TaMADS29 interacts with TaNF-YB1 to synergistically regulate early grain development in bread wheat.

Grain development is a crucial determinant of yield and quality in bread wheat (Triticum aestivum L.). However, the regulatory mechanisms underlying wheat grain development remain elusive. Here we report how TaMADS29 interacts with TaNF-YB1 to synergistically regulate early grain development in bread wheat. The tamads29 mutants generated by CRISPR/Cas9 exhibited severe grain filling deficiency, coupled with excessive accumulation of reactive oxygen species (ROS) and abnormal programmed cell death that occurred in early developing grains, while overexpression of TaMADS29 increased grain width and 1,000-kernel weight. Further analysis revealed that TaMADS29 interacted directly with TaNF-YB1; null mutation in TaNF-YB1 caused grain developmental deficiency similar to tamads29 mutants. The regulatory complex composed of TaMADS29 and TaNF-YB1 exercises its possible function that inhibits the excessive accumulation of ROS by regulating the genes involved in chloroplast development and photosynthesis in early developing wheat grains and prevents nucellar projection degradation and endosperm cell death, facilitating transportation of nutrients into the endosperm and wholly filling of developing grains. Collectively, our work not only discloses the molecular mechanism of MADS-box and NF-Y TFs in facilitating bread wheat grain development, but also indicates that caryopsis chloroplast might be a central regulator of grain development rather than merely a photosynthesis organelle. More importantly, our work offers an innovative way to breed high-yield wheat cultivars by controlling the ROS level in developing grains.

Heat Stress Tolerance 2 confers basal heat stress tolerance in allohexaploid wheat (Triticum aestivum L.).

Heat stress substantially reduces the yield potential of wheat (Triticum aestivum L.), one of the most widely cultivated staple crops, and greatly threatens global food security in the context of global warming. However, few studies have explored the heat stress tolerance (HST)-related genetic resources in wheat. Here, we identified and fine-mapped a wheat HST locus, TaHST2, which is indispensable for HST in both the vegetative and reproductive stages of the wheat life cycle. The studied pair of near isogenic lines (NILs) exhibited diverse morphologies under heat stress, based on which we mapped TaHST2 to a 485 kb interval on chromosome arm 4DS. Under heat stress, TaHST2 confers a superior conversion rate from soluble sugars to starch in wheat grains, resulting in faster grain filling and a higher yield potential. A further exploration of genetic resources indicated that TaHST2 underwent strong artificial selection during wheat domestication, suggesting it is an essential locus for basal HST in wheat. Our findings provide deeper insights into the genetic basis of wheat HST and might be useful for global efforts to breed heat-stress-tolerant cultivars.

Cloning and Functional Analysis of TaWRI1Ls, the Key Genes for Grain Fatty Acid Synthesis in Bread Wheat.

WRINKLED1 (WRI1), an APETALA2 (AP2) transcription factor (TF), critically regulates the processes related to fatty acid synthesis, storage oil accumulation, and seed development in plants. However, the WRI1 genes remain unknown in allohexaploid bread wheat (Triticum aestivum L.). In this study, based on the sequence of Arabidopsis AtWRI1, two TaWRI1Ls genes of bread wheat, TaWRI1L1 and TaWRI1L2, were cloned. TaWRI1L2 was closely related to monocotyledons and clustered in one subgroup with AtWRI1, while TaWRI1L1 was clustered in another subgroup with AtWRI3 and AtWRI4. Both were expressed highly in the developmental grain, subcellular localized in the nucleus, and showed transcriptional activation activity. TaWRI1L2, rather than TaWRI1L1, promoted oil body accumulation and significantly increased triglyceride (TAG) content in tobacco leaves. Overexpression of TaWRI1L2 compensated for the functional loss of AtWRI1 in an Arabidopsis mutant and restored the wild-type phenotypes of seed shape, generation, and fatty acid synthesis and accumulation. Knockout of TaWRI1L2 reduced grain size, 1000 grain weight, and grain fatty acid synthesis in bread wheat. Conclusively, TaWRI1L2, rather than TaWRI1L1, was the key transcriptional factor in the regulation of grain fatty acid synthesis in bread wheat. This study lays a foundation for gene regulation and genetic manipulation of fatty acid synthesis in wheat genetic breeding programs.

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.

Identification and Validation of Stable Quantitative Trait Loci for SDS-Sedimentation Volume in Common Wheat (Triticum aestivum L.).

Sodium dodecyl sulfate-sedimentation volume is an important index to evaluate the gluten strength of common wheat and is closely related to baking quality. In this study, a total of 15 quantitative trait locus (QTL) for sodium dodecyl sulfate (SDS)-sedimentation volume (SSV) were identified by using a high-density genetic map including 2,474 single-nucleotide polymorphism (SNP) markers, which was constructed with a doubled haploid (DH) population derived from the cross between Non-gda3753 (ND3753) and Liangxing99 (LX99). Importantly, four environmentally stable QTLs were detected on chromosomes 1A, 2D, and 5D, respectively. Among them, the one with the largest effect was identified on chromosome 1A (designated as QSsv.cau-1A.1) explaining up to 39.67% of the phenotypic variance. Subsequently, QSsv.cau-1A.1 was dissected into two QTLs named as QSsv.cau-1A.1.1 and QSsv.cau-1A.1.2 by saturating the genetic linkage map of the chromosome 1A. Interestedly, favorable alleles of these two loci were from different parents. Due to the favorable allele of QSsv.cau-1A.1.1 was from the high-value parents ND3753 and revealed higher genetic effect, which explained 25.07% of the phenotypic variation, mapping of this locus was conducted by using BC3F1 and BC3F2 populations. By comparing the CS reference sequence, the physical interval of QSsv.cau-1A.1.1 was delimited into 14.9 Mb, with 89 putative high-confidence annotated genes. SSVs of different recombinants between QSsv.cau-1A.1.1 and QSsv.cau-1A.1 detected from DH and BC3F2 populations showed that these two loci had an obvious additive effect, of which the combination of two favorable loci had the high SSV, whereas recombinants with unfavorable loci had the lowest. These results provide further insight into the genetic basis of SSV and QSsv.cau-1A.1.1 will be an ideal target for positional cloning and wheat breeding programs.

Genome-wide association study of six quality-related traits in common wheat (Triticum aestivum L.) under two sowing conditions.

KEY MESSAGE: We identified genomic regions associated with six quality-related traits in wheat under two sowing conditions and analyzed the effects of multienvironment-significant SNPs on the stability of these traits. Grain quality affects the nutritional and commercial value of wheat (Triticum aestivum L.) and is a critical factor influencing consumer preferences for specific wheat varieties. Climate change is predicted to increase environmental stress and thereby reduce wheat quality. Here, we performed a genotyping assay involving the use of the wheat 90 K array in a genome-wide association study of six quality-related traits in 486 wheat accessions under two sowing conditions (normal and late sowing) over 4 years. We identified 64 stable quantitative trait loci (QTL), including 10 for grain protein content, 9 for wet gluten content, 4 for grain starch content, 14 for water absorption, 15 for dough stability time and 12 for grain hardness in wheat under two sowing conditions. These QTL harbored 175 single nucleotide polymorphisms (SNPs), explaining approximately 3-13% of the phenotypic variation in multiple environments. Some QTL on chromosomes 6A and 5D were associated with multiple traits simultaneously, and two (QNGPC.cau-6A, QNGH.cau-5D) harbored known genes, such as NAM-A1 for grain protein content and Pinb for grain hardness, whereas other QTL could facilitate gene discovery. Forty-three SNPs that were detected under late or both normal and late sowing conditions appear to be related to phenotypic stability. The effects of these SNP alleles were confirmed in the association population. The results of this study will be useful for further dissecting the genetic basis of quality-related traits in wheat and developing new wheat cultivars with desirable alleles to improve the stability of grain quality.

Over-Expressing TaSPA-B Reduces Prolamin and Starch Accumulation in Wheat (Triticum aestivum L.) Grains.

Starch and prolamin composition and content are important indexes for determining the processing and nutritional quality of wheat (Triticum aestivum L.) grains. Several transcription factors (TFs) regulate gene expression during starch and protein biosynthesis in wheat. Storage protein activator (TaSPA), a member of the basic leucine zipper (bZIP) family, has been reported to activate glutenin genes and is correlated to starch synthesis related genes. In this study, we generated TaSPA-B overexpressing (OE) transgenic wheat lines. Compared with wild-type (WT) plants, the starch content was slightly reduced and starch granules exhibited a more polarized distribution in the TaSPA-B OE lines. Moreover, glutenin and ω- gliadin contents were significantly reduced, with lower expression levels of related genes (e.g., By15, Dx2, and ω-1,2 gliadin gene). RNA-seq analysis identified 2023 differentially expressed genes (DEGs). The low expression of some DEGs (e.g., SUSase, ADPase, Pho1, Waxy, SBE, SSI, and SS II a) might explain the reduction of starch contents. Some TFs involved in glutenin and starch synthesis might be regulated by TaSPA-B, for example, TaPBF was reduced in TaSPA-B OE-3 lines. In addition, dual-luciferase reporter assay indicated that both TaSPA-B and TaPBF could transactivate the promoter of ω-1,2 gliadin gene. These results suggest that TaSPA-B regulates a complex gene network and plays an important role in starch and protein biosynthesis in wheat.

Plant-mediated gene silencing of an essential olfactory-related Gqα gene enhances resistance to grain aphid in common wheat in greenhouse and field.

BACKGROUND: Grain aphid (Sitobion avenae F.) is a dominant pest that limits cereal crop production around the globe. Gq proteins have important roles in signal transduction in insect olfaction. Plant-mediated RNA interference (RNAi) has been widely studied in insect control, but its application for the control wheat aphid in the field requires further study. Here, we used double-stranded (ds)RNA feeding to verify the potential of selected Gqα fragments for host-mediated RNAi, and then evaluated the effect of RNAi on aphid olfaction in transgenic wheat in the greenhouse and field. RESULTS: Gqα gene was expressed in the aphid life cycle, and a 540 bp fragment shared 98.1% similarity with the reported sequence. dsGqα feeding reduced the expression of Gqα, and both reproduction and molting in the grain aphid. Feeding transgenic lines in the greenhouse downregulated expression of aphid Gqα, and significantly reduced reproduction and molting numbers. Furthermore, our field results indicate that transgenic lines have lower aphid numbers and higher 1000-grain weight than an unsprayed wild-type control. CONCLUSION: Plant-mediated silencing of an essential olfactory-related Gqα gene could enhance resistance to grain aphid in common wheat in both the greenhouse and the field. © 2018 Society of Chemical Industry.

Plant-mediated RNAi of grain aphid CHS1 gene confers common wheat resistance against aphids.

BACKGROUND: Chitin is an important component of the insect exoskeleton and peritrophic membrane. Chitin synthase 1 (CHS1) is a key enzyme in the chitin synthesis pathway, and has a role in insect molting and growth. Plant-mediated RNA interference (RNAi) has been used as a more target-specific and environmentally safe approach to prevent and control agricultural insects. The aims of this study were to use grain aphid (Sitobion avanae) CHS1 as the target gene and to produce transgenic wheat lines for aphid control via plant-mediated RNAi. RESULTS: Expression levels of CHS1 changed at different developmental stages. After feeding on the representative T3 transgenic lines Tb5-2 and Tb10-3, CHS1 expression levels in grain aphid decreased by 50.29% and 45.32%, respectively; and total and molting aphid numbers reduced significantly, compared with controls. Consistent with this, aphid numbers in mixed natural populations reduced significantly in the respective T4 and T5 transgenic lines under field conditions, and T5 transgenic lines had higher grain weight compared with the unsprayed insecticide wild-type and insecticide-sprayed wild-type. CONCLUSION: These results indicate that plant-mediated RNAi of the grain aphid CHS1 gene confers common wheat resistance against aphids. © 2018 Society of Chemical Industry.

Expression of Pinellia pedatisecta Lectin Gene in Transgenic Wheat Enhances Resistance to Wheat Aphids.

Wheat aphids are major pests during the seed filling stage of wheat. Plant lectins are toxic to sap-sucking pests such as wheat aphids. In this study, Pinellia pedatisecta agglutinin (ppa), a gene encoding mannose binding lectin, was cloned, and it shared 92.69% nucleotide similarity and 94% amino acid similarity with Pinellia ternata agglutinin (pta). The ppa gene, driven by the constitutive and phloem-specific ribulose bisphosphate carboxylase small subunit gene (rbcs) promoter in pBAC-rbcs-ppa expression vector, was transferred into the wheat cultivar Baofeng104 (BF104) by particle bombardment transformation. Fifty-four T0 transgenic plants were generated. The inheritance and expression of the ppa gene were confirmed by PCR and RT-PCR analysis respectively, and seven homozygous transgenic lines were obtained. An aphid bioassay on detached leaf segments revealed that seven ppa transgenic wheat lines had lower aphid growth rates and higher inhibition rates than BF104. Furthermore, two-year aphid bioassays in isolated fields showed that aphid numbers per tiller of transgenic lines were significantly decreased, compared with wild type BF104. Therefore, ppa could be a strong biotechnological candidate to produce aphid-resistant wheat.

Differential effects of a post-anthesis heat stress on wheat (Triticum aestivum L.) grain proteome determined by iTRAQ.

Heat stress, a major abiotic stressor of wheat (Triticum aestivum L.), often results in reduced yield and decreased quality. In this study, a proteomic method, Tags for Relative and Absolute Quantitation Isobaric (iTRAQ), was adopted to analyze the protein expression profile changes among wheat cultivar Jing411 under heat stress. Results indicated that there were 256 different proteins expressed in Jing411 under heat stress. According to the result of gene annotation and functional classification, 239 proteins were annotated by 856 GO function entries, including growth and metabolism proteins, energy metabolism proteins, processing and storage proteins, defense-related proteins, signal transduction, unknown function proteins and hypothetical proteins. GO enrichment analysis suggested that the differentially expressed proteins in Jing411 under heat stress were mainly involved in stimulus response (67), abiotic stress response (26) and stress response (58), kinase activity (12), and transferase activity (12). Among the differentially expressed proteins in Jing411, 115 were attributed to 119 KEGG signaling/metabolic pathways. KEGG pathway enrichment analysis in Jing411 showed that heat stress mainly affected the starch and sucrose metabolism as well as protein synthesis pathway in the endoplasmic reticulum. The protein interaction network indicated that there were 8 differentially expressed proteins that could form an interaction network in Jing411.

Silencing of a lipase maturation factor 2-like gene by wheat-mediated RNAi reduces the survivability and reproductive capacity of the grain aphid, Sitobion avenae.

Lipase maturation factor (LMF) family proteins are required for the maturation and transport of active lipoprotein lipases. However, the specific roles of LMF2 remain unknown. In this study, a grain aphid lmf2-like gene fragment was cloned and was highly similar in sequence to a homologous gene in the pea aphid, Acyrthosiphon pisum. An RNAi vector was constructed with this fragment and used for wheat transformation. The expression of the lmf2-like gene in aphid, as well as the growth and reproduction of the aphids, was analyzed after feeding on the transgenic wheat. There were no significant differences in the expression of the lmf2-like gene over development. The expression of the lmf2-like gene was significantly reduced by 27.6% on the fifth day, and 57.6% on the 10th day after feeding. The total number of aphids produced on the transgenic plants was less than the number produced on control plants, and the difference became significant or after 2 weeks. The molting numbers were also reduced in the aphids reared on the transgenic plants. Our findings indicate that lmf2-like genes may have potential as a target gene for the control of grain aphids and show that feeding aphids with wheat expressing lmf2-like RNAi resulted in significant reductions in survival and reproduction.

Silencing of an aphid carboxylesterase gene by use of plant-mediated RNAi impairs Sitobion avenae tolerance of Phoxim insecticides.

RNA interference (RNAi) describes the ability of double-stranded RNA (dsRNA) to inhibit homologous gene expression at the RNA level. Its specificity is sequence-based and depends on the sequence of one strand of the dsRNA corresponding to part or all of a specific gene transcript. In this study we adopted plant-mediated RNAi technology that targets Sitobion avenae (S. avenae) to enable gene silencing in the aphid and to minimize handling of the insects during experiments. S. avenae was selected for this study because it causes serious economic losses to wheat throughout the world. The carboxylesterase (CbE E4) gene in S. avenae was homologously cloned, which increased synthesis of a protein known to be critical to the resistance (tolerance) this species has developed to a wide range of pesticides. A plant RNAi vector was constructed, and transgenic Triticum aestivum (dsCbE1-5 and dsCbE2-2 lines) expressing CbE E4 dsRNA were developed. S. avenae were fed on dsCbE1-5 and dsCbE2-2 lines stably producing the CbE E4 dsRNA. CbE E4 gene expression in S. avenae was reduced by up to 30-60%. The number of aphids raised on dsCbE1-5 and dsCbE2-2 was lower than the number raised on non-transgenic plants. A solution of CbE E4 enzyme from S. avenae fed on dsCbE1-5 and dsCbE2-2 plants hydrolyzed only up to 20-30% Phoxim solution within 40 min whereas a solution of the enzyme from CbE E4 fed on control plants hydrolyzed 60% of Phoxim solution within 40 min. CbE E4 gene silencing was achieved by our wheat-mediated RNAi approach. This plant-mediated RNAi approach for addressing degradation-based pesticide resistance mechanisms in aphids and may prove useful in pest management for diverse agro-ecosystems.

[Maize transformation using xylose isomerase gene as a selection marker].

The xylA gene, encoding xylose isomerase, was cloned as a 1342-bp BamHI/SacI fragment from the E. coli. As a selection marker, the xylA gene was fused between the enhanced CaMV 35S promoter (E35S) and terminator (35St) in pBAC413 (Fig.2). pBAC413 was constructed to prevent the expression of sbeIIb in maize. PDS1000/He was used to bombard maize calli, which were induced to form by the elite inbred lines. The selection was carried out on the media containing concentrations of xylose from 0 to 100%. The results showed that the media containing 50% to 100% D-xylose were better, but differed with the genotype of maize (Tables 1 and 2). Successful integration of xylA gene into the maize genome was confirmed by DNA dot blotting, PCR and PCR-Southern hybridization (Figs.4 to 6). A method was established in which transformed maize cells were successively screened on a medium containing xylose instead of antibiotic and herbicide for bio-safety.

Induced expression of DREB transcriptional factor and study on its physiological effects of drought tolerance in transgenic wheat.

Expression vector pBAC128F, which carries DREB transcriptional factor gene driven by drought inducing promoter rd29B and bar gene driven by CaMV 35S promoter and maize Adh1 gene first intron, was transferred into the explants of immature inflorescence and immature embryos of hexaploid winter wheat cv. 8901, 5-98, 99-92 and 104 by particle bombardment. More than 70 resistant transgenic plants were obtained. Genomic PCR and RNA dot blotting analyses showed that DREB gene had been integrated into wheat genome of the transgenic plants (T0 and T1) and was well expressed in offspring seed of different transgenic lines. The content of proline in leaves and seeds of T2 transgenic lines was analyzed. Among 16 tested transgenic lines, 10 transgenic lines exhibited more than two fold of proline level in leaves as compared with CK plants. Under drought condition, after stopping water for 15 days the leaves of transgenic lines were still green, while CK were faded. After rewatering for 10 days, the leaves of transgenic lines maintained their green, while all CK plants were dead. Our research suggested that introducing a novel DREB transcriptional factor into wheat is an effective way to improve its drought-tolerance ability.

[Molecular identification of wheat granule bound starch synthase gene polymorphism].

Fourteen wheat cultivars were identified into six types of Wx proteins combinations using 6% SDS-PAGE. PCR primers were designed according to the three Wx genes sequences and their mutants, respectively. A 327 bp-band was amplified from the Wx-A1 mutant,while the band was absent for the normal alleles at the Wx-A1 locus,as well as the presence or absence of a 187 bp PCR fragment at the Wx-B1 locus and a 700 bp PCR fragment at the Wx-D1 locus, respectively, corresponding to the normal and mutant alleles. Compared with the former studies, shorter and more different PCR products at three loci, amplified by the primers designed for Wx-B1 gene can be separated in 2% agarose gel, which enables screening breeding lines for noodle use faster and effectively.

Betaine improves LA-PCR amplification.

PCR is a powerful tool for the amplification of genetic sequences. It has been widely applied in molecular biology. It is generally used to amplify short segments (several hundreds basepairs to several kilobasepairs). It is difficult to amplify a long DNA segment. Based on the sequenced genes, it is known that most intact genes are very long. And intact gene is very important for the gene to express specially and effectively. Long PCR is a very useful tool to amplify intact genes for constructing special expression vectors. We have tried several chemicals to optimize long PCR system and found betaine was the best. Betaine, as an amino acid analogue with small tetraalkylammonium ions, could remarkably improve the amplification of long targets from the plant genome. The suitable concentration of betaine was between 1.0 mol/L and 2.5mol/L. We could effectively amplify a 9 kb DNA segment from maize genome DNA and a 16 kb DNA segment from plasmid. It was shown that different primers and different targets (different GC content) needed different concentrations of betaine. Betaine can reduce or eliminate non-special amplification. In the meantime we tried other additive chemicals, such as DMSO, glycerin, formamide. They were no notable results in long PCR.

[Genetic diversity of D-genome revealed by SSR markers in synthesized hexaploid wheat introduced from CIMMYT].

Simple sequence repeat (SSR) molecular marker was used to measure the genetic diversity of D-genome in 26 synthesized hexaploid wheat (AABBDD) introduced from CIMMYT. Twenty-three D-genome specific SSR primers were selected for PCR amplification, among which 22 primers can detect polymorphism. A total of 92 alleles were identified at 23 loci using the above SSR primers, with an average of 4 alleles per locus. The 92 alleles were used to calculate Nei's similarity index (GS) and the genetic distance (GD). It was also found that the mean genetic distance between 26 synthesized hexaploid wheat was 0.4955, which was obviously high. From the above results, it can be indicated that the genetic variation of D-genome in synthesized hexaploid wheat was abundant and could be used to improve the genetic diversity in wheat breeding. Interestingly, synthesized hexaploid wheat 17 and 18 shared the same D-genome donor, but three of 23 detected SSR loci were polymorphic between the two materials. Therefore, during the period of allopolyploidization, there was genetic differentiation in repeat region of donor genome.