Molecular characterization and validation of adult-plant stripe rust resistance gene Yr86 in Chinese wheat cultivar Zhongmai 895.

KEY MESSAGE: Adult-plant stripe rust resistance gene Yr86 in Chinese wheat cultivar Zhongmai 895 was mapped to the physical interval 710.2-713.2 Mb on the long arm of chromosome 2A. Adult-plant resistance to stripe rust is generally more durable than all-stage resistance. Chinese wheat cultivar Zhongmai 895 showed stable stripe rust resistance at the adult-plant stage. To map the genetic loci underlying its resistance, 171 doubled haploid (DH) lines from a Yangmai 16/Zhongmai 895 cross were genotyped with the wheat 660 K SNP chip. Disease severities of the DH population and parents were assessed in four environments. A major QTL designated QYryz.caas-2AL was mapped to interval 703.7-715.3 Mb on the long arm of chromosome 2A using both chip-based and KASP (kompetitive allele-specific PCR) marker-based methods, explaining 31.5 to 54.1% of the phenotypic variances. The QTL was further validated in an F2 population of cross Emai 580/Zhongmai 895 with 459 plants and a panel of 240 wheat cultivars using KASP markers. Three reliable KASP markers predicted a low frequency (7.2-10.5%) of QYryz.caas-2AL in the test panel and remapped the gene to the physical interval 710.2-713.2 Mb. Based on different physical positions or genetic effects from known genes or QTL on chromosome arm 2AL, the gene was predicted to be a new one for adult-plant stripe rust resistance and was named Yr86. Twenty KASP markers linked to Yr86 were developed in this study based on wheat 660 K SNP array and genome re-sequencing. Three of them are significantly associated with stripe rust resistance in natural population. These markers should be useful for marker-assisted selection and also provide a starting point for fine mapping and map-based cloning of the new resistance gene.

Multiple roles of wheat calmodulin genes during stress treatment and TaCAM2-D as a positive regulator in response to drought and salt tolerance.

Calmodulin (CaM) and calmodulin-like (CML) proteins are the most prominent calcium (Ca2+) sensing proteins involved in Ca2+-signaling processes. However, the function of these calcium sensors in wheat remains unclear. In this study, 15 TaCAMs and 113 TaCMLs were identified from the wheat reference genome. The analysis of cis-acting elements and expression patterns showed that TaCAMs might play an important role in response to abiotic and biotic stresses. TaCAM2-D gene was found to be significantly upregulated under drought and salt stresses, and thus, it was selected to further explore the biological function. Moreover, TaCAM2-D was observed to be localized in the nucleus, membrane and cytoplasm. Overexpression of TaCAM2-D in Arabidopsis conferred greater tolerance to drought and salt. The prediction analysis, the yeast two-hybrid analysis, and bimolecular fluorescence complementation assay indicated that TaCAM2-D interacted with TaMPK8, which is one of the wheat mitogen-activated protein kinases. Thus, the current study provides insights into the understanding of the TaCAM and TaCML genes in wheat.

Investigation and Genome-Wide Association Analysis of Fusarium Seedling Blight Resistance in Chinese Elite Wheat Lines.

Fusarium seedling blight (FSB) is an important disease of wheat occurring as part of the Fusarium disease complex consisting also of Fusarium head blight (FHB). 240 Chinese elite cultivars and lines were evaluated in greenhouse experiments for FSB resistance and genotyped using the wheat 90 K single nucleotide polymorphism arrays. Among them, 23 accessions had an average lesion length of less than 0.6 cm, exhibiting potential for breeding for FSB resistance in wheat. Jingfumai 1 and Yangmai 11 had a relatively high resistance to both FSB and FHB simultaneously. Six relatively stable quantitative trait loci (QTLs) were detected on chromosome arms 1DL, 3AS, 3BL, 6BL, 7AL, and Un using the mixed linear model approach, interpreting 4.83-7.53% of phenotypic variation. There was a negative correlation between the average FSB lesion length and the BLUE FHB index with a low coefficient, and resistance to both diseases appeared to be conferred by different QTLs across the same population. Four KASP markers were detected on 1DL, 3AS, 3BL, and 6BL in QTLs to facilitate marker-assisted selection. Combined with transcriptome data analysis, eight defense-related genes were considered as candidates for mapping QTLs. The resistant elite germplasm, mapped QTLs, and KASP markers developed in this study are useful resources for enhancing Fusarium seedling blight in wheat breeding.

Effects of three fertilizer application patterns on wheat sowed by machinery in slope cropland of Western Hubei, China.

Unscientific fertilization, unstable grain quality, and low profit are the key problems on wheat production in slope cropland of Western Hubei. To solve these problems, three optimized planting patterns (high nitrogen and potassium reduction, HNPR; medium nitrogen and potassium reductionm, MNPR; low nitrogen and potassium reduction, LNPR) were conducted during two consecutive years to assess their effects on wheat yield, quality, profit, and fertilizer use efficiency in Danjiangkou Reservoir area, a typical slope cropland region with wheat-maize rotation. The results showed that the application of chemical fertilizer significantly increased grain yield (GY) and wet gluten content (WGC) of wheat. Compared with the conventional planting pattern (CK), the partial factor productivity (PFPK) and agricultural fertilizer use efficiency (AFUEK) of potassium were significantly improved in the three optimized planting patterns. The dry matter amount (DMA), GY, and crude protein content (CPC) were the highest under HNPR, which increased by 9.4%, 19.4%, and 7.8% than CK, respectively. Such a result indicated that HNPR benefited wheat to exploit high yield potential. WGC and falling number (FN) were the highest under MNPR, and increased by 3.9%, and 9.3% than CK, respectively, which was suitable for high-efficiency production of medium-gluten wheat. PFPN, AFUEN, PFPK, AFUEK, and net profit were the highest under LNPR, which increased by 15.7%, 134.1%, 131.3%, 368.2%, and 37.3% than CK, respectively, while the CPC and WGC were decreased by 2.1% and 2.6% than CK, respectively, suggesting it was suitable for environment-friendly and simplified production of weak-gluten wheat production. Our results could provide a reference for wheat production in the slope cropland.

Genome-wide identification and expression analysis of ADP-ribosylation factors associated with biotic and abiotic stress in wheat (Triticum aestivum L.).

The ARF gene family plays important roles in intracellular transport in eukaryotes and is involved in conferring tolerance to biotic and abiotic stresses in plants. To explore the role of these genes in the development of wheat (Triticum aestivum L.), 74 wheat ARF genes (TaARFs; including 18 alternate transcripts) were identified and clustered into seven sub-groups. Phylogenetic analysis revealed that TaARFA1 sub-group genes were strongly conserved. Numerous cis-elements functionally associated with the stress response and hormones were identified in the TaARFA1 sub-group, implying that these TaARFs are induced in response to abiotic and biotic stresses in wheat. According to available transcriptome data and qRT-PCR analysis, the TaARFA1 genes displayed tissue-specific expression patterns and were regulated by biotic stress (powdery mildew and stripe rust) and abiotic stress (cold, heat, ABA, drought and NaCl). Protein interaction network analysis further indicated that TaARFA1 proteins may interact with protein phosphatase 2C (PP2C), which is a key protein in the ABA signaling pathway. This comprehensive analysis will be useful for further functional characterization of TaARF genes and the development of high-quality wheat varieties.

Molecular Mapping of Quantitative Trait Loci for Fusarium Head Blight Resistance in a Doubled Haploid Population of Chinese Bread Wheat.

Fusarium head blight (FHB) is a destructive disease of wheat worldwide, particularly in China. To map genetic loci underlying FHB resistance, a doubled haploid (DH) population consisting of 174 lines was developed from a cross between widely grown Chinese cultivars Yangmai 16 and Zhongmai 895. The DH population and parents were evaluated in field nurseries at Wuhan in 2016 to 2017 and 2017 to 2018 crop seasons with both spray inoculation and natural infection, and at Jingzhou in 2017 to 2018 crop season with grain-spawn inoculation. The DH lines were genotyped with a wheat 660K SNP array. The FHB index, plant height, anther extrusion, and days to anthesis were recorded and used for quantitative trait loci (QTL) analysis. Seven QTL for FHB resistance were mapped to chromosome arms 3BL, 4AS, 4BS, 4DS, 5AL, 6AL, and 6BS in at least two environments. QFhb.caas-4BS and QFhb.caas-4DS co-located with semi-dwarfing alleles Rht-B1b and Rht-D1b, respectively, and were associated with anther extrusion. The other five QTL were genetically independent of the agronomic traits, indicating their potential value when breeding for FHB resistance. Based on correlations between FHB indices and agronomic traits in this population, we concluded that increasing plant height to some extent would enhance FHB resistance, that anther extrusion had a more important role in environments with less severe FHB, and that days to anthesis were independent of the FHB response when viewed across years. PCR-based markers were developed for the 3BL and 5AL QTL, which were detected in more than three environments. The InDel marker InDel_AX-89588684 for QFhb.caas-5AL was also validated on a wheat panel, confirming its effectiveness for marker-assisted breeding for improvements in FHB resistance.

Genome-wide association analysis of stripe rust resistance in modern Chinese wheat.

BACKGROUND: Stripe rust (yellow rust) is a significant disease for bread wheat (Triticum aestivum L.) worldwide. A genome-wide association study was conducted on 240 Chinese wheat cultivars and elite lines genotyped with the wheat 90 K single nucleotide polymorphism (SNP) arrays to decipher the genetic architecture of stripe rust resistance in Chinese germplasm. RESULTS: Stripe rust resistance was evaluated at the adult plant stage in Pixian and Xindu in Sichuan province in the 2015-2016 cropping season, and in Wuhan in Hubei province in the 2013-2014, 2016-2017 and 2018-2019 cropping seasons. Twelve stable loci for stripe rust resistance were identified by GWAS using TASSEL and GAPIT software. These loci were distributed on chromosomes 1B, 1D, 2A, 2B, 3A, 3B, 4B (3), 4D, 6D, and 7B and explained 3.6 to 10.3% of the phenotypic variation. Six of the loci corresponded with previously reported genes/QTLs, including Sr2/Yr30/Lr27, while the other six (QYr.hbaas-1BS, QYr.hbaas-2BL, QYr.hbaas-3AL, QYr.hbaas-4BL.3, QYr.hbaas-4DL, and QYr.hbaas-6DS) are probably novel. The results suggest high genetic diversity for stripe rust resistance in this population. The resistance alleles of QYr.hbaas-2AS, QYr.hbaas-3BS, QYr.hbaas-4DL, and QYr.hbaas-7BL were rare in the present panel, indicating their potential use in breeding for stripe rust resistance in China. Eleven penta-primer amplification refractory mutation system (PARMS) markers were developed from SNPs significantly associated with seven mapped QTLs. Twenty-seven genes were predicted for mapped QTLs. Six of them were considered as candidates for their high relative expression levels post-inoculation. CONCLUSION: The resistant germplasm, mapped QTLs, and PARMS markers developed in this study are resources for enhancing stripe rust resistance in wheat breeding.

Novel Soil Bacterium Strain Desulfitobacterium sp. PGC-3-9 Detoxifies Trichothecene Mycotoxins in Wheat via De-Epoxidation under Aerobic and Anaerobic Conditions.

Trichothecenes are the most common mycotoxins contaminating small grain cereals worldwide. The C12,13 epoxide group in the trichothecenes was identified as a toxic group posing harm to humans, farm animals, and plants. Aerobic biological de-epoxidation is considered the ideal method of controlling these types of mycotoxins. In this study, we isolated a novel trichothecene mycotoxin-de-epoxidating bacterium, Desulfitobacterium sp. PGC-3-9, from a consortium obtained from the soil of a wheat field known for the occurrence of frequent Fusarium head blight epidemics under aerobic conditions. Along with MMYPF media, a combination of two antibiotics (sulfadiazine and trimethoprim) substantially increased the relative abundance of Desulfitobacterium species from 1.55% (aerobic) to 29.11% (aerobic) and 28.63% (anaerobic). A single colony purified strain, PGC-3-9, was isolated and a 16S rRNA sequencing analysis determined that it was Desulfitobacterium. The PGC-3-9 strain completely de-epoxidated HT-2, deoxynivalenol (DON), nivalenol and 15-acetyl deoxynivalenol, and efficiently eliminated DON in wheat grains under aerobic and anaerobic conditions. The strain PGC-3-9 exhibited high DON de-epoxidation activity at a wide range of pH (6-10) and temperature (15-50 °C) values under both conditions. This strain may be used for the development of detoxification agents in the agriculture and feed industries and the isolation of de-epoxidation enzymes.

Genome-Wide Association Analysis of Fusarium Head Blight Resistance in Chinese Elite Wheat Lines.

Fusarium head blight (FHB) is a devastating wheat disease worldwide. To decipher the genetic architecture of FHB resistance in Chinese germplasm, a Wheat Association Panel for Scab Research (WAPS) consisting of 240 leading Chinese wheat cultivars and elite lines was genotyped using the 90K single nucleotide polymorphism (SNP) arrays. The FHB response was evaluated in the field nurseries in Wuhan in Hubei Province over four consecutive years from 2014 to 2017. Five quantitative trait loci (QTL) were consistently identified on chromosome arms 1AS, 2DL, 5AS, 5AL, and 7DS using a mixed linear model (MLM), explaining 5.6, 10.3, 5.7, 5.4, and 5.6% of phenotypic variation, respectively. The QTL on 5AS, 5AL, and 7DS QTL are probably novel. The allelic frequency analysis indicated that cultivars from the Middle and Lower Yangtze River Valleys harbored more favorable alleles and were therefore more resistant than those from other regions. To facilitate in-house germplasm screening and marker-assisted selection (MAS), SNP-derived PCR markers were developed for the QTL regions on 1AS, 5AS, and 5AL QTL. In addition to the above five QTL, the WAPS population had a very low frequency of Fhb1, confirming that the gene is not widely used in Chinese wheat breeding programs. The resistant lines and molecular markers developed in this study are resources and information for enhancing FHB resistance in breeding populations by marker-assisted recurrent selection and gene stacking.

Biomass digestibility is predominantly affected by three factors of wall polymer features distinctive in wheat accessions and rice mutants.

BACKGROUND: Wheat and rice are important food crops with enormous biomass residues for biofuels. However, lignocellulosic recalcitrance becomes a crucial factor on biomass process. Plant cell walls greatly determine biomass recalcitrance, thus it is essential to identify their key factors on lignocellulose saccharification. Despite it has been reported about cell wall factors on biomass digestions, little is known in wheat and rice. In this study, we analyzed nine typical pairs of wheat and rice samples that exhibited distinct cell wall compositions, and identified three major factors of wall polymer features that affected biomass digestibility. RESULTS: Based on cell wall compositions, ten wheat accessions and three rice mutants were classified into three distinct groups each with three typical pairs. In terms of group I that displayed single wall polymer alternations in wheat, we found that three wall polymer levels (cellulose, hemicelluloses and lignin) each had a negative effect on biomass digestibility at similar rates under pretreatments of NaOH and H2SO4 with three concentrations. However, analysis of six pairs of wheat and rice samples in groups II and III that each exhibited a similar cell wall composition, indicated that three wall polymer levels were not the major factors on biomass saccharification. Furthermore, in-depth detection of the wall polymer features distinctive in rice mutants, demonstrated that biomass digestibility was remarkably affected either negatively by cellulose crystallinity (CrI) of raw biomass materials, or positively by both Ara substitution degree of non-KOH-extractable hemicelluloses (reverse Xyl/Ara) and p-coumaryl alcohol relative proportion of KOH-extractable lignin (H/G). Correlation analysis indicated that Ara substitution degree and H/G ratio negatively affected cellulose crystallinity for high biomass enzymatic digestion. It was also suggested to determine whether Ara and H monomer have an interlinking with cellulose chains in the future. CONCLUSIONS: Using nine typical pairs of wheat and rice samples having distinct cell wall compositions and wide biomass saccharification, Ara substitution degree and monolignin H proportion have been revealed to be the dominant factors positively determining biomass digestibility upon various chemical pretreatments. The results demonstrated the potential of genetic modification of plant cell walls for high biomass saccharification in bioenergy crops.

Expression, purification and antimicrobial activity of puroindoline A protein and its mutants.

Wheat puroindoline proteins, PINA and PINB, play key roles in determining wheat grain hardness as well as in defending the plant against pathogens. PINA has much greater membrane-binding property and antimicrobial activity because it contains more tryptophan residues in the unique tryptophan-rich domain (TRD). In order to obtain proteins with higher antimicrobial activity, mutants of PINA containing two or three copies of TRD, designated ABBC and ABBBC, respectively, were constructed and expressed in E. coli Rosetta-gami (DE3). Metal affinity chromatography was used to purify the soluble affinity-tagged recombinant proteins. The secondary structures of the recombinant proteins were predicted by the online program Protein Homology/analog Y Recognition Engine v2.0 and experimentally assessed using circular dichroism. Minimum inhibition concentration tests and fluorescence microscope analyses were employed to evaluate the antimicrobial activities of the mutants. The results showed that the purified recombinant ABBC was correctly folded and presented significantly higher antimicrobial activities against E. coli and S. aureus than wild-type PINA, suggesting its potential use as an antimicrobial agent. The results also confirmed that TRD is a determinant of the antimicrobial activity of PINA and demonstrated that it is feasible to enhance the antimicrobial activity of PINA by adding one copy of TRD.