A membrane associated tandem kinase from wild emmer wheat confers broad-spectrum resistance to powdery mildew.

Crop wild relatives offer natural variations of disease resistance for crop improvement. Here, we report the isolation of broad-spectrum powdery mildew resistance gene Pm36, originated from wild emmer wheat, that encodes a tandem kinase with a transmembrane domain (WTK7-TM) through the combination of map-based cloning, PacBio SMRT long-read genome sequencing, mutagenesis, and transformation. Mutagenesis assay reveals that the two kinase domains and the transmembrane domain of WTK7-TM are critical for the powdery mildew resistance function. Consistently, in vitro phosphorylation assay shows that two kinase domains are indispensable for the kinase activity of WTK7-TM. Haplotype analysis uncovers that Pm36 is an orphan gene only present in a few wild emmer wheat, indicating its single ancient origin and potential contribution to the current wheat gene pool. Overall, our findings not only provide a powdery mildew resistance gene with great potential in wheat breeding but also sheds light into the mechanism underlying broad-spectrum resistance.

Cloning of the broadly effective wheat leaf rust resistance gene Lr42 transferred from Aegilops tauschii.

The wheat wild relative Aegilops tauschii was previously used to transfer the Lr42 leaf rust resistance gene into bread wheat. Lr42 confers resistance at both seedling and adult stages, and it is broadly effective against all leaf rust races tested to date. Lr42 has been used extensively in the CIMMYT international wheat breeding program with resulting cultivars deployed in several countries. Here, using a bulked segregant RNA-Seq (BSR-Seq) mapping strategy, we identify three candidate genes for Lr42. Overexpression of a nucleotide-binding site leucine-rich repeat (NLR) gene AET1Gv20040300 induces strong resistance to leaf rust in wheat and a mutation of the gene disrupted the resistance. The Lr42 resistance allele is rare in Ae. tauschii and likely arose from ectopic recombination. Cloning of Lr42 provides diagnostic markers and over 1000 CIMMYT wheat lines carrying Lr42 have been developed documenting its widespread use and impact in crop improvement.

Identifying features of source and message that influence the retweeting of health information on social media during the COVID-19 pandemic.

BACKGROUND: Social media has become an essential tool to implement risk communication, giving health information could gain more exposure by retweeting during the COVID-19 pandemic. METHODS: Content analysis was conducted to scrutinize the official (national and provincial) public health agencies' Weibo posts (n = 4396) to identify features of information sources and message features (structure, style content). The Zero-Inflated Negative Binomial (ZINB) model was adopted to analyze the association between these features and the frequency of the retweeted messages. RESULTS: Results indicated that features of source and health information, such as structure, style, and content, were correlated to retweeting. The results of IRR further suggested that compared to provincial accounts, messages from national health authorities' accounts gained more retweeting. Regarding the information features, messages with hashtags#, picture, video have been retweeted more often than messages without any of these features respectively, while messages with hyperlinks received fewer retweets than messages without hyperlinks. In terms of the information structure, messages with the sentiment (!) have been retweeted more frequently than messages without sentiment. Concerning content, messages containing severity, reassurance, efficacy, and action frame have been retweeted with higher frequency, while messages with uncertainty frames have been retweeted less often. CONCLUSIONS: Health organizations and medical professionals should pay close attention to the features of health information sources, structures, style, and content to satisfy the public's information needs and preferences to promote the public's health engagement. Designing suitable information systems and promoting health communication strategies during different pandemic stages may improve public awareness of the COVID-19, alleviate negative emotions, and promote preventive measures to curb the spread of the virus.

Functional characterization of powdery mildew resistance gene MlIW172, a new Pm60 allele and its allelic variation in wild emmer wheat.

Wild emmer wheat (Triticum dicoccoides, WEW) is an immediate progenitor of both the cultivated tetraploid and hexaploid wheats and it harbors rich genetic diversity against powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt). A powdery mildew resistance gene MlIW172 originated from WEW accession IW172 (G-797-M) is fine mapped in a 0.048 centimorgan (cM) genetic interval on 7AL, corresponding to a genomic region spanning 233 kb, 1 Mb and 800 kb in Chinese Spring, WEW Zavitan, and T. urartu G1812, respectively. MlIW172 encodes a typical NLR protein NLRIW172 and physically locates in an NBS-LRR gene cluster. NLRIW172 is subsequently identified as a new allele of Pm60, and its function is validated by EMS mutagenesis and transgenic complementation. Haplotype analysis of the Pm60 alleles reveals diversifications in sequence variation in the locus and presence and absence variations (PAV) in WEW populations. Four common single nucleotide variations (SNV) are detected between the Pm60 alleles from WEW and T. urartu, indicative of speciation divergence between the two different wheat progenitors. The newly identified Pm60 alleles and haplotypes in WEW are anticipated to be valuable for breeding powdery mildew resistance wheat cultivars via marker-assisted selection.

Fine mapping of powdery mildew resistance gene MlWE74 derived from wild emmer wheat (Triticum turgidum ssp. dicoccoides) in an NBS-LRR gene cluster.

KEY MESSAGE: Powdery mildew resistance gene MlWE74, originated from wild emmer wheat accession G-748-M, was mapped in an NBS-LRR gene cluster of chromosome 2BS. Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a globally devastating disease. Wild emmer wheat (Triticum turgidum var. dicoccoides) is a valuable genetic resource for improving disease resistance in common wheat. A powdery mildew resistance gene was transferred to hexaploid wheat line WE74 from wild emmer accession G-748-M. Genetic analysis revealed that the powdery mildew resistance in WE74 is controlled by a single dominant gene, herein temporarily designated MlWE74. Bulked segregant analysis (BSA) and molecular mapping delimited MlWE74 to the terminal region of chromosome 2BS flanking by markers WGGBD412 and WGGBH346 within a genetic interval of 0.25 cM and corresponding to 799.9 kb genomic region in the Zavitan reference sequence. Sequence annotation revealed two phosphoglycerate mutase-like genes, an alpha/beta-hydrolases gene, and five NBS-LRR disease resistance genes that could serve as candidates for map-based cloning of MlWE74. The geographical location analysis indicated that MlWE74 is mainly distributed in Rosh Pinna and Amirim regions, in the northern part of Israel, where environmental conditions are favorable to the occurrence of powdery mildew. Moreover, the co-segregated marker WGGBD425 is helpful in marker-assisted transfer of MlWE74 into elite cultivars.

Molecular Characterization of All-Stage and Adult-Plant Resistance Loci Against Powdery Mildew in Winter Wheat Cultivar Liangxing 99 Using BSR-Seq Technology.

Winter wheat cultivar Liangxing 99, which carries gene Pm52, is resistant to powdery mildew at both seedling and adult-plant stages. An F2:6 recombinant inbred line population from cross Liangxing 99 × Zhongzuo 9504 was phenotyped with Blumeria graminis f. sp. tritici isolate Bgt27 at the adult-plant stage in four field tests and the seedling stage in a greenhouse test. The analysis of bulk segregant RNA sequencing (BSR-Seq) identified a single-nucleotide polymorphism-enriched locus, Qaprpm.caas.2B, on chromosome 2BL in the same genomic interval of Pm52 associated with the all-stage resistance (ASR) and Qaprpm.caas.7A on chromosome 7AL associated with the adult-plant resistance (APR) against the disease. Qaprpm.caas.2B was detected in a 1.3 cM genetic interval between markers Xicscl726 and XicsK128 in which Pm52 was placed with a range of logarithm of odds (LOD) values from 28.1 to 34.6, and the phenotype variations explained in terms of maximum disease severity (MDS) ranged from 45 to 52%. The LOD peak of Qaprpm.caas.7A was localized in a 4.6 cM interval between markers XicsK7A8 and XicsK7A26 and explained the phenotypic variation of MDS ranging from 13 to 16%. The results of this study confirmed Pm52 for ASR and identified Qaprpm.caas.7A for APR to powdery mildew in Liangxing 99.

Exploring How Media Influence Preventive Behavior and Excessive Preventive Intention during the COVID-19 Pandemic in China.

In the context of global fighting against the unexpected COVID-19 pandemic, how to promote the public implementation of preventive behavior is the top priority of pandemic prevention and control. This study aimed at probing how the media would affect the public's preventive behavior and excessive preventive intention accordingly. Data were collected from 653 respondents in the Chinese mainland through online questionnaires and further analyzed by using partial least squares structural equation modeling (PLS-SEM). Taking risk perception, negative emotions, and subjective norms as mediators, this study explored the impact of mass media exposure and social networking services involvement on preventive behavior and excessive preventive intention. Based on differences in the severity of the pandemic, the samples were divided into the Wuhan group and other regions group for multi-group comparison. The results showed that mass media exposure had a significant positive impact on subjective norms; moreover, mass media exposure could significantly enhance preventive behavior through subjective norms, and social networking services involvement had a significant positive impact on negative emotions; meanwhile, social networking services involvement promoted excessive preventive intention through negative emotions.

A CNL protein in wild emmer wheat confers powdery mildew resistance.

Powdery mildew, a fungal disease caused by Blumeria graminis f. sp. tritici (Bgt), has a serious impact on wheat production. Loss of resistance in cultivars prompts a continuing search for new sources of resistance. Wild emmer wheat (Triticum turgidum ssp. dicoccoides, WEW), the progenitor of both modern tetraploid and hexaploid wheats, harbors many powdery mildew resistance genes. We report here the positional cloning and functional characterization of Pm41, a powdery mildew resistance gene derived from WEW, which encodes a coiled-coil, nucleotide-binding site and leucine-rich repeat protein (CNL). Mutagenesis and stable genetic transformation confirmed the function of Pm41 against Bgt infection in wheat. We demonstrated that Pm41 was present at a very low frequency (1.81%) only in southern WEW populations. It was absent in other WEW populations, domesticated emmer, durum, and common wheat, suggesting that the ancestral Pm41 was restricted to its place of origin and was not incorporated into domesticated wheat. Our findings emphasize the importance of conservation and exploitation of the primary WEW gene pool, as a valuable resource for discovery of resistance genes for improvement of modern wheat cultivars.

A rare single nucleotide variant in Pm5e confers powdery mildew resistance in common wheat.

Powdery mildew poses severe threats to wheat production. The most sustainable way to control this disease is through planting resistant cultivars. We report the map-based cloning of the powdery mildew resistance allele Pm5e from a Chinese wheat landrace. We applied a two-step bulked segregant RNA sequencing (BSR-Seq) approach in developing tightly linked or co-segregating markers to Pm5e. The first BSR-Seq used phenotypically contrasting bulks of recombinant inbred lines (RILs) to identify Pm5e-linked markers. The second BSR-Seq utilized bulks of genetic recombinants screened from a fine-mapping population to precisely quantify the associated genomic variation in the mapping interval, and identified the Pm5e candidate genes. The function of Pm5e was validated by transgenic assay, loss-of-function mutants and haplotype association analysis. Pm5e encodes a nucleotide-binding domain leucine-rich-repeat-containing (NLR) protein. A rare nonsynonymous single nucleotide variant (SNV) within the C-terminal leucine rich repeat (LRR) domain is responsible for the gain of powdery mildew resistance function of Pm5e, an allele endemic to wheat landraces of Shaanxi province of China. Results from this study demonstrate the value of landraces in discovering useful genes for modern wheat breeding. The key SNV associated with powdery mildew resistance will be useful for marker-assisted selection of Pm5e in wheat breeding programs.

Identification and fine mapping of spot blotch (Bipolaris sorokiniana) resistance gene Sb4 in wheat.

KEY MESSAGE: A new spot blotch (Bipolaris sorokiniana) resistance gene Sb4 was mapped in a genomic interval of 1.34 Mb on wheat chromosome 4BL. Spot blotch, caused by Bipolaris sorokiniana, has emerged as a serious concern for cultivation of wheat in warmer and humid regions of the world, which results in substantial yield losses and descends with quality. In this study, we identified and mapped a spot blotch resistance gene, designated as Sb4, against B. sorokiniana in wheat. Bulked segregant RNA-Seq (BSR-Seq) analysis and single-nucleotide polymorphism mapping showed that Sb4 is located on the long arm of chromosome 4B. A genetic linkage map of Sb4 was constructed using an F4 mapping population developed from the cross between 'GY17' and 'Zhongyu1211,' and Sb4 was delimited in a 7.14-cM genetic region on 4BL between markers B6811 and B6901. Using the Chinese Spring reference sequences of chromosome arm 4BL, 13 new polymorphic markers were developed. Finally, Sb4 was mapped in a 1.19-cM genetic interval corresponding to a 1.34-Mb physical genomic region of Chinese Spring chromosome 4BL containing 21 predicted genes. This study provides a foundational step for further cloning of Sb4 using a map-based approach.

Regenerative Metaplastic Clones in COPD Lung Drive Inflammation and Fibrosis.

Chronic obstructive pulmonary disease (COPD) is a progressive condition of chronic bronchitis, small airway obstruction, and emphysema that represents a leading cause of death worldwide. While inflammation, fibrosis, mucus hypersecretion, and metaplastic epithelial lesions are hallmarks of this disease, their origins and dependent relationships remain unclear. Here we apply single-cell cloning technologies to lung tissue of patients with and without COPD. Unlike control lungs, which were dominated by normal distal airway progenitor cells, COPD lungs were inundated by three variant progenitors epigenetically committed to distinct metaplastic lesions. When transplanted to immunodeficient mice, these variant clones induced pathology akin to the mucous and squamous metaplasia, neutrophilic inflammation, and fibrosis seen in COPD. Remarkably, similar variants pre-exist as minor constituents of control and fetal lung and conceivably act in normal processes of immune surveillance. However, these same variants likely catalyze the pathologic and progressive features of COPD when expanded to high numbers.

Cloning of ground-state intestinal stem cells from endoscopic biopsy samples.

'Adult' or 'somatic' stem cells harbor an intrinsic ability to regenerate tissues. Heterogeneity of such stem cells along the gastrointestinal tract yields the known segmental specificity of this organ and may contribute to the pathology of certain enteric conditions. Here we detail technology for the generation of 'libraries' of clonogenic cells from 1-mm-diamter endoscopic biopsy samples from the human gastrointestinal tract. Each of the 150-300 independent clones in a typical stem cell library can be clonally expanded to billions of cells in a few weeks while maintaining genomic stability and the ability to undergo multipotent differentiation to the specific epithelia from which the sample originated. The key to this methodology is the intrinsic immortality of normal intestinal stem cells (ISCs) and culture systems that maintain them as highly immature, ground-state ISCs marked by a single-cell clonogenicity of 70% and a corresponding 250-fold proliferative advantage over spheroid technologies. Clonal approaches such as this enhance the resolution of molecular genetics, make genome editing easier, and may be useful in regenerative medicine, unravelling heterogeneity in disease, and facilitating drug discovery.

A rare gain of function mutation in a wheat tandem kinase confers resistance to powdery mildew.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most destructive diseases that pose a great threat to wheat production. Wheat landraces represent a rich source of powdery mildew resistance. Here, we report the map-based cloning of powdery mildew resistance gene Pm24 from Chinese wheat landrace Hulutou. It encodes a tandem kinase protein (TKP) with putative kinase-pseudokinase domains, designated WHEAT TANDEM KINASE 3 (WTK3). The resistance function of Pm24 was validated by transgenic assay, independent mutants, and allelic association analyses. Haplotype analysis revealed that a rare 6-bp natural deletion of lysine-glycine codons, endemic to wheat landraces of Shaanxi Province, China, in the kinase I domain (Kin I) of WTK3 is critical for the resistance function. Transgenic assay of WTK3 chimeric variants revealed that only the specific two amino acid deletion, rather than any of the single or more amino acid deletions, in the Kin I of WTK3 is responsible for gaining the resistance function of WTK3 against the Bgt fungus.

Identification of a Recessive Gene PmQ Conferring Resistance to Powdery Mildew in Wheat Landrace Qingxinmai Using BSR-Seq Analysis.

Wheat powdery mildew is caused by Blumeria graminis f. sp. tritici (Bgt), a biotrophic fungal species. It is very important to mine new powdery mildew (Pm) resistance genes for developing resistant wheat cultivars to reduce the deleterious effects of the disease. This study was carried out to characterize the Pm gene in Qingxinmai, a winter wheat landrace from Xinjiang, China. Qingxinmai is resistant to many Bgt isolates collected from different wheat fields in China. F1, F2, and F2:3 generations of the cross between Qingxinmai and powdery mildew susceptible line 041133 were developed. It was confirmed that a single recessive gene, PmQ, conferred the seedling resistance to a Bgt isolate in Qingxinmai. Bulked segregant analysis-RNA-Seq (BSR-Seq) was performed on the bulked homozygous resistant and susceptible F2:3 families, which detected 57 single nucleotide polymorphism (SNP) variants that were enriched in a 40 Mb genomic interval on chromosome arm 2BL. Based on the flanking sequences of the candidate SNPs extracted from the Chinese Spring reference genome, 485 simple sequence repeat (SSR) markers were designed. Six polymorphic SSR markers, together with nine markers that were anchored on chromosome arm 2BL, were used to construct a genetic linkage map for PmQ. This gene was placed in a 1.4 cM genetic interval between markers Xicsq405 and WGGBH913 corresponding to 4.9 Mb physical region in the Chinese Spring reference genome. PmQ differed from most of the other Pm genes identified on chromosome arm 2BL based on its position and/or origin. However, this gene and Pm63 from an Iranian common wheat landrace were located in a similar genomic region, so they may be allelic.

Resistance to Heterodera filipjevi and H. avenae in Winter Wheat is Conferred by Different QTL.

The coexistence of cereal cyst nematode (CCN) species Heterodera avenae and H. filipjevi, often involving multiple pathotypes, is a limiting factor for wheat production in China. Some of the known genes for resistance to CCN are not effective against both nematode species, hence complicating breeding efforts to develop CCN-resistant wheat cultivars. Here, we demonstrate that the CCN resistance in wheat cultivar Madsen to both Heterodera spp. is controlled by different genetic loci, both of which originated from Aegilops ventricosa. A new quantitative trait locus (QTL), QCre-ma7D, was identified and localized in a 3.77-Mb genomic region on chromosome arm 7DL, which confers resistance to H. filipjevi. QCre-ma2A on chromosome arm 2AS corresponds to CCN resistance gene Cre5 and confers resistance to H. avenae. This QTL is a new locus on chromosome arm 7DL and is designated Cre9. Three Kompetitive allele-specific PCR markers (BS00150072, BS00021745, and BS00154302) were developed for molecular marker-assisted selection of Cre9 and locally adapted wheat lines with resistance to both nematode species were developed. QCre-ma2A on chromosome arm 2AS corresponds to CCN resistance gene Cre5 and confers resistance to H. avenae. The identification of different loci underlying resistance to H. filipjevi and H. avenae and the development of adapted resistant entries will facilitate breeding of wheat cultivars that are resistant to these devastating nematodes in China.

Alternative splicing of a barley gene results in an excess-tillering and semi-dwarf mutant.

KEY MESSAGE: An excess-tillering semi-dwarf gene Hvhtd was identified from an EMS-induced mutant in barley and alternative splicing results in excess-tillering semi-dwarf traits. Tillering and plant height are important traits determining plant architecture and grain production in cereal crops. This study identified an excess-tillering semi-dwarf mutant (htd) from an EMS-treated barley population. Genetic analysis of the F1, F2, and F2:3 populations showed that a single recessive gene controlled the excess-tillering semi-dwarf in htd. Using BSR-Seq and gene mapping, the Hvhtd gene was delimited within a 1.8 Mb interval on chromosome 2HL. Alignment of the RNA-Seq data with the functional genes in the interval identified a gene HORVU2Hr1G098820 with alternative splicing between exon2 and exon3 in the mutant, due to a G to A single-nucleotide substitution at the exon and intron junction. An independent mutant with a similar phenotype confirmed the result, with alternative splicing between exon3 and exon4. In both cases, the alternative splicing resulted in a non-functional protein. And the gene HORVU2Hr1G098820 encodes a trypsin family protein and may be involved in the IAA signaling pathway and differs from the mechanism of Green Revolution genes in the gibberellic acid metabolic pathway.

Correction to: Alternative splicing of a barley gene results in an excess­tillering and semi­dwarf mutant.

In the original publication of this article, the acknowledgement section has been missed to publish. Now the same has been provided in this correction.

Development of SNP, KASP, and SSR Markers by BSR-Seq Technology for Saturation of Genetic Linkage Map and Efficient Detection of Wheat Powdery Mildew Resistance Gene Pm61.

The gene Pm61 that confers powdery mildew resistance has been previously identified on chromosome arm 4AL in Chinese wheat landrace Xuxusanyuehuang (XXSYH). To facilitate the use of Pm61 in breeding practices, the bulked segregant analysis-RNA-Seq (BSR-Seq) analysis, in combination with the information on the Chinese Spring reference genome sequence, was performed in the F2:3 mapping population of XXSYH × Zhongzuo 9504. Two single nucleotide polymorphism (SNP), two Kompetitive Allele Specific PCR (KASP), and six simple sequence repeat (SSR) markers, together with previously identified polymorphic markers, saturated the genetic linkage map for Pm61, especially in the proximal side of the target gene that was short of gene-linked markers. In the newly established genetic linkage map, Pm61 was located in a 0.71 cM genetic interval and can be detected in a high throughput scale by the KASP markers Xicsk8 and Xicsk13 or by the standard PCR-based markers Xicscx497 and Xicsx538. The newly saturated genetic linkage map will be useful in molecular marker assisted-selection of Pm61 in breeding for disease resistant cultivar and in its map-based cloning.

Development of Molecular Markers Linked to Powdery Mildew Resistance Gene Pm4b by Combining SNP Discovery from Transcriptome Sequencing Data with Bulked Segregant Analysis (BSR-Seq) in Wheat.

Powdery mildew resistance gene Pm4b, originating from Triticum persicum, is effective against the prevalent Blumeria graminis f. sp. tritici (Bgt) isolates from certain regions of wheat production in China. The lack of tightly linked molecular markers with the target gene prevents the precise identification of Pm4b during the application of molecular marker-assisted selection (MAS). The strategy that combines the RNA-Seq technique and the bulked segregant analysis (BSR-Seq) was applied in an F2:3 mapping population (237 families) derived from a pair of isogenic lines VPM1/7∗Bainong 3217 F4 (carrying Pm4b) and Bainong 3217 to develop more closely linked molecular markers. RNA-Seq analysis of the two phenotypically contrasting RNA bulks prepared from the representative F2:3 families generated 20,745,939 and 25,867,480 high-quality read pairs, and 82.8 and 80.2% of them were uniquely mapped to the wheat whole genome draft assembly for the resistant and susceptible RNA bulks, respectively. Variant calling identified 283,866 raw single nucleotide polymorphisms (SNPs) and InDels between the two bulks. The SNPs that were closely associated with the powdery mildew resistance were concentrated on chromosome 2AL. Among the 84 variants that were potentially associated with the disease resistance trait, 46 variants were enriched in an about 25 Mb region at the distal end of chromosome arm 2AL. Four Pm4b-linked SNP markers were developed from these variants. Based on the sequences of Chinese Spring where these polymorphic SNPs were located, 98 SSR primer pairs were designed to develop distal markers flanking the Pm4b gene. Three SSR markers, Xics13, Xics43, and Xics76, were incorporated in the new genetic linkage map, which located Pm4b in a 3.0 cM genetic interval spanning a 6.7 Mb physical genomic region. This region had a collinear relationship with Brachypodium distachyon chromosome 5, rice chromosome 4, and sorghum chromosome 6. Seven genes associated with disease resistance were predicted in this collinear genomic region, which included C2 domain protein, peroxidase activity protein, protein kinases of PKc_like super family, Mlo family protein, and catalytic domain of the serine/threonine kinases (STKc_IRAK like super family). The markers developed in the present study facilitate identification of Pm4b during its MAS practice.