Characterization of a powdery mildew resistance gene in wheat breeding line Jingzi 102 using BSR-Seq.

Wheat (Triticum aestivum L.) is one of the most important crops worldwide. Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a destructive disease threatening wheat yield and quality. The utilization of resistant genes and cultivars is considered the most economical, environmentally-friendly, and effective method to control powdery mildew. Wheat breeding line Jingzi 102 was highly resistant to powdery mildew at both seedling and adult plant stages. Genetic analysis of F1, F2, and F2:3 populations of "Jingzi 102 × Shixin 828" showed that the resistance of Jingzi 102 against powdery mildew isolate E09 at the seedling stage was controlled by a single dominant gene, temporarily designated PmJZ. Using bulked segregant RNA-Seq combined with molecular markers analysis, PmJZ was located on the long arm of chromosome 2B and flanked by markers BJK695-1 and CIT02g-20 with the genetic distances of 1.2 and 0.5 cM, respectively, corresponding to the bread wheat genome of Chinese Spring (IWGSC RefSeq v2.1) 703.8-707.6 Mb. PmJZ is most likely different from the documented Pm genes on chromosome 2BL based on their physical positions, molecular markers analysis, and resistance spectrum. Based on the gene annotation information, five genes related to disease resistance could be considered as the candidate genes of PmJZ. To accelerate the application of PmJZ, the flanking markers BJK695-1 and CIT02g-20 can serve for marker-assisted selection of PmJZ in wheat disease resistance breeding.

Genetic basis of an elite wheat cultivar Guinong 29 with harmonious improvement between multiple diseases resistance and other comprehensive traits.

Fungal diseases, such as powdery mildew and rusts, significantly affect the quality and yield of wheat. Pyramiding diverse types of resistance genes into cultivars represents the preferred strategy to combat these diseases. Moreover, achieving collaborative improvement between diseases resistance, abiotic stress, quality, and agronomic and yield traits is difficult in genetic breeding. In this study, the wheat cultivar, Guinong 29 (GN29), showed high resistance to powdery mildew and stripe rust at both seedling and adult plant stages, and was susceptible to leaf rust at the seedling stage but slow resistance at the adult-plant stage. Meanwhile, it has elite agronomic and yield traits, indicating promising coordination ability among multiple diseases resistance and other key breeding traits. To determine the genetic basis of these elite traits, GN29 was tested with 113 molecular markers for 98 genes associated with diseases resistance, stress tolerance, quality, and adaptability. The results indicated that two powdery mildew resistance (Pm) genes, Pm2 and Pm21, confirmed the outstanding resistance to powdery mildew through genetic analysis, marker detection, genomic in situ hybridization (GISH), non-denaturing fluorescence in situ hybridization (ND-FISH), and homology-based cloning; the stripe rust resistance (Yr) gene Yr26 and leaf rust resistance (Lr) genes Lr1 and Lr46 conferred the stripe rust and slow leaf rust resistance in GN29, respectively. Meanwhile, GN29 carries dwarfing genes Rht-B1b and Rht-D1a, vernalization genes vrn-A1, vrn-B1, vrn-D1, and vrn-B3, which were consistent with the phenotypic traits in dwarf characteristic and semi-winter property; carries genes Dreb1 and Ta-CRT for stress tolerance to drought, salinity, low temperature, and abscisic acid (ABA), suggesting that GN29 may also have elite stress-tolerance ability; and carries two low-molecular-weight glutenin subunit genes Glu-B3b and Glu-B3bef which contributed to high baking quality. This study not only elucidated the genetic basis of the elite traits in GN29 but also verified the capability for harmonious improvement in both multiple diseases resistance and other comprehensive traits, offering valuable information for breeding breakthrough-resistant cultivars.

Structural and physicochemical effects on the starch quality of the high-quality wheat genotype caused by delayed sowing.

Background: Bread wheat is one of the most important food crops associated with ensuring food security and human nutritional health. The starch quality is an important index of high-quality wheat. It is affected by a complex series of factors; among which, suitable sowing time is a key factor. Aim and methods: To analyze the integrative effects of sowing time on the starch quality of high-quality wheat, in the present study, we selected a high-quality bread wheat cultivar Jinan 17 and investigated the effect of different sowing times on the starch properties and the related genes by analyzing X-ray diffraction patterns, apparent amylose content, thermal properties, pasting properties, in vitro starch digestibility, and qRT-PCR. Meanwhile, we also investigated the agronomic and yield performance that may be associated with the starch properties. Results: Delayed sowing had little effect on starch crystalline morphology, but there was a tendency to reduce the formation of crystals within wheat starch granules: (1) delayed sowing for 15 days altered the thermal properties of starch, including onset, peak and termination temperatures, and enthalpy changes; (2) delayed sowing for 30 days changed the thermal characteristics of starch relatively insignificant; (3) significant differences in pasting characteristics occurred: peak viscosity and hold-through viscosity increased, while final viscosity, breakdown viscosity, and setback viscosity tended to increase and then decrease, suggesting that delayed sowing caused changes in the surface of the starch granules resulting in a decrease in digestibility. Analysis of related genes showed that several key enzymes in starch biosynthesis were significantly affected by delayed sowing, leading to a reduction in apparent straight-chain starch content. In addition to starch properties, thousand-kernel weight also increased under delayed sowing conditions compared with normal sowing. Conclusion: The impact of delayed sowing on starch quality is multifaceted and complex, from the fine structure, and functional properties of the starch to the regulation of key gene expression. Our study holds significant practical value for optimizing wheat planting management and maximizing the potential in both quality and yield.

Bulked segregant RNA-seq reveals complex resistance expression profile to powdery mildew in wild emmer wheat W762.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most destructive fungal diseases threatening global wheat production. Exploring powdery mildew resistance (Pm) gene(s) and dissecting the molecular mechanism of the host resistance are critical to effectively and reasonably control this disease. Durum wheat (Triticum turgidum L. var. durumDesf.) is an important gene donor for wheat improvement against powdery mildew. In this study, a resistant durum wheat accession W762 was used to investigate its potential resistance component(s) and profile its expression pattern in responding to Bgt invasion using bulked segregant RNA-Seq (BSR-Seq) and further qRT-PCR verification. Genetic analysis showed that the powdery mildew resistance in W762 did not meet monogenic inheritance and complex genetic model might exist within the population of W762 × Langdon (susceptible durum wheat). After BSR-Seq, 6,196 consistently different single nucleotide polymorphisms (SNPs) were called between resistant and susceptible parents and bulks, and among them, 763 SNPs were assigned to the chromosome arm 7B. Subsequently, 3,653 differentially expressed genes (DEGs) between resistant and susceptible parents and bulks were annotated and analyzed by Gene Ontology (GO), Cluster of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. The potential regulated genes were selected and analyzed their temporal expression patterns following Bgt inoculation. As a result, nine disease-related genes showed distinctive expression profile after Bgt invasion and might serve as potential targets to regulate the resistance against powdery mildew in W762. Our study could lay a foundation for analysis of the molecular mechanism and also provide potential targets for the improvement of durable resistance against powdery mildew.

Characterization of the powdery mildew resistance locus in wheat breeding line Jimai 809 and its breeding application.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a severe disease that affects the yield and quality of wheat. Popularization of resistant cultivars in production is the preferred strategy to control this disease. In the present study, the Chinese wheat breeding line Jimai 809 showed excellent agronomic performance and high resistance to powdery mildew at the whole growth stage. To dissect the genetic basis for this resistance, Jimai 809 was crossed with the susceptible wheat cultivar Junda 159 to produce segregation populations. Genetic analysis showed that a single dominant gene, temporarily designated PmJM809, conferred the resistance to different Bgt isolates. PmJM809 was then mapped on the chromosome arm 2BL and flanked by the markers CISSR02g-1 and CIT02g-13 with genetic distances 0.4 and 0.8 cM, respectively, corresponding to a physical interval of 704.12-708.24 Mb. PmJM809 differed from the reported Pm genes on chromosome arm 2BL in origin, resistance spectrum, physical position and/or genetic diversity of the mapping interval, also suggesting PmJM809 was located on a complex interval with multiple resistance genes. To analyze and screen the candidate gene(s) of PmJM809, six genes related to disease resistance in the candidate interval were evaluated their expression patterns using an additional set of wheat samples and time-course analysis post-inoculation of the Bgt isolate E09. As a result, four genes were speculated as the key candidate or regulatory genes. Considering its comprehensive agronomic traits and resistance findings, PmJM809 was expected to be a valuable gene resource in wheat disease resistance breeding. To efficiently transfer PmJM809 into different genetic backgrounds, 13 of 19 closely linked markers were confirmed to be suitable for marker-assisted selection. Using these markers, a series of wheat breeding lines with harmonious disease resistance and agronomic performance were selected from the crosses of Jimai 809 and several susceptible cultivars. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01467-8.

Evaluation and identification of powdery mildew-resistant genes in 137 wheat relatives.

Powdery mildew is one of the most severe diseases affecting wheat yield and quality and is caused by Blumeria graminis f. sp. tritici (Bgt). Host resistance is the preferred strategy to prevent this disease. However, the narrow genetic basis of common wheat has increased the demand for diversified germplasm resources against powdery mildew. Wheat relatives, especially the secondary gene pool of common wheat, are important gene donors in the genetic improvement of common wheat because of its abundant genetic variation and close kinship with wheat. In this study, a series of 137 wheat relatives, including 53 Triticum monococcum L. (2n = 2x = 14, AA), 6 T. urartu Thumanjan ex Gandilyan (2n = 2x = 14, AA), 9 T. timopheevii Zhuk. (2n = 4x = 28, AAGG), 66 T. aestivum subsp. spelta (2n = 6x = 42, AABBDD), and 3 Aegilops speltoides (2n = 2x = 14, SS) were systematically evaluated for their powdery mildew resistance and composition of Pm genes. Out of 137 (60.58%) accessions, 83 were resistant to Bgt isolate E09 at the seedling stage, and 116 of 137 (84.67%) wheat relatives were resistant to the mixture of Bgt isolates at the adult stage. This indicates that these accessions show a high level of resistance to powdery mildew. Some 31 markers for 23 known Pm genes were used to test these 137 accessions, and, in the results, only Pm2, Pm4, Pm6, Pm58, and Pm68 were detected. Among them, three Pm4 alleles (Pm4a, Pm4b, and Pm4f) were identified in 4 T. subsp. spelta accessions. q-RT PCR further confirmed that Pm4 alleles played a role in disease resistance in these four accessions. The phylogenetic tree showed that the kinship of Pm4 was close to Pm24 and Sr62. This study not only provides reference information and valuable germplasm resources for breeding new wheat varieties with disease resistance but also lays a foundation for enriching the genetic basis of wheat resistance to powdery mildew.

Evaluation and Identification of Powdery Mildew Resistance Genes in Aegilops tauschii and Emmer Wheat Accessions.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a serious threat to wheat (Triticum aestivum L.) production. Narrow genetic basis of common wheat boosted the demand for diversified donors against powdery mildew. Aegilops tauschii Coss (2n = 2x = DD) and emmer wheat (2n = 4x = AABB), as the ancestor species of common wheat, are important gene donors for genetic improvement of common wheat. In this study, a total of 71 Ae. tauschii and 161 emmer wheat accessions were first evaluated for their powdery mildew resistance using the Bgt isolate E09. Thirty-three Ae. tauschii (46.5%) and 108 emmer wheat accessions (67.1%) were resistant. Then, all these accessions were tested by the diagnostic markers for 21 known Pm genes. The results showed that Pm2 alleles were detected in all the 71 Ae. tauschii and only Pm4 alleles were detected in 20 of 161 emmer wheat accessions. After haplotype analysis, we identified four Pm4 alleles (Pm4a, Pm4b, Pm4d, and Pm4f) in the emmer wheat accessions and three Pm2 alleles (Pm2d, Pm2e, and Pm2g) in the Ae. tauschii. Further resistance spectrum analysis indicated that these resistance accessions displayed different resistance reactions to different Bgt isolates, implying they may have other Pm genes apart from Pm2 and/or Pm4 alleles. Notably, a new Pm2 allele, Pm2S, was identified in Ae. tauschii, which contained a 64-bp deletion in the first exon and formed a new termination site at the 513th triplet of the shifted reading frame compared with reported Pm2 alleles. The phylogenetic tree of Pm2S showed that the kinship of Pm2S was close to Pm2h. To efficiently and accurately detect Pm2S and distinguish with other Pm2 alleles in Ae. tauschii background, a diagnostic marker, YTU-QS-3, was developed, and its effectiveness was verified. This study provided valuable Pm alleles and enriched the genetic diversity of the powdery mildew resistance in wheat improvement.

Two functional CC-NBS-LRR proteins from rye chromosome 6RS confer differential age-related powdery mildew resistance to wheat.

Rye (Secale cereale), a valuable relative of wheat, contains abundant powdery mildew resistance (Pm) genes. Using physical mapping, transcriptome sequencing, barley stripe mosaic virus-induced gene silencing, ethyl methane sulfonate mutagenesis, and stable transformation, we isolated and validated two coiled-coil, nucleotide-binding site and leucine-rich repeat (CC-NBS-LRR) alleles, PmTR1 and PmTR3, located on rye chromosome 6RS from different triticale lines. PmTR1 confers age-related resistance starting from the three-leaf stage, whereas its allele, PmTR3, confers typical all-stage resistance, which may be associated with their differential gene expression patterns. Overexpression in Nicotiana benthamiana showed that the CC, CC-NBS, and CC-LRR fragments of PMTR1 induce cell death, whereas in PMTR3 the CC and full-length fragments perform this function. Luciferase complementation imaging and pull-down assays revealed distinct interaction activities between the CC and NBS fragments. Our study elucidates two novel rye-derived Pm genes and their derivative germplasm resources and provides novel insights into the mechanism of age-related resistance, which can aid the improvement of resistance against wheat powdery mildew.

Development of novel wheat-rye 6RS small fragment translocation lines with powdery mildew resistance and physical mapping of the resistance gene PmW6RS.

KEY MESSAGE: Novel wheat-rye 6RS small fragment translocation lines with powdery mildew resistance were developed, and the resistance gene PmW6RS was physically mapped onto 6RS-0.58-0.66-bin corresponding to 18.38 Mb in Weining rye. Rye (Secale cereale L., RR) contains valuable genes for wheat improvement. However, most of the rye resistance genes have not been successfully used in wheat cultivars. Identification of new rye resistance genes and transfer of these genes to wheat by developing small fragment translocation lines will make these genes more usable for wheat breeding. In this study, a broad-spectrum powdery mildew resistance gene PmW6RS was localized on rye chromosome arm 6RS using a new set of wheat-rye disomic and telosomic addition lines. To further study and use PmW6RS, 164 wheat-rye 6RS translocation lines were developed by 60Coγ-ray irradiation. Seedling and adult stage powdery mildew resistance analysis showed that 106 of the translocation lines were resistant. A physical map of 6RS was constructed using the 6RS translocation and deletion lines, and PmW6RS was localized in the 6RS-0.58-0.66-bin, flanked by markers X6RS-3 and X6RS-10 corresponding to the physical interval of 50.23-68.61 Mb in Weining rye genome. A total of 23 resistance-related genes were annotated. Nine markers co-segregate with the 6RS-0.58-0.66-bin, which can be used to rapidly trace the 6RS fragment carrying PmW6RS. Small fragment translocation lines with powdery mildew resistance were backcrossed with wheat cultivars, and 39 agronomically acceptable homozygous 6RS small fragment translocation lines were obtained. In conclusion, this study not only provides novel gene source and germplasms for wheat resistance breeding, but also laid a solid foundation for cloning of PmW6RS.

Fighting wheat powdery mildew: from genes to fields.

KEY MESSAGE: Host resistance conferred by Pm genes provides an effective strategy to control powdery mildew. The study of Pm genes helps modern breeding develop toward more intelligent and customized. Powdery mildew of wheat is one of the most destructive diseases seriously threatening the crop yield and quality worldwide. The genetic research on powdery mildew (Pm) resistance has entered a new era. Many Pm genes from wheat and its wild and domesticated relatives have been mined and cloned. Meanwhile, modern breeding strategies based on high-throughput sequencing and genome editing are emerging and developing toward more intelligent and customized. This review highlights mining and cloning of Pm genes, molecular mechanism studies on the resistance and avirulence genes, and prospects for genomic-assisted breeding for powdery mildew resistance in wheat.

Genetic Analysis and Molecular Identification of the Powdery Mildew Resistance in 116 Elite Wheat Cultivars/Lines.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a destructive disease worldwide. Host resistance is the preferred method for limiting the disease epidemic, protecting the environment, and minimizing economic losses. In the present study, the reactions to powdery mildew for a collection of 600 wheat cultivars and breeding lines from different wheat-growing regions were tested using the Bgt isolate E09. Next, 116 resistant genotypes were identified and then crossed with susceptible wheat cultivars/lines to produce segregating populations for genetic analysis. Among them, 87, 19, and 10 genotypes displayed single, dual, and multiple genic inheritance, respectively. To identify the Pm gene(s) in those resistant genotypes, 16 molecular markers for 13 documented Pm genes were used to test the resistant and susceptible parents and their segregating populations. Of the 87 wheat genotypes that fitted the monogenic inheritance, 75 carried the Pm2a allele. Three, two, one, and two genotypes carried Pm21, Pm6, Pm4, and the recessive genes pm6 and pm42, respectively. Four genotypes did not carry any of the tested genes, suggesting that they might have other uncharacterized or new genes. The other 29 wheat cultivars/lines carried two or more of the tested Pm genes and/or other untested genes, including Pm2, Pm5, Pm6, and/or pm42. It was obvious that Pm2 was widely used in wheat production, whereas Pm1, Pm24, Pm33, Pm34, Pm35, Pm45, and Pm47 were not detected in any of these resistant wheat genotypes. This study clarified the genetic basis of the powdery mildew resistance of these wheat cultivars/lines to provide information for their rational utilization in different wheat-growing regions. Moreover, some wheat genotypes which may have novel Pm gene(s) were mined to enrich the diversity of resistance source.

Characterization and identification of the powdery mildew resistance gene in wheat breeding line ShiCG15-009.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a serious fungal disease that critically threatens the yield and quality of wheat. Utilization of host resistance is the most effective and economical method to control this disease. In our study, a wheat breeding line ShiCG15-009, released from Hebei Province, was highly resistant to powdery mildew at all stages. To dissect its genetic basis, ShiCG15-009 was crossed with the susceptible cultivar Yannong 21 to produce F1, F2 and F2:3 progenies. After genetic analysis, a single dominant gene, tentatively designated PmCG15-009, was proved to confer resistance to Bgt isolate E09. Further molecular markers analysis showed that PmCG15-009 was located on chromosome 2BL and flanked by markers XCINAU130 and XCINAU143 with the genetic distances 0.2 and 0.4 cM, respectively, corresponding to a physic interval of 705.14-723.48 Mb referred to the Chinese Spring reference genome sequence v2.1. PmCG15-009 was most likely a new gene differed from the documented Pm genes on chromosome 2BL since its different origin, genetic diversity, and physical position. To analyze and identify the candidate genes, six genes associated with disease resistance in the candidate interval were confirmed to be associated with PmCG15-009 via qRT-PCR analysis using the parents ShiCG15-009 and Yannong 21 and time-course analysis post-inoculation with Bgt isolate E09. To accelerate the transfer of PmCG15-009 using marker-assisted selection (MAS), 18 closely or co-segregated markers were evaluated and confirmed to be suitable for tracing PmCG15-009, when it was transferred into different wheat cultivars.

Identification of a Pm4 Allele Conferring Powdery Mildew Resistance in Wheat Breeding Line GR18-1.

Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a serious fungal wheat disease of wheat worldwide. Host resistance is considered to be the most environmentally friendly and efficient approach against this disease. Wheat breeding line GR18-1 showed resistance to powdery mildew at both seedling and adult stages for several years. Genetic analysis indicated that a single dominant gene, tentatively designated as PmGR-18, conferred powdery mildew resistance in GR18-1. Bulked segregant analysis and marker analysis showed that PmGR-18 was located in the Pm4 interval on chromosome arm 2AL and was flanked by the markers Xwgrc763 and Xwgrc872, respectively, with genetic distances of 0.5 and 1.0 cM corresponding to a physical interval of 1.13 Mb based on the Chinese Spring reference genome sequence v2.1. Using homology-based cloning and Sanger sequencing, we found that the sequence of PmGR-18 was totally consistent with that of Pm4d. qRT-PCR analysis showed that the expression levels of two splicing variants Pm4d_V1 and Pm4d_V2 in GR18-1 were significantly upregulated after inoculating with Bgt isolate E09, and the level of Pm4d_V2 was significantly lower than that of Pm4d_V1 at most of the time points, suggesting a different resistance pattern may be involved in the genotype. To facilitate the transfer of PmGR-18 in marker-assisted selection (MAS) breeding, the flanked markers Xwgrc763 and Xwgrc872 and the functional marker JS717/JS718 were tested and confirmed to enable the tracking of PmGR-18 when it transferred into those susceptible cultivars.

Identification of the major QTL QPm.cas-7D for adult plant resistance to wheat powdery mildew.

Developing effective and durable host plant resistance is crucial for controlling powdery mildew, a devastating disease caused by Blumeria graminis f. sp. tritici (Bgt). In the present study, we dissected the genetic basis of the adult plant resistance to powdery mildew using a recombinant inbred line (RIL) composed of 176 F9 RILs population derived from a cross between PuBing 3228 (P3228) and susceptible cultivar Gao 8901. P3228 exhibits stable adult-plant resistance to powdery mildew in the field over consecutive years. We identified two QTLs on chromosomes 7DS (QPm.cas-7D) and 1AL (QPm.cas-1A) contributed by P3228, and one QTL on 3DS (QPm.cas-3D) contributed by Gao 8901, which could explain 65.44%, 3.45%, and 2.18% of the phenotypic variances, respectively. By analyzing the annotated genes in the 1.168 Mb physical interval of the major QTL QPm.cas-7D, we locked a previously cloned adult-plant resistance gene Pm38 that was most probably the candidate gene of QPm.cas-7D. Sequence alignment analysis revealed that the candidate gene of QPm.cas-7D in P3228 was identical to the reported Pm38 sequence. Two haplotypes QPm-7D-R and QPm-7D-S were identified in the whole Pm38 genomic regions between P3228 and Gao 8901. To apply QPm.cas-7D in wheat breeding, we developed a kompetitive allele-specific PCR (KASP) marker Kasp5249 that is closely linked with these haplotypes. It is worth mentioning that the QPm-7D-R haplotype significantly decreased TKW and underwent negative selection for higher yields in China wheat breeding. In this study, we identified a major QTL QPm.cas-7D and revealed the relationship between its resistance and yield, which could be beneficial for further applications in wheat disease resistance and high-yield breeding.

Characterization of a new splicing variant of powdery mildew resistance gene Pm4 in synthetic hexaploid wheat YAV249.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a destructive fungal disease of wheat throughout the world. Utilization of effective powdery mildew resistance genes and cultivars is considered as the most economic, efficient, and environmental-friendly method to control this disease. Synthetic hexaploid wheat (SHW), which was developed through hybridization of diploid Aegilops and tetraploid wheat, is a valuable genetic resource for resistance to powdery mildew. SHW line YAV249 showed high levels of resistance to powdery mildew at both the seedling and adult stages. Genetic analysis indicated that the resistance was controlled by a single dominant gene, temporarily designated PmYAV. Bulked segregant analysis with wheat 660K single nucleotide polymorphism (SNP) array scanning and marker analysis showed that PmYAV was located on chromosome 2AL and flanked by markers Xgdm93 and Xwgrc763, respectively, with genetic distances of 0.8 cM and 1.2 cM corresponding to a physic interval of 1.89 Mb on the Chinese Spring reference genome sequence v1.0. Sequence alignment analysis demonstrated that the sequence of PmYAV was consistent with that of Pm4a but generated an extra splicing event. When inoculated with different Bgt isolates, PmYAV showed a significantly different spectrum from Pm4a, hence it might be a new resistant resource for improvement of powdery mildew resistance. The flanked markers GDM93 and WGRC763, and the co-segregated markers BCD1231 and JS717/JS718 were confirmed to be easily performed in marker-assisted selection (MAS) of PmYAV. Using MAS strategy, PmYAV was transferred into the commercial cultivar Kenong 199 (KN199) and a wheat line YK13 was derived at generation BC3F3 from the population of YAV249/4*KN199 due to its excellent agronomic traits and resistance to powdery mildew. In conclusion, an alternative splicing variant of Pm4 was identified in this study, which informed the regulation of Pm4 gene function.

PM2b, a CC-NBS-LRR protein, interacts with TaWRKY76-D to regulate powdery mildew resistance in common wheat.

Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a destructive disease of wheat throughout the world. Host resistance is considered the most sustainable way to control this disease. Powdery mildew resistance gene Pm2b was mapped to the same genetic interval with Pm2a and PmCH1357 cloned previously, but showed different resistance spectra from them, indicating that they might be caused by different resistance genes or alleles. In this study, Pm2b was delimited to a 1.64 Mb physical interval using a large segregating population containing 4,354 F2:3 families of resistant parent KM2939 and susceptible cultivar Shimai 15. In this interval, TraesCS5D03G0111700 encoding the coiled-coil nucleotide-binding site leucine-rich repeat protein (CC-NBS-LRR) was determined as the candidate gene of Pm2b. Silencing by barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) technology and two independent mutants analysis in KM2939 confirmed the candidate gene TraesCS5D03G0111700 was Pm2b. The sequence of Pm2b was consistent with Pm2a/PmCH1357. Subcellular localization showed Pm2b was located on the cell nucleus and plasma membrane. Pm2b had the highest expression level in leaves and was rapidly up-regulated after inoculating with Bgt isolate E09. The yeast two-hybrid (Y2H) and luciferase complementation imaging assays (LCI) showed that PM2b could self-associate through the NB domain. Notably, we identified PM2b interacting with the transcription factor TaWRKY76-D, which depended on the NB domain of PM2b and WRKY domain of TaWRKY76-D. TaWRKY76-D negatively regulated the resistance to powdery mildew in wheat. The specific KASP marker K529 could take the advantage of high-throughput and high-efficiency for detecting Pm2b and be useful in molecular marker assisted-selection breeding. In conclusion, cloning and disease resistance mechanism analysis of Pm2b provided an example to emphasize a need of the molecular isolation of resistance genes, which has implications in marker assisted wheat breeding.

Genetic basis analysis of key Loci in 23 Yannong series wheat cultivars/lines.

Fungal diseases, drought, pre-harvest sprouting (PHS) and other biotic and abiotic stresses have seriously affected the quality and yield in wheat production. Identifying related genes/loci in released cultivars/lines can provide reference information and theoretical basis for wheat improvement. Yannong series wheat cultivars/lines have distinctive characteristics in wheat cultivars and play an important role in genetic improvement and production of Chinese wheat production system. To dissect their genetic basis of the stress-resistant traits, in this study, 23 representative Yannong series wheat cultivars/lines were tested by 58 molecular markers for 40 genes related to adaptability, disease resistance and stress tolerance to clarify the genetic composition of the key loci. The results showed that most of the tested wheat accessions carried dwarfing genes RhtB1b/RhtD1b/Rht8 and recessive vernalization genes vrn-A1/vrn-B1/vrn-D1/vrn-B3. It was also consistent with the phenotypic traits of tested Yannong series wheat which were dwarf and winter or semi winter wheat. In addition, the overall level of seedling powdery mildew resistance in 23 Yannong wheat cultivars/lines was moderate or inadequate. Eleven accessions carried none of the tested Pm genes and twelve accessions carried Pm2, Pm6, Pm42 and Pm52 singly or in combination. Then, 23 wheat cultivars/lines were also tested by 17 diagnostic markers for 14 Yr genes. The results showed that 16 wheat cultivars/lines were likely to carry one or more of tested Yr genes, whereas Yannong 15, Yannong 17, Yannong 23, Yannong 24, Yannong 377, Yannong 572 and Yannong 999 carried none of the tested Yr genes. Moreover, in our study, nine markers for four genes related to drought tolerance and PHS were used to evaluate the stress tolerance of the 23 wheat cultivars/lines. The results indicated that all 23 wheat cultivars/lines carried drought resistance genes Ta-Dreb1/TaCRT-D, indicating that they had the drought resistance to the extent. Except for Yannong 30, Yannong 377, Yannong 390, Yannong 745 and Yannong 1766, other wheat cultivars/lines carried one to three elite PHS-resistant alleles Vp-1Bc/Vp-1Bf/TaAFP-1Bb.

Identification of Resistant Germplasm and Detection of Genes for Resistance to Powdery Mildew and Leaf Rust from 2,978 Wheat Accessions.

Powdery mildew and leaf rust, caused by Blumeria graminis f. sp. tritici and Puccinia triticina, respectively, are widespread diseases of wheat worldwide. The use of resistant cultivars is considered the most economical, environment-friendly, and effective method to control these diseases. In the present study, a collection of 2,978 wheat accessions consisting of 1,394 advanced breeding lines, 1,078 Chinese cultivars, 291 introduced cultivars, 132 lines containing alien chromosomes, and 83 landraces was tested for reactions to powdery mildew and leaf rust. The results indicated that 659 wheat accessions (22.1%) were highly resistant to a widely prevalent B. graminis f. sp. tritici isolate, E09, at the seedling stage, and 390 were consistently resistant to the mixture of B. graminis f. sp. tritici isolates at the adult plant stage. Meanwhile, 63 accessions (2.1%) were highly resistant to leaf rust at the adult plant stage, of which 54 were resistant to a predominant and highly virulent P. triticina race, THTT, at the seedling stage. Notably, 17 accessions were resistant to both powdery mildew and leaf rust. To detect known genes for resistance to powdery mildew and leaf rust, these accessions were tested with gene-specific or tightly linked markers for seven powdery mildew genes (Pm genes; Pm2, Pm4, Pm5, Pm6, Pm8, Pm21, and Pm24) and 10 Lr genes (Lr1, Lr9, Lr10, Lr19, Lr20, Lr24, Lr26, Lr34, Lr37, and Lr46). Of the 659 powdery mildew-resistant accessions, 328 might carry single Pm genes and 191 carry combined Pm genes. Pm2 was detected at the highest frequency of 59.6%, followed by Pm8, Pm6, Pm21, Pm4, and Pm5, whereas Pm24 was not detected. In addition, 139 accessions might contain unknown Pm genes different from those tested in this study. In the 63 accessions resistant to leaf rust, four leaf rust genes (Lr genes; Lr1, Lr10, Lr26, and Lr34) were detected in 41 accessions singly or in combination, whereas six genes (Lr9, Lr19, Lr20, Lr24, Lr37, and Lr46) were not detected. Twenty-two accessions might contain unknown Lr genes different from those tested in this study. This study not only provided important information for rationally distributing resistance genes in wheat breeding programs, but also identified resistant germplasm that might have novel genes to enrich the diversity of resistance sources.

E3 ubiquitin ligase TRIM24 deficiency promotes NLRP3/caspase-1/IL-1ß-mediated pyroptosis in endometriosis.

Endometriosis is an inflammation-dependent disease that shares similarities with malignant tumors including attachment and infiltration. Tripartite motif-containing 24 (TRIM24) has been illustrated in inflammatory responses and gynecological tumors, and Nod-like receptor protein 3 (NLRP3) inflammasome has been implicated in endometriosis. However, the involvement of TRIM24 and the role of NLRP3/caspase-1/interleukin-1ß (IL-1ß)-mediated pyroptosis in endometriosis remain obscure. In this study, we originally detected the decreased expression of TRIM24 in the ectopic endometrium of endometriosis compared with the normal endometrium. Then we measured the promoted protein expression of pyroptotic biomarkers (NLRP3, procaspase-1, caspase-1, pro-IL-1ß, and IL-1ß) using Western blot analysis and the stimulated secretion of IL-1ß and IL-18 by enzyme-linked immunosorbent assay in ectopic human endometrial stromal cells (hESC) compared with normal hESC. TRIM24-small-interfering RNA (siTRIM24) was used to silence TRIM24, whereas TRIM24-pcDNA3.1 was used for overexpressing TRIM24. The migration of hESC was determined by a Transwell migration assay. Coimmunoprecipitation and ubiquitination analyses were conducted to explore the interaction between TRIM24 and NLRP3. Subsequently, we found that TRIM24 negatively regulated NLRP3/caspase-1/IL-1ß-mediated pyroptosis and cell migration of hESC, and CY-09, the specific inhibitor of NLRP3, could reverse the promoted pyroptosis and cell migration induced by siTRIM24. Furthermore, TRIM24 interacted with NLRP3 and the upregulation of TRIM24 facilitated the ubiquitination of NLRP3 in ectopic hESC. Our findings suggest that TRIM24 may participate in the progression of endometriosis through the NLRP3/caspase-1/IL-1ß-mediated pyroptotic pathway via ubiquitination of NLRP3, which reveals the significant molecular mechanism underlying endometriosis.

Identification of an Elite Wheat-Rye T1RS·1BL Translocation Line Conferring High Resistance to Powdery Mildew and Stripe Rust.

Wheat-rye T1RS·1BL translocations have been widely used worldwide in wheat production for multiple disease resistance and superior yield traits. However, many T1RS·1BL translocations have successively lost their resistance to pathogens due to the coevolution of pathogen virulence with host resistance. Because of the extensive variation in rye (Secale cereale L.) as a naturally cross-pollinating relative of wheat, it still has promise to widen the variation of 1RS and to fully realize its application value in wheat improvement. In the present study, the wheat-rye breeding line R2207 was characterized by comprehensive analyses using genomic in situ hybridization (GISH), multicolor fluorescence in situ hybridization with multiple probes, multicolor GISH, and molecular marker analysis, and then was proven to be a cytogenetically stable wheat-rye T1RS·1BL translocation line. Based on the disease responses to different isolates of powdery mildew and genetic analysis, R2207 appears to possess a novel variation for resistance, which was confirmed to be located on the rye chromosome arm 1RS. Line R2207 also exhibited high levels of resistance to stripe rust at both seedling and adult stages, as well as enhanced agronomic performance, so it has been transferred into a large number of commercial cultivars using an efficient 1RS-specific kompetitive allele specific PCR marker for marker-assisted selection.