characteristics by using transcriptome data. More than 40.87 Mb of clean reads including at least 36.61 Mb of unique mapped reads were obtained for each biological sample in this project. We utilized our transcriptome data to identify 124,971 genes. Among these genes, 4,340 differentially expressed genes (DEG) were identified between the qd and wild-type (WT) plants. Compared to their WT counterparts, qd plants expressed 2,462 DEGs with downregulated expression levels and 1878 DEGs with upregulated expression levels. Using DEXSeq, we identified 2,391 counting bins corresponding to 1,148 genes, and 289 of them were also found in the DEG analysis, demonstrating differences between qd and WT. The 5,199 differentially expressed genes between qd and WT were employed for GO and KEGG analyses. Biological processes, including protein-DNA complex subunit organization, protein-DNA complex assembly, nucleosome organization, nucleosome assembly, and chromatin assembly, were significantly enriched by GO analysis. However, only benzoxazinoid biosynthesis pathway-associated genes were enriched by KEGG analysis. Genes encoding the benzoxazinoid biosynthesis enzymes Bx1, Bx3, Bx4, Bx5, and Bx8_9 were confirmed to be differentially expressed between qd and WT. Our results suggest that benzoxazinoids could play critical roles in regulating the stem elongation phenotype of qd.

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.

Abstract 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.

Abstract 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.

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.

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.

Mutated genetic resources play an important role in gene/allele characterization. Currently, there are few hexaploid winter wheat mutated resources available. Here, we developed a hexaploid winter wheat resource by inducing mutations via EMS treatment by the single seed descent method. A broad mutation spectrum with high mutation frequency (∼19%) on phenotypic variations was identified. These mutations included spike, leaf and seed morphology, plant architecture, and heading date variations. To evaluate the efficiency of the resource for reverse genetic analysis, allelic variations in the TaAGP.L-B1 gene, encoding the AGPase large subunit, were screened by the TILLING approach. Four missense mutations were identified and one allele in line E3-1-3, resulted in an amino acid change predicated to have severe effects on gene function. The other three mutations were predicted to have no effect. Results of gene expression patterns and grain starch content demonstrated that the novel allele in E3-1-3 altered the function of TaAGP.L-B1. Our results indicated that this mutated genetic wheat resource contained broad spectrum phenotypic and genotypic variations, that may be useful for wheat improvement, gene discovery, and functional genomics.

Chloroplast development is an integral part of plant survival and growth, and occurs in parallel with chlorophyll biosynthesis. However, little is known about the mechanisms underlying chloroplast development in hexaploid wheat. Here, we obtained a spaceflight-induced wheat albino mutant mta. Chloroplast ultra-structural observation showed that chloroplasts of mta exhibit abnormal morphology and distribution compared to wild type. Photosynthetic pigments content was also significantly decreased in mta. Transcriptome and chloroplast proteome profiling of mta and wild type were done to identify differentially expressed genes (DEGs) and proteins (DEPs), respectively. In total 4,588 DEGs including 1,980 up- and 2,608 down-regulated, and 48 chloroplast DEPs including 15 up- and 33 down-regulated were identified in mta. Classification of DEGs revealed that most were involved in chloroplast development, chlorophyll biosynthesis, or photosynthesis. Besides, transcription factors such as PIF3, GLK and MYB which might participate in those pathways were also identified. The correlation analysis between DEGs and DEPs revealed that the transcript-to-protein in abundance was functioned into photosynthesis and chloroplast relevant groups. Real time qPCR analysis validated that the expression level of genes encoding photosynthetic proteins was significantly decreased in mta. Together, our results suggest that the molecular mechanism for albino leaf color formation in mta is a thoroughly regulated and complicated process. The combined analysis of transcriptome and proteome afford comprehensive information for further research on chloroplast development mechanism in wheat. And spaceflight provides a potential means for mutagenesis in crop breeding.