Reducing brassinosteroid signalling enhances grain yield in semi-dwarf wheat.

Modern green revolution varieties of wheat (Triticum aestivum L.) confer semi-dwarf and lodging-resistant plant architecture owing to the Reduced height-B1b (Rht-B1b) and Rht-D1b alleles1. However, both Rht-B1b and Rht-D1b are gain-of-function mutant alleles encoding gibberellin signalling repressors that stably repress plant growth and negatively affect nitrogen-use efficiency and grain filling2-5. Therefore, the green revolution varieties of wheat harbouring Rht-B1b or Rht-D1b usually produce smaller grain and require higher nitrogen fertilizer inputs to maintain their grain yields. Here we describe a strategy to design semi-dwarf wheat varieties without the need for Rht-B1b or Rht-D1b alleles. We discovered that absence of Rht-B1 and ZnF-B (encoding a RING-type E3 ligase) through a natural deletion of a haploblock of about 500 kilobases shaped semi-dwarf plants with more compact plant architecture and substantially improved grain yield (up to 15.2%) in field trials. Further genetic analysis confirmed that the deletion of ZnF-B induced the semi-dwarf trait in the absence of the Rht-B1b and Rht-D1b alleles through attenuating brassinosteroid (BR) perception. ZnF acts as a BR signalling activator to facilitate proteasomal destruction of the BR signalling repressor BRI1 kinase inhibitor 1 (TaBKI1), and loss of ZnF stabilizes TaBKI1 to block BR signalling transduction. Our findings not only identified a pivotal BR signalling modulator but also provided a creative strategy to design high-yield semi-dwarf wheat varieties by manipulating the BR signal pathway to sustain wheat production.

Positional cloning and characterization reveal the role of TaSRN-3D and TaBSR1 in the regulation of seminal root number in wheat.

Seminal roots play a critical role in water and nutrient absorption, particularly in the early developmental stages of wheat. However, the genes responsible for controlling SRN in wheat remain largely unknown. Genetic mapping and functional analyses identified a candidate gene (TraesCS3D01G137200, TaSRN-3D) encoding a Ser/Thr kinase glycogen synthase kinase 3 (STKc_GSK3) that regulated SRN in wheat. Additionally, experiments involving hormone treatment, nitrate absorption and protein interaction were conducted to explore the regulatory mechanism of TaSRN-3D. Results showed that the TaSRN-3D4332 allele inhibited seminal roots initiation and development, while loss-of-function mutants showed significantly higher seminal root number (SRN). Exogenous application of epi-brassinolide could increase the SRN in a HS2-allelic background. Furthermore, chlorate sensitivity and 15N uptake assays revealed that a higher number of seminal roots promoted nitrate accumulation. TaBSR1 (BIN2-related SRN Regulator 1, orthologous to OsGRF4/GL2 in rice) acts as an interactor of TaSRN-3D and promotes TaBSR1 degradation to reduce SRN. This study provides valuable insights into understanding the genetic basis and regulatory network of SRN in wheat, highlighting their roles as potential targets for root-based improvement in wheat breeding.

ggComp enables dissection of germplasm resources and construction of a multiscale germplasm network in wheat.

Accurate germplasm characterization is a vital step for accelerating crop genetic improvement, which remains largely infeasible for crops such as bread wheat (Triticum aestivum L.), which has a complex genome that undergoes frequent introgression and contains many structural variations. Here, we propose a genomic strategy called ggComp, which integrates resequencing data with copy number variations and stratified single-nucleotide polymorphism densities to enable unsupervised identification of pairwise germplasm resource-based Identity-By-Descent (gIBD) blocks. The reliability of ggComp was verified in wheat cultivar Nongda5181 by dissecting parental-descent patterns represented by inherited genomic blocks. With gIBD blocks identified among 212 wheat accessions, we constructed a multi-scale genomic-based germplasm network. At the whole-genome level, the network helps to clarify pedigree relationship, demonstrate genetic flow, and identify key founder lines. At the chromosome level, we were able to trace the utilization of 1RS introgression in modern wheat breeding by hitchhiked segments. At the single block scale, the dissected germplasm-based haplotypes nicely matched with previously identified alleles of "Green Revolution" genes and can guide allele mining and dissect the trajectory of beneficial alleles in wheat breeding. Our work presents a model-based framework for precisely evaluating germplasm resources with genomic data. A database, WheatCompDB (http://wheat.cau.edu.cn/WheatCompDB/), is available for researchers to exploit the identified gIBDs with a multi-scale network.

A Single Amino Acid Substitution in STKc_GSK3 Kinase Conferring Semispherical Grains and Its Implications for the Origin of Triticum sphaerococcum.

Six subspecies of hexaploid wheat (Triticum aestivum) have been identified, but the origin of Indian dwarf wheat (Triticum sphaerococcum), the only subspecies with round grains, is currently unknown. Here, we isolated the grain-shape gene Tasg-D1 in T sphaerococcum via positional cloning. Tasg-D1 encodes a Ser/Thr protein kinase glycogen synthase kinase3 (STKc_GSK3) that negatively regulates brassinosteroid signaling. Expression of TaSG-D1 and the mutant form Tasg-D1 in Arabidopsis (Arabidopsis thaliana) suggested that a single amino acid substitution in the Thr-283-Arg-284-Glu-285-Glu-286 domain of TaSG-D1 enhances protein stability in response to brassinosteroids, likely leading to formation of round grains in wheat. This gain-of-function mutation has pleiotropic effects on plant architecture and exhibits incomplete dominance. Haplotype analysis of 898 wheat accessions indicated that the origin of T sphaerococcum in ancient India involved at least two independent mutations of TaSG-D1 Our results demonstrate that modest genetic changes in a single gene can induce dramatic phenotypic changes.

Quantitative trait loci for rolled leaf in a wheat EMS mutant from Jagger.

KEY MESSAGE: Two QTLs with major effects on rolled leaf trait were consistently detected on chromosomes 1A (QRl.hwwg-1AS) and 5A (QRl.hwwg-5AL) in the field experiments. Rolled leaf (RL) is a morphological strategy to protect plants from dehydration under stressed field conditions. Identification of quantitative trait loci (QTLs) underlining RL is essential to breed drought-tolerant wheat cultivars. A mapping population of 154 recombinant inbred lines was developed from the cross between JagMut1095, a mutant of Jagger, and Jagger to identify quantitative trait loci (QTLs) for the RL trait. A linkage map of 3106 cM was constructed with 1003 unique SNPs from 21 wheat chromosomes. Two consistent QTLs were identified for RL on chromosomes 1A (QRl.hwwg-1AS) and 5A (QRl.hwwg-5AL) in all field experiments. QRl.hwwg-1AS explained 24-56% of the phenotypic variation and QRl.hwwg-5AL explained up to 20% of the phenotypic variation. The combined percent phenotypic variation associated with the two QTLs was up to 61%. Analyses of phenotypic and genotypic data of recombinants generated from heterogeneous inbred families of JagMut1095 × Jagger delimited QRl.hwwg-1AS to a 6.04 Mb physical interval. This work lays solid foundation for further fine mapping and map-based cloning of QRl.hwwg-1AS.

A single nucleotide deletion in the third exon of FT-D1 increases the spikelet number and delays heading date in wheat (Triticum aestivum L.).

The spikelet number and heading date are two crucial and correlated traits for yield in wheat. Here, a quantitative trait locus (QTL) analysis was conducted in F8 recombinant inbred lines (RILs) derived from crossing two common wheats with different spikelet numbers. A total of 15 stable QTL influencing total spikelet number (TSN) and heading date (HD) were detected. Notably, FT-D1, a well-known flowering time gene in wheat, was located within the finely mapped interval of a major QTL on 7DS (QTsn/Hd.cau-7D). A causal indel of one G in the third exon of FT-D1 was significantly associated with total spikelet number and heading date. Consistently, CRISPR/Cas9 mutant lines with homozygous mutations in FT-D1 displayed an increase in total spikelet number and heading date when compared with wild type. Moreover, one simple and robust marker developed according to the polymorphic site of FT-D1 revealed that this one G indel had been preferentially selected to adapt to different environments. Collectively, these data provide further insights into the genetic basis of spikelet number and heading date, and the diagnostic marker of FT-D1 will be useful for marker-assisted pyramiding in wheat breeding.

FRIZZY PANICLE defines a regulatory hub for simultaneously controlling spikelet formation and awn elongation in bread wheat.

Grain yield in bread wheat (Triticum aestivum L.) is largely determined by inflorescence architecture. Zang734 is an endemic Tibetan wheat variety that exhibits a rare triple spikelet (TRS) phenotype with significantly increased spikelet/floret number per spike. However, the molecular basis underlying this specific spike morphology is completely unknown. Through map-based cloning, the causal genes for TRS trait in Zang734 were isolated. Furthermore, using CRISPR/Cas9-based gene mutation, transcriptome sequencing and protein-protein interaction, the downstream signalling networks related to spikelet formation and awn elongation were defined. Results showed that the null mutation in WFZP-A together with deletion of WFZP-D led to the TRS trait in Zang734. More interestingly, WFZP plays a dual role in simultaneously repressing spikelet formation gene TaBA1 and activating awn development genes, basically through the recruitments of chromatin remodelling elements and the Mediator complex. Our findings provide insights into the molecular bases by which WFZP suppresses spikelet formation but promotes awn elongation and, more importantly, define WFZP-D as a favourable gene for high-yield crop breeding.

Phenotypic characterization of the glossy1 mutant and fine mapping of GLOSSY1 in common wheat (Triticum aestivum L.).

KEY MESSAGE: A novel wax locus GLOSSY1 was finely mapped to an approximately 308.1-kbp genomic interval on chromosome 2DS of wheat. The epicuticular wax, the outermost layer of aerial organs, gives plants their bluish-white (glaucous) appearance. Epicuticular wax is ubiquitous and provides an essential protective function against environmental stresses. In this study, we identified the glossy1 mutant on the basis of its glossy glume from an EMS population in the elite wheat (Triticum aestivum L.) cultivar Jimai22. The mutant had a dramatically different profile in total wax load and composition of individual wax constituents relative to the wild type, resulting in the increased cuticle permeability of glumes. The glossy glume phenotype was controlled by a single, semidominant locus mapping to the short arm of chromosome 2D, within a 308.1-kbp genomic interval that contained ten annotated protein-coding genes. These results pave the way for an in-depth analysis of the underlying genetic basis of wax formation patterns and enrich our understanding of mechanisms regulating wax metabolism.

The semidominant mutation w5 impairs epicuticular wax deposition in common wheat (Triticum aestivum L.).

KEY MESSAGE: The semidominant EMS-induced mutant w5 affects epicuticular wax deposition and mapped to an approximately 194-kb region on chromosome 7DL. Epicuticular wax is responsible for the glaucous appearance of plants and protects against many biotic and abiotic stresses. In wheat (Triticum aestivum L.), ß-diketone is a major component of epicuticular wax in adult plants and contributes to the glaucousness of the aerial organs. In the present study, we identified an ethyl methanesulfonate-induced epicuticular wax-deficient mutant from the elite wheat cultivar Jimai22. Compared to wild-type Jimai22, the mutant lacked ß-diketone and failed to form the glaucous coating on all aerial organs. The mutant also had significantly increased in cuticle permeability, based on water loss and chlorophyll efflux. Genetic analysis indicated that the mutant phenotype is controlled by a single, semidominant gene on the long arm of chromosome 7D, which was not allelic to the known wax gene loci W1-W4, and was therefore designated W5. W5 was finely mapped to an ~ 194-kb region (flanked by the molecular markers SSR2 and STARP11) that harbored four annotated genes according to the reference genome of Chinese Spring (RefSeq v1.0). Collectively, these data will broaden the knowledge of the genetic basis underlying epicuticular wax deposition in wheat.

Pleiotropic QTL influencing spikelet number and heading date in common wheat (Triticum aestivum L.).

KEY MESSAGE: Three pleiotropic QTL regions associated with spikelet number and heading date were identified, with FT-A1 considered the candidate gene for QTspn/Hd.cau-7A. Spikelet number traits and heading date (HD) play key roles in yield improvement of wheat and its wide adaptation to different environments. Here, we used a Recombinant Inbred Lines population derived from a cross between Yi5029 (5029) and Nongda4332 (4332) to construct a high-density genetic linkage map and identify quantitative trait loci (QTL) associated with total spikelet number per spike (TSPN), fertile spikelet number per spike (FSPN), sterile spikelet number per spike (SSPN) and HD. A total of 22 environmentally stable QTL for TSPN, FSPN, SSPN and HD were identified. Notably, three pleiotropic QTL regions for TSPN and HD were detected on chromosomes 2A, 7A and 7D. The QTL associated with TSPN and HD on chromosome 7AS was designated QTspn/Hd.cau-7A. Furthermore, the candidate gene FT-A1 located in the region of QTspn/Hd.cau-7A had a single-nucleotide polymorphism (T-G) within the third exon, which might be the cause of diversity in spikelet number and HD between the two parents. Additionally, we developed a semi-thermal asymmetric reverse PCR (STARP) marker to analyze the geographical distribution and evolution of FT-A1 (T or G) alleles. This study contributes to our understanding of the molecular mechanisms of the four traits (TSPN, FSPN, SSPN and HD) and provides further insights into the genetic relationship between spikelet number traits and HD in wheat.

Dissection of genetic factors underlying grain size and fine mapping of QTgw.cau-7D in common wheat (Triticum aestivum L.).

KEY MESSAGE: Thirty environmentally stable QTL controlling grain size and/or plant height were identified, among which QTgw.cau-7D was delimited into the physical interval of approximately 4.4 Mb. Grain size and plant height (PHT) are important agronomic traits in wheat breeding. To dissect the genetic basis of these traits, we conducted a quantitative trait locus (QTL) analysis using recombinant inbred lines (RILs). In total, 30 environmentally stable QTL for thousand grain weight (TGW), grain length (GL), grain width (GW) and PHT were detected. Notably, one major pleiotropic QTL on chromosome arm 3DS explained the highest phenotypic variance for TGW, GL and PHT, and two stable QTL (QGw.cau-4B and QGw.cau-7D) on chromosome arms 4BS and 7DS contributed greater effects for GW. Furthermore, the stable QTL controlling grain size (QTgw.cau-7D and QGw.cau-7D) were delimited into the physical interval of approximately 4.4 Mb harboring 56 annotated genes. The elite NILs of QTgw.cau-7D increased TGW by 12.79-21.75% and GW by 4.10-8.47% across all three environments. Collectively, these results provide further insight into the genetic basis of TGW, GL, GW and PHT, and the fine-mapped QTgw.cau-7D will be an attractive target for positional cloning and marker-assisted selection in wheat breeding programs.

Correction to: Dissection of two quantitative trait loci with pleiotropic effects on plant height and spike length linked in coupling phase on the short arm of chromosome 2D of common wheat (Triticum aestivum L.).

Theoretical and Applied Genetics.

Dissection of two quantitative trait loci with pleiotropic effects on plant height and spike length linked in coupling phase on the short arm of chromosome 2D of common wheat (Triticum aestivum L.).

KEY MESSAGE: Two QTL with pleiotropic effects on plant height and spike length linked in coupling phase on chromosome 2DS were dissected, and diagnostic marker for each QTL was developed. Plant height (PHT) is a crucial trait related to plant architecture and yield potential, and dissection of its underlying genetic basis would help to improve the efficiency of designed breeding in wheat. Here, two quantitative trait loci (QTL) linked in coupling phase on the short arm of chromosome 2D with pleiotropic effects on PHT and spike length, QPht/Sl.cau-2D.1 and QPht/Sl.cau-2D.2, were separated and characterized. QPht/Sl.cau-2D.1 is a novel QTL located between SNP makers BS00022234_51 and BobWhite_rep_c63957_1472. QPht/Sl.cau-2D.2 is mapped between two SSR markers, SSR-2062 and Xgwm484, which are located on the same genomic interval as Rht8. Moreover, the diagnostic marker tightly linked with each QTL was developed for the haplotype analysis using diverse panels of wheat accessions. The frequency of the height-reduced allele of QPht/Sl.cau-2D.1 is much lower than that of QPht/Sl.cau-2D.2, suggesting that this novel QTL may be an attractive target for genetic improvement. Consistent with a previous study of Rht8, a significant difference in cell length was observed between the NILs of QPht/Sl.cau-2D.2. By contrast, there was no difference in cell length between NILs of QPht/Sl.cau-2D.1, indicating that the underlying molecular mechanism for these two QTL may be different. Collectively, these data provide a new example of QTL dissection, and the developed diagnostic markers will be useful in marker-assisted pyramiding of QPht/Sl.cau-2D.1 and/or QPht/Sl.cau-2D.2 with the other genes in wheat breeding.

Dissection of two quantitative trait loci with pleiotropic effects on plant height and spike length linked in coupling phase on the short arm of chromosome 2D of common wheat (Triticum aestivum L.).

KEY MESSAGE: Two QTL with pleiotropic effects on plant height and spike length linked in coupling phase on chromosome 2DS were dissected, and diagnostic marker for each QTL was developed. Plant height (PHT) is a crucial trait related to plant architecture and yield potential, and dissection of its underlying genetic basis would help to improve the efficiency of designed breeding in wheat. Here, two quantitative trait loci (QTL) linked in coupling phase on the short arm of chromosome 2D with pleiotropic effects on PHT and spike length, QPht/Sl.cau-2D.1 and QPht/Sl.cau-2D.2, were separated and characterized. QPht/Sl.cau-2D.1 is a novel QTL located between SNP makers BS00022234_51 and BobWhite_rep_c63957_1472. QPht/Sl.cau-2D.2 is mapped between two SSR markers, SSR-2062 and Xgwm484, which are located on the same genomic interval as Rht8. Moreover, the diagnostic marker tightly linked with each QTL was developed for the haplotype analysis using diverse panels of wheat accessions. The frequency of the height-reduced allele of QPht/Sl.cau-2D.1 is much lower than that of QPht/Sl.cau-2D.2, suggesting that this novel QTL may be an attractive target for genetic improvement. Consistent with a previous study of Rht8, a significant difference in cell length was observed between the NILs of QPht/Sl.cau-2D.2. By contrast, there was no difference in cell length between NILs of QPht/Sl.cau-2D.1, indicating that the underlying molecular mechanism for these two QTL may be different. Collectively, these data provide a new example of QTL dissection, and the developed diagnostic markers will be useful in marker-assisted pyramiding of QPht/Sl.cau-2D.1 and/or QPht/Sl.cau-2D.2 with the other genes in wheat breeding.

Identification and characterization of a high kernel weight mutant induced by gamma radiation in wheat (Triticum aestivum L.).

BACKGROUND: Inducing mutations are considered to be an effective way to create novel genetic variations and hence novel agronomical traits in wheat. This study was conducted to assess the genetic differences between Shi4185 and its mutant line Fu4185, produced by gamma radiation with larger grain, and to identify quantitative trait loci (QTLs) for thousand kernel weight (TKW). RESULTS: Phenotypic analysis revealed that the TKW of Fu4185 was much higher than that of Shi4185 under five different environments. At the genomic level, 110 of 2019 (5.4%) simple sequence repeats (SSR) markers showed polymorphism between Shi4185 and Fu4185. Notably, 30% (33 out of 110) polymorphic SSR markers were located on the D-genome, which was higher than the percentage of polymorphisms among natural allohexaploid wheat genotypes, indicating that mutations induced by gamma radiation could be a potential resource to enrich the genetic diversity of wheat D-genome. Moreover, one QTL, QTkw.cau-5D, located on chromosome 5DL, with Fu4185 contributing favorable alleles, was detected under different environments, especially under high temperature conditions. CONCLUSIONS: QTkw.cau-5D is an environmental stable QTL, which may be a desired target for genetic improvement of wheat kernel weight.

Epigenetic modification contributes to the expression divergence of three TaEXPA1 homoeologs in hexaploid wheat (Triticum aestivum).

Common wheat is a hexaploid species with most of the genes present as triplicate homoeologs. Expression divergences of homoeologs are frequently observed in wheat, as well as in other polyploid plants. However, the mechanisms underlying this phenomenon are poorly understood. Expansin genes play important roles in the regulation of cell size, as well as organ size. We found that all three TaEXPA1 homoeologs were silenced in seedling roots. In seedling leaves, TaEXPA1-A and TaEXPA1-D were expressed, but TaEXPA1-B was silenced. Further analysis revealed that silencing of TaEXPA1-B in leaves occurred after the formation of the hexaploid. Chromatin immunoprecipitation assays revealed that the transcriptional silencing of three TaEXPA1 homoeologs in roots was correlated with an increased level of H3K9 dimethylation and decreased levels of H3K4 trimethylation and H3K9 acetylation. Reactivation of TaEXPA1-A and TaEXPA1-D expression in leaves was correlated with increased levels of H3K4 trimethylation and H3K9 acetylation, and decreased levels of H3K9 dimethylation in their promoters, respectively. Moreover, a higher level of cytosine methylation was detected in the promoter region of TaEXPA1-B, which may contribute to its silencing in leaves. We demonstrated that epigenetic modifications contribute to the expression divergence of three TaEXPA1 homoeologs during wheat development.

TaACTIN7-D regulates plant height and grain shape in bread wheat.

Exploitation of new gene resources and genetic networks contributing to the control of crop yield-related traits, such as plant height, grain size, and shape, may enable us to breed modern high-yielding wheat varieties through molecular methods. In this study, via ethylmethanesulfonate mutagenesis, we identify a wheat mutant plant, mu-597, that shows semi-dwarf plant architecture and round grain shape. Through bulked segregant RNA-seq and map-based cloning, the causal gene for the semi-dwarf phenotype of mu-597 is located. We find that a single-base mutation in the coding region of TaACTIN7-D (TaACT7-D), leading to a Gly-to-Ser (G65S) amino acid mutation at the 65th residue of the deduced TaACT7-D protein, can explain the semi-dwarfism and round grain shape of mu-597. Further evidence shows that the G65S mutation in TaACT7-D hinders the polymerization of actin from monomeric (G-actin) to filamentous (F-actin) status while attenuates wheat responses to multiple phytohormones, including brassinosteroids, auxin, and gibberellin. Together, these findings not only define a new semi-dwarfing gene resource that can be potentially used to design plant height and grain shape of bread wheat but also establish a direct link between actin structure modulation and phytohormone signal transduction.

A natural variation in Ribonuclease H-like gene underlies Rht8 to confer "Green Revolution" trait in wheat.

Rht8 is a gibberellin-sensitive Reduced height (Rht) locus that has been widely used in crop wheat semi-dwarfing breeding. In this study, the authors reported the map-based cloning of Rht8 candidate gene, and confirmed that loss of Ribonuclease H-Like 1 (RNHL-D1) is responsible for Rht8 semi-dwarfing effect.

Ectopic expression of VRT-A2 underlies the origin of Triticum polonicum and Triticum petropavlovskyi with long outer glumes and grains.

Polish wheat (Triticum polonicum) is a unique tetraploid wheat species characterized by an elongated outer glume. The genetic control of the long-glume trait by a single semi-dominant locus, P1 (from Polish wheat), was established more than 100 years ago, but the underlying causal gene and molecular nature remain elusive. Here, we report the isolation of VRT-A2, encoding an SVP-clade MADS-box transcription factor, as the P1 candidate gene. Genetic evidence suggests that in T. polonicum, a naturally occurring sequence rearrangement in the intron-1 region of VRT-A2 leads to ectopic expression of VRT-A2 in floral organs where the long-glume phenotype appears. Interestingly, we found that the intron-1 region is a key ON/OFF molecular switch for VRT-A2 expression, not only because it recruits transcriptional repressors, but also because it confers intron-mediated transcriptional enhancement. Genotypic analyses using wheat accessions indicated that the P1 locus is likely derived from a single natural mutation in tetraploid wheat, which was subsequently inherited by hexaploid T. petropavlovskyi. Taken together, our findings highlight the promoter-proximal intron variation as a molecular basis for phenotypic differentiation, and thus species formation in Triticum plants.