Sequencing 4.3 million mutations in wheat promoters to understand and modify gene expression.

Wheat is an important contributor to global food security, and further improvements are required to feed a growing human population. Functional genetics and genomics tools can help us to understand the function of different genes and to engineer beneficial changes. In this study, we used a promoter capture assay to sequence 2-kb regions upstream of all high-confidence annotated genes from 1,513 mutagenized plants from the tetraploid wheat variety Kronos. We identified 4.3 million induced mutations with an accuracy of 99.8%, resulting in a mutation density of 41.9 mutations per kb. We also remapped Kronos exome capture reads to Chinese Spring RefSeq v1.1, identified 4.7 million mutations, and predicted their effects on annotated genes. Using these predictions, we identified 59% more nonsynonymous substitutions and 49% more truncation mutations than in the original study. To show the biological value of the promoter dataset, we selected two mutations within the promoter of the VRN-A1 vernalization gene. Both mutations, located within transcription factor binding sites, significantly altered VRN-A1 expression, and one reduced the number of spikelets per spike. These publicly available sequenced mutant datasets provide rapid and inexpensive access to induced variation in the promoters and coding regions of most wheat genes. These mutations can be used to understand and modulate gene expression and phenotypes for both basic and commercial applications, where limited governmental regulations can facilitate deployment. These mutant collections, together with gene editing, provide valuable tools to accelerate functional genetic studies in this economically important crop.

Strigolactone and abscisic acid synthesis and signaling pathways are enhanced in the wheat oligo-tillering mutant ot1.

Tiller number greatly contributes to grain yield in wheat. Using ethylmethanesulfonate mutagenesis, we previously discovered the oligo-tillering mutant ot1. The tiller number was significantly lower in ot1 than in the corresponding wild type from the early tillering stage until the heading stage. Compared to the wild type, the thousand-grain weight and grain length were increased by 15.41% and 31.44%, respectively, whereas the plant height and spike length were decreased by 26.13% and 37.25%, respectively. Transcriptomic analysis was conducted at the regreening and jointing stages to identify differential expressed genes (DEGs). Functional enrichment analysis with the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases showed differential expression of genes associated with ADP binding, transmembrane transport, and transcriptional regulation during tiller development. Differences in tiller number in ot1 led to the upregulation of genes in the strigolactone (SL) and abscisic acid (ABA) pathways. Specifically, the SL biosynthesis genes DWARF (D27), D17, D10, and MORE AXILLARY GROWTH 1 (MAX1) were upregulated by 3.37- to 8.23-fold; the SL signal transduction genes D14 and D53 were upregulated by 1.81- and 1.32-fold, respectively; the ABA biosynthesis genes 9-CIS-EPOXICAROTENOID DIOXIGENASE 3 (NCED3) and NCED5 were upregulated by 1.66- and 3.4-fold, respectively; and SNF1-REGULATED PROTEIN KINASE2 (SnRK2) and PROTEIN PHOSPHATASE 2C (PP2C) genes were upregulated by 1.30- to 4.79-fold. This suggested that the tiller number reduction in ot1 was due to alterations in plant hormone pathways. Genes known to promote tillering growth were upregulated, whereas those known to inhibit tillering growth were downregulated. For example, PIN-FORMED 9 (PIN9), which promotes tiller development, was upregulated by 8.23-fold in ot1; Ideal Plant Architecture 1 (IPA1), which inhibits tiller development, was downregulated by 1.74-fold. There were no significant differences in the expression levels of TILLER NUMBER 1 (TN1) or TEOSINTE BRANCHED 1 (TB1), indicating that the tiller reduction in ot1 was not controlled by known genes. Our findings provide valuable data for subsequent research into the genetic bases and regulatory mechanisms of wheat tillering. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01450-3.

Evaluation of altered starch mutants and identification of candidate genes responsible for starch variation in wheat.

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 < 0.05) in starch content within the population. Furthermore, two high starch mutants (JE0173 and JE0218) and two low starch mutants (JE0089 and JE0418) were selected for studying different traits. A multiple comparison test showed that significant variation in all physiological and morphological traits, with respect to the parent variety (J411) in 2019-2020 and 2020-2021. The quantitative expression of starch metabolic genes revealed that eleven genes of JE0173 and twelve genes of JE0218 had consistent expression in high starch mutant lines. Similarly, in low starch mutant lines, eleven genes of JE0089 and thirteen genes of JE0418 had consistent expression in all stages of seed development. An additional two candidate genes showed over-expression (PHO1, PUL) in the high starch mutant lines, indicating that other starch metabolic genes may also contribute to the starch biosynthesis. The overexpression of SSII, SSIII and SBEI in JE0173 may be due to presence of missense mutations in these genes and SSI also showed overexpression which may be due to 3-primer_UTR variant. These mutations can affect the other starch related genes and help to increase the starch content in this mutant line (JE0173). CONCLUSIONS: This study screened a large scale of mutant population and identified mutants, could provide useful genetic resources for the study of starch biosynthesis and genetic improvement of wheat in the future. Further study will help to understand new genes which are responsible for the fluctuation of total starch.

Genetic mapping and identification of Rht8-B1 that regulates plant height in wheat.

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.

A large-scale whole-exome sequencing mutant resource for functional genomics in wheat.

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.

Fine mapping and genetic analysis identified a C2H2-type zinc finger as a candidate gene for heading date regulation in wheat.

KEY MESSAGE: A minor-effect QTL, Qhd.2AS, that affects heading date in wheat was mapped to a genomic interval of 1.70-Mb on 2AS, and gene analysis indicated that the C2H2-type zinc finger protein gene TraesCS2A02G181200 is the best candidate for Qhd.2AS. Heading date (HD) is a complex quantitative trait that determines the regional adaptability of cereal crops, and identifying the underlying genetic elements with minor effects on HD is important for improving wheat production in diverse environments. In this study, a minor QTL for HD that we named Qhd.2AS was detected on the short arm of chromosome 2A by Bulked Segregant Analysis and validated in a recombinant inbred population. Using a segregating population of 4894 individuals, Qhd.2AS was further delimited to an interval of 0.41 cM, corresponding to a genomic region spanning 1.70 Mb (from 138.87 to 140.57 Mb) that contains 16 high-confidence genes based on IWGSC RefSeq v1.0. Analyses of sequence variations and gene transcription indicated that TraesCS2A02G181200, which encodes a C2H2-type zinc finger protein, is the best candidate gene for Qhd.2AS that influences HD. Screening a TILLING mutant library identified two mutants with premature stop codons in TraesCS2A02G181200, both of which exhibited a delay in HD of 2-4 days. Additionally, variations in its putative regulatory sites were widely present in natural accession, and we also identified the allele which was positively selected during wheat breeding. Epistatic analyses indicated that Qhd.2AS-mediated HD variation is independent of VRN-B1 and environmental factors. Phenotypic investigation of homozygous recombinant inbred lines (RILs) and F2:3 families showed that Qhd.2AS has no negative effect on yield-related traits. These results provide important cues for refining HD and therefore improving yield in wheat breeding programs and will deepen our understanding of the genetic regulation of HD in cereal plants.

Gene Mapping and Identification of a Missense Mutation in One Copy of VRN-A1 Affects Heading Date Variation in Wheat.

Heading date (HD) is an important trait for wide adaptability and yield stability in wheat. The Vernalization 1 (VRN1) gene is a key regulatory factor controlling HD in wheat. The identification of allelic variations in VRN1 is crucial for wheat improvement as climate change becomes more of a threat to agriculture. In this study, we identified an EMS-induced late-heading wheat mutant je0155 and crossed it with wide-type (WT) Jing411 to construct an F2 population of 344 individuals. Through Bulk Segregant Analysis (BSA) of early and late-heading plants, we identified a Quantitative Trait Locus (QTL) for HD on chromosome 5A. Further genetic linkage analysis limited the QTL to a physical region of 0.8 Mb. Cloning and sequencing revealed three copies of VRN-A1 in the WT and mutant lines; one copy contained a missense mutation of C changed to T in exon 4 and another copy contained a mutation in intron 5. Genotype and phenotype analysis of the segregation population validated that the mutations in VRN-A1 contributed to the late HD phenotype in the mutant. Expression analysis of C- or T-type alleles in exon 4 of the WT and mutant lines indicated that this mutation led to lower expression of VRN-A1, which resulted in the late-heading of je0155. This study provides valuable information for the genetic regulation of HD and many important resources for HD refinement in wheat breeding programs.

Agronomic Trait Analysis and Genetic Mapping of a New Wheat Semidwarf Gene Rht-SN33d.

Plant height is a key agronomic trait that is closely to the plant morphology and lodging resistance in wheat. However, at present, the few dwarf genes widely used in wheat breeding have narrowed wheat genetic diversity. In this study, we selected a semi-dwarf wheat mutant dwarf33 that exhibits decreased plant height with little serious negative impact on other agronomic traits. Genetic analysis and mutant gene mapping indicated that dwarf33 contains a new recessive semi-dwarf gene Rht-SN33d, which was mapped into ~1.3 Mb interval on the 3DL chromosome. The gibberellin metabolism-related gene TraesCS3D02G542800, which encodes gibberellin 2-beta-dioxygenase, is considered a potential candidate gene of Rht-SN33d. Rht-SN33d reduced plant height by approximately 22.4% in mutant dwarf33. Further study revealed that shorter stem cell length may be the main factor causing plant height decrease. In addition, the coleoptile length of dwarf33 was just 9.3% shorter than that of wild-type Shaannong33. These results will help to expand our understanding of new mechanisms of wheat height regulation, and obtain new germplasm for wheat improvement.

Screening of Induced Mutants Led to the Identification of Starch Biosynthetic Genes Associated with Improved Resistant Starch in Wheat.

Several health benefits are obtained from resistant starch, also known as healthy starch. Enhancing resistant starch with genetic modification has huge commercial importance. The variation of resistant starch content is narrow in wheat, in relation to which limited improvement has been attained. Hence, there is a need to produce a wheat population that has a wide range of variations in resistant starch content. In the present study, stable mutants were screened that showed significant variation in the resistant starch content. A megazyme kit was used for measuring the resistant starch content, digestible starch, and total starch. The analysis of variance showed a significant difference in the mutant population for resistant starch. Furthermore, four diverse mutant lines for resistant starch content were used to study the quantitative expression patterns of 21 starch metabolic pathway genes; and to evaluate the candidate genes for resistant starch biosynthesis. The expression pattern of 21 starch metabolic pathway genes in two diverse mutant lines showed a higher expression of key genes regulating resistant starch biosynthesis (GBSSI and their isoforms) in the high resistant starch mutant lines, in comparison to the parent variety (J411). The expression of SBEs genes was higher in the low resistant starch mutants. The other three candidate genes showed overexpression (BMY, Pho1, Pho2) and four had reduced (SSIII, SBEI, SBEIII, ISA3) expression in high resistant starch mutants. The overexpression of AMY and ISA1 in the high resistant starch mutant line JE0146 may be due to missense mutations in these genes. Similarly, there was a stop_gained mutation for PHO2; it also showed overexpression. In addition, the gene expression analysis of 21 starch metabolizing genes in four different mutants (low and high resistant starch mutants) shows that in addition to the important genes, several other genes (phosphorylase, isoamylases) may be involved and contribute to the biosynthesis of resistant starch. There is a need to do further study about these new genes, which are responsible for the fluctuation of resistant starch in the mutants.

Identification of the vernalization gene VRN-B1 responsible for heading date variation by QTL mapping using a RIL population in wheat.

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.

Functional mutation allele mining of plant architecture and yield-related agronomic traits and characterization of their effects in wheat.

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.

Novel mutant alleles of the starch synthesis gene TaSSIVb-D result in the reduction of starch granule number per chloroplast in wheat.

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.

AhDMT1, a Fe(2+) transporter, is involved in improving iron nutrition and N2 fixation in nodules of peanut intercropped with maize in calcareous soils.

Peanut (Arachis hypogaea L.) is an important legume providing edible proteins and N2 fixation. However, iron deficiency severely reduces peanut growth in calcareous soils. The maize/peanut intercropping effectively improves iron nutrition and N2 fixation of peanut under pot and field conditions on calcareous soils. However, little was known of how intercropping regulates iron transporters in peanut. We identified AhDMT1 as a Fe(2+) transporter which was highly expressed in mature nodules with stronger N2 fixation capacity. Promoter expression analysis indicated that AhDMT1 was localized in the vascular tissues of both roots and nodules in peanut. Short-term Fe-deficiency temporarily induced an AhDmt1 expression in mature nodules in contrast to roots. However, analysis of the correlation between the complex regulation pattern of AhDmt1 expression and iron nutrition status indicated that sufficient iron supply for long term was a prerequisite for keeping AhDmt1 at a high expression level in both, peanut roots and mature nodules. The AhDmt1 expression in peanut intercropped with maize under 3 years greenhouse experiments was similar to that of peanut supplied with sufficient iron in laboratory experiments. Thus, the positive interspecific effect of intercropping may supply sufficient iron to enhance the expression of AhDmt1 in peanut roots and mature nodules to improve the iron nutrition and N2 fixation in nodules. This study may also serve as a paradigm in which functionally important genes and their ecological significance in intercropping were characterized using a candidate gene approach.

Mapping and identification of a reverse mutation of Rht2 that enhances plant height and thousand grain weight in an elite wheat mutant induced by spaceflight.

As climate change continues to negatively impact our farmlands, abiotic factors like salinity and drought stress increasingly threaten global food security. The development of elite germplasms with resistance to multiple abiotic stresses is essential for breeding climate-resilient wheat cultivars. In this study, we determined that the previously reported salt-tolerant st1 mutant, obtained via spaceflight mutagenesis, may also resist to drought stress at the seedling stage. Moreover, our field trial revealed that yield-related traits including plant height, 1000-grain weight, and spike number per plant were significantly increased in st1 compared to the wild type. An F2 population of 334 individuals derived from a cross between the wild type and st1 displayed a bimodal distribution indicating that st1 plant height is controlled by a single major gene. Our Bulked Segregant Analysis and exome capture sequencing indicate that this gene is located on chromosome 4D. Further genetic linkage and gene sequence analysis suggests that a reverse mutation of Rht2 is putatively responsible for plant height variation in st1. Our genotypic and phenotypic analysis of the F2 population and F3 lines indicate that this reverse mutation significantly increases plant height and thousand grain weight but slightly decreases spike number per plant. Together, these results supply helpful information for the utilization of Rht2 in wheat breeding and provide an important material for breeding environmentally resilient, high-yield wheat varieties.

Genetic Analysis and Fine Mapping of QTL for the Erect Leaf in Mutant mths29 Induced through Fast Neutron in Wheat.

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.

Molecular evidence for phytosiderophore-induced improvement of iron nutrition of peanut intercropped with maize in calcareous soil.

Peanut/maize intercropping is a sustainable and effective agroecosystem that evidently enhances the Fe nutrition of peanuts in calcareous soils. So far, the mechanism involved in this process has not been elucidated. In this study, we unravel the effects of phytosiderophores in improving Fe nutrition of intercropped peanuts in peanut/maize intercropping. The maize ys3 mutant, which cannot release phytosiderophores, did not improve Fe nutrition of peanut, whereas the maize ys1 mutant, which can release phytosiderophores, prevented Fe deficiency, indicating an important role of phytosiderophores in improving the Fe nutrition of intercropped peanut. Hydroponic experiments were performed to simplify the intercropping system, which revealed that phytosiderophores released by Fe-deficient wheat promoted Fe acquisition in nearby peanuts and thus improved their Fe nutrition. Moreover, the phytosiderophore deoxymugineic acid (DMA) was detected in the roots of intercropped peanuts. The yellow stripe1-like (YSL) family of genes, which are homologous to maize yellow stripe 1 (ZmYS1), were identified in peanut roots. Further characterization indicated that among five AhYSL genes, AhYSL1, which was localized in the epidermis of peanut roots, transported Fe(III)-DMA. These results imply that in alkaline soil, Fe(III)-DMA dissolved by maize might be absorbed directly by neighbouring peanuts in the peanut/maize intercropping system.

Genetic analysis and mapping of dwarf gene without yield penalty in a γ-ray-induced wheat mutant.

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.

Genome-wide characterization of two homeobox families identifies key genes associated with grain-related traits in wheat.

Homeodomain proteins encoded by BEL1- and KNAT1-type genes are ubiquitously distributed across plant species and play important roles in growth and development, whereby a comprehensive investigation of their molecular interactions and potential functions in wheat is of great significance. In this study, we systematically investigated the phylogenetic relationships, gene structures, conserved domains, and cis-acting elements of 34 TaBEL and 34 TaKNAT genes in the wheat genome. Our analysis revealed these genes evolved under different selective pressures and showed variable transcript levels in different wheat tissues. Subcellular localization analysis further indicated the proteins encoded by these genes were either exclusively located in the nucleus or both in the nucleus and the cytoplasm. Additionally, a comprehensive protein-protein interaction network was constructed with representative genes in which each TaBEL or TaKNAT proteins interact with at least two partners. The evaluation of wheat mutants identified key genes, including TaBEL-5B, TaBEL-4A.4, and TaKNAT6, which are involved in grain-related traits. Finally, haplotype analysis suggests TaKNAT-6B is associated with grain-related traits and is preferentially selected among a large set of wheat accessions. Our study provides important information on BEL1- and KNAT1-type gene families in wheat, and lays the foundation for functional research in the future.

AhNRAMP1 iron transporter is involved in iron acquisition in peanut.

Peanut/maize intercropping is a sustainable and effective agroecosystem to alleviate iron-deficiency chlorosis. Using suppression subtractive hybridization from the roots of intercropped and monocropped peanut which show different iron nutrition levels, a peanut gene, AhNRAMP1, which belongs to divalent metal transporters of the natural resistance-associated macrophage protein (NRAMP) gene family was isolated. Yeast complementation assays suggested that AhNRAMP1 encodes a functional iron transporter. Moreover, the mRNA level of AhNRAMP1 was obviously induced by iron deficiency in both roots and leaves. Transient expression, laser microdissection, and in situ hybridization analyses revealed that AhNRAMP1 was mainly localized on the plasma membrane of the epidermis of peanut roots. Induced expression of AhNRAMP1 in tobacco conferred enhanced tolerance to iron deprivation. These results suggest that the AhNRAMP1 is possibly involved in iron acquisition in peanut plants.

Physiological and Differential Proteomic Analysis at Seedling Stage by Induction of Heavy-Ion Beam Radiation in Wheat Seeds.

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.