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.
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 .
Wheat is an important food crop, but adversity stresses are becoming more common, resulting in signi cant yield losses. Accelerating molecularly assisted resistance breeding is critical. Through statistical analysis of published loci in wheat over the last two decades, we selected 60 loci with main breeding objectives, high heritability, and reliable genotyping, such as stress resistance, yield, plant height, and resistance to spike germination. Then, using genotyping by target sequencing(GBTS) technology, we developed a liquid phase chip based on 101 functional or closely linked markers. The genotyping of 42 loci was con rmed in an extensive collection of Chinese wheat cultivars, indicating that the chip can be used in molecular-assisted selection (MAS) for target breeding goals. Besides, we can perform the preliminary parentage analysis with the genotype data. The most signi cant contribution of this paper is to evaluate the target traits of breeding materials without conducting eld experiments. Breeders can quickly screen germplasm resources, parental breeding materials, and intermediate materials for the presence of excellent allelic variants using the genotyping data by this chip, which is high throughput, convenient, reliable, and cost-e cient.
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.
Wheat is an important food crop, but adversity stresses are becoming more common, resulting in significant yield losses. Accelerating molecularly assisted resistance breeding is critical. Through statistical analysis of published loci in wheat over the last two decades, we selected 60 loci with main breeding objectives, high heritability, and reliable genotyping, such as stress resistance, yield, plant height, and resistance to spike germination. Then, using genotyping by target sequencing(GBTS) technology, we developed a liquid phase chip based on 101 functional or closely linked markers. The genotyping of 42 loci was confirmed in an extensive collection of Chinese wheat cultivars, indicating that the chip can be used in molecular-assisted selection (MAS) for target breeding goals. Besides, we can perform the preliminary parentage analysis with the genotype data. The most significant contribution of this paper is to evaluate the target traits of breeding materials without conducting field experiments. Breeders can quickly screen germplasm resources, parental breeding materials, and intermediate materials for the presence of excellent allelic variants using the genotyping data by this chip, which is high throughput, convenient, reliable, and cost-efficient.
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