High density mapping of wheat stripe rust resistance gene QYrXN3517-1BL using QTL mapping, BSE-Seq and candidate gene analysis.

KEY MESSAGE: Fine mapping of a major stripe rust resistance locus QYrXN3517-1BL to a 336 kb region that includes 12 candidate genes. Utilization of genetic resistance is an effective strategy to control stripe rust disease in wheat. Cultivar XINONG-3517 (XN3517) has remained highly resistant to stripe rust since its release in 2008. To understand the genetic architecture of stripe rust resistance, Avocet S (AvS) × XN3517 F6 RIL population was assessed for stripe rust severity in five field environments. The parents and RILs were genotyped by using the GenoBaits Wheat 16 K Panel. Four stable QTL from XINONG-3517 were detected on chromosome arms 1BL, 2AL, 2BL, and 6BS, named as QYrXN3517-1BL, QYrXN3517-2AL, QYrXN3517-2BL, and QYrXN3517-6BS, respectively. Based on the Wheat 660 K array and bulked segregant exome sequencing (BSE-Seq), the most effective QTL on chromosome 1BL is most likely different from the known adult plant resistance gene Yr29 and was mapped to a 1.7 cM region [336 kb, including twelve candidate genes in International Wheat Genome Sequencing Consortium (IWGSC) RefSeq version 1.0]. The 6BS QTL was identified as Yr78, and the 2AL QTL was probably same as QYr.caas-2AL or QYrqin.nwafu-2AL. The novel QTL on 2BL was effective in seedling stage against the races used in phenotyping. In addition, allele-specifc quantitative PCR (AQP) marker nwafu.a5 was developed for QYrXN3517-1BL to assist marker-assisted breeding.

Slow stripe rusting in Chinese wheat Jimai 44 conferred by Yr29 in combination with a major QTL on chromosome arm 6AL.

KEY MESSAGE: YrJ44, a more effective slow rusting gene than Yr29, was localized to a 3.5-cM interval between AQP markers AX-109373479 and AX-109563479 on chromosome 6AL. "Slow rusting" (SR) is a type of adult plant resistance (APR) that can provide non-specific durable resistance to stripe rust in wheat. Chinese elite wheat cultivar Jimai 44 (JM44) has maintained SR to stripe rust in China since its release despite exposure to a changing and variable pathogen population. An F2:6 population comprising 295 recombinant inbred lines (RILs) derived from a cross between JM44 and susceptible cultivar Jimai 229 (JM229) was used in genetic analysis of the SR. The RILs and parental lines were evaluated for stripe rust response in five field environments and genotyped using the Affymetrix Wheat55K SNP array and 13 allele-specific quantitative PCR-based (AQP) markers. Two stable QTL on chromosome arms 1BL and 6AL were identified by inclusive composite interval mapping. The 1BL QTL was probably the pleiotropic gene Lr46/Yr29/Sr58. QYr.nwafu-6AL (hereafter named YrJ44), mapped in a 3.5-cM interval between AQP markers AX-109373479 and AX-109563479, was more effective than Yr29 in reducing disease severity and relative area under the disease progress curve (rAUDPC). RILs harboring both YrJ44 and Yr29 displayed levels of SR equal to the resistant parent JM44. The AQP markers linked with YrJ44 were polymorphic and significantly correlated with stripe rust resistance in a panel of 1,019 wheat cultivars and breeding lines. These results suggested that adequate SR resistance can be obtained by combining YrJ44 and Yr29 and the AQP markers can be used in breeding for durable stripe rust resistance.

Reduction of Rhizoctonia cerealis Infection on Wheat Through Host- and Spray-Induced Gene Silencing of an Orphan Secreted Gene.

Rhizoctonia cerealis is a soilborne fungus that can cause sharp eyespot in wheat, resulting in massive yield losses found in many countries. Due to the lack of resistant cultivars, fungicides have been widely used to control this pathogen. However, chemical control is not environmentally friendly and is costly. Meanwhile, the lack of genetic transformation tools has hindered the functional characterization of virulence genes. In this study, we attempted to characterize the function of virulence genes by two transient methods, host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS), which use RNA interference to suppress the pathogenic development. We identified ten secretory orphan genes from the genome. After silencing these ten genes, only the RcOSP1 knocked-down plant significantly inhibited the growth of R. cerealis. We then described RcOSP1 as an effector that could impair wheat biological processes and suppress pathogen-associated molecular pattern-triggered immunity in the infection process. These findings confirm that HIGS and SIGS can be practical tools for researching R. cerealis virulence genes. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genome-wide Mapping for Stripe Rust Resistance Loci in Common Wheat Cultivar Qinnong 142.

Stripe rust caused by Puccinia striiformis f. sp. tritici threatens worldwide wheat production. Growing resistant cultivars is the best way to control this disease. Chinese wheat cultivar Qinnong 142 (QN142) has a high level of adult-plant resistance to stripe rust. To identify quantitative trait loci (QTLs) related to stripe rust resistance, we developed a recombinant inbred line (RIL) population from a cross between QN142 and susceptible cultivar Avocet S. The parents and 165 F6 RILs were evaluated in terms of their stripe rust infection type and disease severity in replicated field tests with six site-year environments. The parents and RILs were genotyped with single-nucleotide polymorphism (SNP) markers. Four stable QTLs were identified in QN142 and mapped to chromosome arms 1BL, 2AL, 2BL, and 6BS. The 1BL QTL was probably the known resistance gene Yr29, the 2BL QTL was in a resistance gene-rich region, and the 2AL and 6BS QTLs might be new. Kompetitive allele specific polymerase chain reaction markers developed from the SNP markers flanking these QTLs were highly polymorphic in a panel of 150 wheat cultivars and breeding lines. These markers could be used in marker-assisted selection for incorporating the stripe rust resistance QTL into new wheat cultivars.

Identification of Long Intergenic Noncoding RNAs in Rhizoctonia cerealis following Inoculation of Wheat.

Wheat sharp eyespot caused by Rhizoctonia cerealis is primarily a severe threat to worldwide wheat production. Currently, there are no resistant wheat cultivars, and the use of fungicides is the primary method for controlling this disease. Elucidating the mechanisms of R. cerealis pathogenicity can accelerate the pace of the control of this disease. Long intergenic noncoding RNAs (lincRNAs) that function in plant-pathogen interactions might provide a new perspective. We systematically analyzed lincRNAs and identified a total of 1,319 lincRNAs in R. cerealis. We found that lincRNAs are involved in various biological processes, as shown by differential expression analysis and weighted correlation network analysis (WGCNA). Next, one of nine hub lincRNAs in the blue module that was related to infection and growth processes, MSTRG.4380.1, was verified to reduce R. cerealis virulence on wheat by a host-induced gene silencing (HIGS) assay. Following that, RNA sequencing (RNA-Seq) analysis revealed that the significantly downregulated genes in the MSTRG.4380.1 knockdown lines were associated mainly with infection-related processes, including hydrolase, transmembrane transporter, and energy metabolism activities. Additionally, 23 novel microRNAs (miRNAs) were discovered during small RNA (sRNA) sequencing (sRNA-Seq) analysis of MSTRG.4380.1 knockdown, and target prediction of miRNAs suggested that MSTRG.4380.1 does not act as a competitive endogenous RNA (ceRNA). This study performed the first genome-wide identification of R. cerealis lincRNAs and miRNAs. It confirmed the involvement of a lincRNA in the infection process, providing new insights into the mechanism of R. cerealis infection and offering a new approach for protecting wheat from R. cerealis. IMPORTANCE Rhizoctonia cerealis, the primary causal agent of wheat sharp eyespot, has caused significant losses in worldwide wheat production. Since no resistant wheat cultivars exist, chemical control is the primary method. However, this approach is environmentally unfriendly and costly. RNA interference (RNAi)-mediated pathogenicity gene silencing has been proven to reduce the growth of Rhizoctonia and provides a new perspective for disease control. Recent studies have shown that lincRNAs are involved in various biological processes across species, such as biotic and abiotic stresses. Therefore, verifying the function of lincRNAs in R. cerealis is beneficial for understanding the infection mechanism. In this study, we reveal that lincRNAs could contribute to the virulence of R. cerealis, which provides new insights into controlling this pathogen.

CRISPR-Cas12a-Based Diagnostics of Wheat Fungal Diseases.

Fusarium head blight (FHB) of wheat, mainly caused by Fusarium graminearum (F. graminearum) infection, reduces crop yield and contaminates grain with mycotoxins. We report a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a-based nucleic acid assay for an early and rapid diagnosis of wheat FHB. Guide RNA (gRNA) was screened for highly specific recognition of polymerase chain reaction (PCR) amplicon of the internal transcribed spacer (ITS) region and the transcription elongation factor 1α (EF1α) of F. graminearum. The trans-activation of Cas12a protein cleaves the single-stranded DNA probes with the terminal fluorophore and quencher groups, thus allowing us to report the presence of ITS and EF1α of F. graminearum. Owing to the dual recognition process through PCR primers and gRNA hybridization, the approach realized specific discrimination of F. graminearum from other pathogenic fungi. It also allowed us to detect as low as 1 fg/µL total DNA from F. graminearum, which is sufficient to diagnose a 4 day F. graminearum infection. CRISPR-Cas12a-based nucleic acid assay promises the molecular diagnosis of crop diseases and broadens the application of CRISPR tools.

Characterization of the Wheat-Psathyrostachys huashania Keng 2Ns/2D Substitution Line H139: A Novel Germplasm With Enhanced Resistance to Wheat Take-All.

Take-all is a devastating soil-borne disease that affects wheat production. The continuous generation of disease-resistance germplasm is an important aspect of the management of this pathogen. In this study, we characterized the wheat-Psathyrostachys huashania Keng (P. huashania)-derived progeny H139 that exhibits significantly improved resistance to wheat take-all disease compared with its susceptible parent 7182. Sequential genomic in situ hybridization (GISH) and multicolor fluorescence in situ hybridization (mc-FISH) analyses revealed that H139 is a stable wheat-P. huashania disomic substitution line lacking wheat chromosome 2D. Expressed sequence tag-sequence tagged site (EST-STS) marker and Wheat Axiom 660K Genotyping Array analysis further revealed that H139 was a novel wheat-P. huashania 2Ns/2D substitution line. In addition, the H139 line was shown to be cytologically stable with a dwarf phenotype and increased spikelet number. These results indicate that H139, with its enhanced wheat take-all disease resistance and desirable agronomic traits, provides valuable genetic resources for wheat chromosome engineering breeding.

QTL Mapping and Validation of Adult Plant Resistance to Stripe Rust in Chinese Wheat Landrace Humai 15.

Stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is a devastating foliar disease that affects common wheat and barley throughout the world. The reasonable deployment of adult plant resistance (APR) wheat varieties is one of the best methods for controlling this disease. Wheat landraces are valuable resources for identifying the genes/QTLs responsible for disease resistance. Humai 15 is a Chinese spring wheat landrace and it has exhibited adequate levels of APR to the prevalent Pst races in field environments for many years. In this study, a population of 177 recombinant inbred lines (RILs) was derived from Humai 15 × Mingxian 169. After screening based on a 90K chip array using 45 RILs and Kompetitive Allelic Specific PCR marker genotyping for the population of RILs, a major effect QTL in Humai 15 was located on the centromere of chromosome 2B, where it accounted for up to 47.2% of the phenotypic variation. Two other minor QTL genes from Humai 15 were located on chromosome arms 3BS and 4BL. The Yr18 gene was identified on chromosome arm 7DS in Mingxian 169.

Construction and characterization of a bacterial artificial chromosome library for the hexaploid wheat line 92R137.

For map-based cloning of genes conferring important traits in the hexaploid wheat line 92R137, a bacterial artificial chromosome (BAC) library, including two sublibraries, was constructed using the genomic DNA of 92R137 digested with restriction enzymes HindIII and BamHI. The BAC library was composed of total 765,696 clones, of which 390,144 were from the HindIII digestion and 375,552 from the BamHI digestion. Through pulsed-field gel electrophoresis (PFGE) analysis of 453 clones randomly selected from the HindIII sublibrary and 573 clones from the BamHI sublibrary, the average insert sizes were estimated as 129 and 113 kb, respectively. Thus, the HindIII sublibrary was estimated to have a 3.01-fold coverage and the BamHI sublibrary a 2.53-fold coverage based on the estimated hexaploid wheat genome size of 16,700 Mb. The 765,696 clones were arrayed in 1,994 384-well plates. All clones were also arranged into plate pools and further arranged into 5-dimensional (5D) pools. The probability of identifying a clone corresponding to any wheat DNA sequence (such as gene Yr26 for stripe rust resistance) from the library was estimated to be more than 99.6%. Through polymerase chain reaction screening the 5D pools with Xwe173, a marker tightly linked to Yr26, six BAC clones were successfully obtained. These results demonstrate that the BAC library is a valuable genomic resource for positional cloning of Yr26 and other genes of interest.

Fine mapping of wheat stripe rust resistance gene Yr26 based on collinearity of wheat with Brachypodium distachyon and rice.

The Yr26 gene, conferring resistance to all currently important races of Puccinia striiformis f. sp. tritici (Pst) in China, was previously mapped to wheat chromosome deletion bin C-1BL-6-0.32 with low-density markers. In this study, collinearity of wheat to Brachypodium distachyon and rice was used to develop markers to saturate the chromosomal region containing the Yr26 locus, and a total of 2,341 F2 plants and 551 F2∶3 progenies derived from Avocet S×92R137 were used to develop a fine map of Yr26. Wheat expressed sequence tags (ESTs) located in deletion bin C-1BL-6-0.32 were used to develop sequence tagged site (STS) markers. The EST-STS markers flanking Yr26 were used to identify collinear regions of the rice and B. distachyon genomes. Wheat ESTs with significant similarities in the two collinear regions were selected to develop conserved markers for fine mapping of Yr26. Thirty-one markers were mapped to the Yr26 region, and six of them cosegregated with the resistance gene. Marker orders were highly conserved between rice and B. distachyon, but some rearrangements were observed between rice and wheat. Two flanking markers (CON-4 and CON-12) further narrowed the genomic region containing Yr26 to a 1.92 Mb region in B. distachyon chromosome 3 and a 1.17 Mb region in rice chromosome 10, and two putative resistance gene analogs were identified in the collinear region of B. distachyon. The markers developed in this study provide a potential target site for further map-based cloning of Yr26 and should be useful in marker assisted selection for pyramiding the gene with other resistance genes.