Transcriptome analysis of Gossypium hirsutum cultivar Zhongzhimian No.2 uncovers the gene regulatory networks involved in defense against Verticillium dahliae.

BACKGROUND: Cotton is globally important crop. Verticillium wilt (VW), caused by Verticillium dahliae, is the most destructive disease in cotton, reducing yield and fiber quality by over 50% of cotton acreage. Breeding resistant cotton cultivars has proven to be an efficient strategy for improving the resistance of cotton to V. dahliae. However, the lack of understanding of the genetic basis of VW resistance may hinder the progress in deploying elite cultivars with proven resistance. RESULTS: We planted the VW-resistant Gossypium hirsutum cultivar Zhongzhimian No.2 (ZZM2) in an artificial greenhouse and disease nursery. ZZM2 cotton was subsequently subjected to transcriptome sequencing after Vd991 inoculation (6, 12, 24, 48, and 72 h post-inoculation). Several differentially expressed genes (DEGs) were identified in response to V. dahliae infection, mainly involved in resistance processes, such as flavonoid and terpenoid quinone biosynthesis, plant hormone signaling, MAPK signaling, phenylpropanoid biosynthesis, and pyruvate metabolism. Compared to the susceptible cultivar Junmian No.1 (J1), oxidoreductase activity and reactive oxygen species (ROS) production were significantly increased in ZZM2. Furthermore, gene silencing of cytochrome c oxidase subunit 1 (COX1), which is involved in the oxidation-reduction process in ZZM2, compromised its resistance to V. dahliae, suggesting that COX1 contributes to VW resistance in ZZM2. CONCLUSIONS: Our data demonstrate that the G. hirsutum cultivar ZZM2 responds to V. dahliae inoculation through resistance-related processes, especially the oxidation-reduction process. This enhances our understanding of the mechanisms regulating the ZZM2 defense against VW.

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.

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.

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.

The TaTCP4/10-B1 cascade regulates awn elongation in wheat (Triticum aestivum L.).

Awns are important morphological markers for wheat and exert a strong physiological effect on wheat yield. The awn elongation suppressor B1 has recently been cloned through association and linkage analysis in wheat. However, the mechanism of awn inhibition centered around B1 remains to be clarified. Here, we identified an allelic variant in the coding region of B1 through analysis of re-sequencing data; this variant causes an amino acid substitution and premature termination, resulting in a long-awn phenotype. Transcriptome analysis indicated that B1 inhibited awn elongation by impeding cytokinin- and auxin-promoted cell division. Moreover, B1 directly repressed the expression of TaRAE2 and TaLks2, whose orthologs have been reported to promote awn development in rice or barley. More importantly, we found that TaTCP4 and TaTCP10 synergistically inhibited the expression of B1, and a G-to-A mutation in the B1 promoter attenuated its inhibition by TaTCP4/10. Taken together, our results reveal novel mechanisms of awn development and provide genetic resources for trait improvement in wheat.

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.

Pinb-D1p is an elite allele for improving end-use quality in wheat (Triticum aestivum L.).

KEY MESSAGE: We identified ten QTLs controlling SDS-SV trait in a RIL population derived from ND3331 and Zang1817. Pinb-D1p is an elite allele from Tibetan semi­wild wheat for good end-use quality. Gluten strength is an important factor for wheat processing and end-product quality and is commonly characterized using the sodium dodecyl sulfate-sedimentation volume (SDS-SV) test. The objective of this study was to identify quantitative trait loci (QTLs) associated with wheat SDS-SV traits using a recombinant inbred line (RIL) population derived from common wheat line NongDa3331 (ND3331) and Tibetan semi-wild wheat accession Zang1817. We detected 10 QTLs controlling SDS-SV on chromosomes 1A, 1B, 3A, 4A, 4B, 5A, 5D, 6B and 7A, with individual QTLs explaining 2.02% to 15.53% of the phenotypic variation. They included four major QTLs, Qsdss-1A, Qsdss-1B.1, Qsdss-1B.2, and Qsdss-5D, whose effects on SDS-SV were due to the Glu-A1 locus encoding the high-molecular-weight glutenin subunit 1Ax1, the 1B/1R translocation, 1Bx7 + 1By8 at the Glu-B1 locus, and the hardness-controlling loci Pina-D1 and Pinb-D1, respectively. We developed KASP markers for the Glu-A1, Glu-B1, and Pinb-D1 loci. Importantly, we showed for the first time that the hardness allele Pinb-D1p positively affects SDS-SV, making it a good candidate for wheat quality improvement. These results broaden our understanding of the genetic characterization of SDS-SV, and the QTLs identified are potential target regions for fine-mapping and marker-assisted selection in wheat breeding programs.

Dispersed emergence and protracted domestication of polyploid wheat uncovered by mosaic ancestral haploblock inference.

Major crops are all survivors of domestication bottlenecks. Studies have focused on the genetic loci related to the domestication syndrome, while the contribution of ancient haplotypes remains largely unknown. Here, an ancestral genomic haploblock dissection method is developed and applied to a resequencing dataset of 386 tetraploid/hexaploid wheat accessions, generating a pan-ancestry haploblock map. Together with cytoplastic evidences, we reveal that domesticated polyploid wheat emerged from the admixture of six founder wild emmer lineages, which contributed the foundation of ancestral mosaics. The key domestication-related loci, originated over a wide geographical range, were gradually pyramided through a protracted process. Diverse stable-inheritance ancestral haplotype groups of the chromosome central zone are identified, revealing the expanding routes of wheat and the trends of modern wheat breeding. Finally, an evolution model of polyploid wheat is proposed, highlighting the key role of wild-to-crop and interploidy introgression, that increased genomic diversity following bottlenecks introduced by domestication and polyploidization.

Unprocessed wheat γ-gliadin reduces gluten accumulation associated with the endoplasmic reticulum stress and elevated cell death.

Along with increasing demands for high yield, elite processing quality and improved nutrient value in wheat, concerns have emerged around the effects of gluten in wheat-based foods on human health. However, knowledge of the mechanisms regulating gluten accumulation remains largely unexplored. Here we report the identification and characterization of a wheat low gluten protein 1 (lgp1) mutant that shows extremely low levels of gliadins and glutenins. The lgp1 mutation in a single γ-gliadin gene causes defective signal peptide cleavage, resulting in the accumulation of an excessive amount of unprocessed γ-gliadin and a reduced level of gluten, which alters the endoplasmic reticulum (ER) structure, forms the autophagosome-like structures, leads to the delivery of seed storage proteins to the extracellular space and causes a reduction in starch biosynthesis. Physiologically, these effects trigger ER stress and cell death. This study unravels a unique mechanism that unprocessed γ-gliadin reduces gluten accumulation associated with ER stress and elevated cell death in wheat. Moreover, the reduced gluten level in the lgp1 mutant makes it a good candidate for specific diets for patients with diabetes or kidney diease.

Identification of a novel major QTL from Chinese wheat cultivar Ji5265 for Fusarium head blight resistance in greenhouse.

KEY MESSAGE: A novel major QTL for FHB resistance was mapped to a 6.8 Mb region on chromosome 2D in a Chinese wheat cultivar Ji5265, and diagnostic KASP markers were developed for detecting it in a worldwide wheat collection. Fusarium head blight (FHB) is a serious disease in wheat (Triticum aestivum L.) and causes significant reductions in grain yield and quality worldwide. Breeding for FHB resistance is the most effective strategy to minimize the losses caused by FHB; therefore, identification of major quantitative trait loci (QTLs) conferring FHB resistance and development of diagnostic markers for the QTLs are prerequisites for marker-assisted selection (MAS). Ji5265 is a Chinese wheat cultivar resistant to FHB in multiple environments. An F6 population of 179 recombinant inbred lines (RILs) was developed from Ji5265 × Wheaton. The population was genotyped by genotyping-by-sequencing (GBS) and phenotyped for FHB Type II resistance in greenhouses. A major QTL, designated as QFhb-2DL, was mapped in a 6.8 Mb region between the markers GBS10238 and GBS12056 on the long arm of chromosome 2D in Ji5265 and explained ~ 30% of the phenotypic variation for FHB resistance. The effect of QFhb-2DL on FHB resistance was validated using near-isogenic lines (NILs) derived from residual heterozygotes from an F6 RIL of Ji5265 × Wheaton. The two flanking markers were converted into Kompetitive allele-specific PCR (KASP) markers (KASP10238 and KASP12056) and validated to be diagnostic in a collection of 2,065 wheat accessions. These results indicate that QFhb-2DL is a novel major QTL for resistance to FHB spread within a spike (Type II) and the two KASP markers can be used for MAS to improve wheat FHB resistance in wheat breeding programs.

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.

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 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.

Identification and characterization of QTL for spike morphological traits, plant height and heading date derived from the D genome of natural and resynthetic allohexaploid wheat.

KEY MESSAGE: QHd.cau-7D.1 for heading date was delimited into the physical interval of approximately 17.38 Mb harboring three CONSTANS-like zinc finger genes. Spike morphological traits, plant height and heading date play important roles in yield improvement of wheat. To reveal the genetic factors that controlling spike morphological traits, plant height and heading date on the D genome, we conducted analysis of quantitative traits locus (QTL) using 198 F7:8 recombinant inbred lines (RILs) derived from a cross between the common wheat TAA10 and resynthesized allohexaploid wheat XX329 with similar AABB genomes. A total of 23 environmentally stable QTL on the D sub-genome for spike length (SL), fertile spikelet number per spike (FSN), sterile spikelet number per spike (SSN), total spikelet number per spike (TSN), spike compactness (SC), plant height (PHT) and heading date (HD) were detected, among which eight appeared to be novel QTL. Furthermore, QHd.cau-7D.1 and QPht.cau-7D.2 shared identical confidence interval and were delimited into the physical interval of approximately 17.38 Mb with 145 annotated genes, including three CONSTANS-like zinc finger genes (TraesCS7D02G209000, TraesCS7D02G213000 and TraesCS7D02G220300). This study will help elucidate the molecular mechanism of the seven traits (SL, FSN, SSN, TSN, SC, PHT and HD) and provide a potentially valuable resource for genetic improvement.

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.

QMrl-7B Enhances Root System, Biomass, Nitrogen Accumulation and Yield in Bread Wheat.

Genetic improvement of root systems is an efficient approach to improve yield potential and nitrogen use efficiency (NUE) of crops. QMrl-7B was a major stable quantitative trait locus (QTL) controlling the maximum root length in wheat (Triticum aestivum L). Two types of near isogenic lines (A-NILs with superior and B-NILs with inferior alleles) were used to specify the effects of QMrl-7B on root, grain output and nitrogen-related traits under both low nitrogen (LN) and high nitrogen (HN) environments. Trials in two consecutive growing seasons showed that the root traits, including root length (RL), root area (RA) and root dry weight (RDW), of the A-NILs were higher than those of the B-NILs at seedling stage (SS) before winter, jointing stage (JS), 10 days post anthesis (PA10) and maturity (MS), respectively. Under the LN environment, in particular, all the root traits showed significant differences between the two types of NILs (p < 0.05). In contrast, there were no critical differences in aerial biomass and aerial N accumulation (ANA) between the two types of NILs at SS and JS stages. At PA10 stage, the aerial biomass and ANA of the A-NILs were significantly higher than those of the B-NILs under both LN and HN environments (p < 0.05). At MS stage, the A-NILs also exhibited significantly higher thousand-grain weight (TGW), plot grain yield, harvest index (HI), grain N accumulation (GNA), nitrogen harvest index (NHI) and nitrogen partial factor productivity (NPFP) than the B-NILs under the corresponding environments (p < 0.05). In summary, the QMrl-7B A-NILs manifested larger root systems compared to the B-NILs which is favorable to N uptake and accumulation, and eventually enhanced grain production. This research provides valuable information for genetic improvement of root traits and breeding elite wheat varieties with high yield potential and NPFP.