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Identification of Rice Blast Loss-of-Function Mutant Alleles in the Wheat Genome as a New Strategy for Wheat Blast Resistance Breeding.

ABSTRACT: Blast is caused by the host-specific lineages of the fungus Magnaporthe oryzae and is the most important destructive disease in major crop plants, including rice and wheat. The first wheat blast outbreak that occurred in Bangladesh in 2016 and the recent epidemic in Zambia were caused by the M. oryzae Triticum (MoT) pathotype, a fungal lineage belonging to M. oryzae. Although a few reported wheat cultivars show modest resistance to MoT, the patterns of genetic variation and diversity of this pathotype make it crucial to identify additional lines of resistant wheat germplasm. Nearly 40 rice blast resistant and susceptible genes have so far been cloned. Here, we used BLAST analysis to locate two rice blast susceptible genes in the wheat reference genome, bsr-d1 and bsr-k1, and identified six identical homologous genes located on subgenomes A, B, and D. We uncovered a total of 171 single nucleotide polymorphisms (SNPs) in an ethyl methanesulfonate (EMS)-induced population, with mutation densities ranging from 1/1107.1 to 1/230.7 kb through Targeting Induced Local Lesions IN Genomes (TILLING) by sequencing. These included 81 SNPs located in exonic and promoter regions, and 13 coding alleles that are predicted to have severe effects on protein function, including two pre-mature mutants that might affect wheat blast resistance. The loss-of-function alleles identified in this study provide insights into new wheat blast resistant lines, which represent a valuable breeding resource.

SUBMITTER: Guo H

PROVIDER: S-EPMC8170139 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Utilization of rice blast-resistance (R) genes is the most economical and environmentally friendly method to control blast disease. However, rice varieties with R genes influence the outcome of genetic architectures of Magnaporthe oryzae (M. oryzae), and mutations in avirulence (AVR) genes of M. oryzae may cause dysfunction of the corresponding R genes in rice varieties. Although monitoring and characterizing rice R genes and pathogen AVR genes in field populations may facilitate the implementation of effective R genes, little is known about the changes of R genes over time and their ultimate impact on pathogen AVR genes. In this study, 117 main cultivated rice varieties over the past five decades and 35 M. oryzae isolates collected from those diseased plants were analyzed by PCR using gene-specific markers of the nine R genes and six primer pairs targeting the coding sequence or promoter of AVR genes, respectively. The R genes Pigm, Pi9, Pi2, Piz-t, Pi-ta, Pik, Pi1, Pikp, and Pikm were identified in 5, 0, 1, 4, 18, 0, 2, 1, and 0 cultivars, respectively. Significantly, none of these R genes had significant changes that correlated to their application periods of time. Among the four identified AVR genes, AVR-Pik had the highest amplification frequency (97.14%) followed by AVR-Pita (51.43%) and AVR-Pi9 (48.57%); AVR-Piz-t had the lowest frequency (28.57%). All these AVR genes except AVR-Pi9 had 1-2 variants. Inoculation mono-genic lines contained functional genes of Pi2/9 and Pik loci with 14 representative isolates from those 35 ones revealed that the presence of certain AVR-Piz-t, AVR-Pita variants, and AVR-Pik-E + AVR-Pik-D in M. oryzae populations, and these variants negated the ability of the corresponding R genes to confer resistance. Importantly, Pi2, Pi9, and Pigm conferred broad-spectrum resistance to these local isolates. These findings reveal that the complex genetic basis of M. oryzae and some effective blast R genes should be considered in future rice blast-resistance breeding programs.

Project description:BackgroundAs rice (Oryza sativa) is the staple food of more than half the world's population, rice production contributes greatly to global food security. Rice blast caused by the fungus Magnaporthe oryzae (M. oryzae) is a devastating disease that affects rice yields and grain quality, resulting in substantial economic losses annually. Because the fungus evolves rapidly, the resistance conferred by most the single blast-resistance genes is broken after a few years of intensive agricultural use. Therefore, effective resistance breeding in rice requires continual enrichment of the reservoir of resistance genes, alleles, or QTLs. Seed banks represent a rich source of genetic diversity; however, they have not been extensively used to identify novel genes and alleles.ResultsWe carried out a large-scale screen for novel blast-resistance alleles in 1883 rice varieties from major rice-producing areas across China. Of these, 361 varieties showed at least moderate resistance to natural infection by rice blast at rice blast nurseries in Enshi and Yichang, Hubei Province. We used sequence-based allele mining to amplify and sequence the allelic variants of the major rice blast-resistance genes at the Pi2/Pi9 locus of chromosome 6 from the 361 blast-resistant varieties, and the full-length coding region of this gene could be amplified from 107 varieties. Thirteen novel Pi9 alleles (named Pi9-Type1 to Pi9-Type13) were identified in these 107 varieties based on comparison to the Pi9 referenced sequence. Based on the sequencing results, the Pi2/Pi9 locus of the 107 varieties was divided into 15 genotypes (including three different genotypes of Pi9-Type5). Fifteen varieties, each representing one genotype, were evaluated for resistance to 34 M. oryzae isolates. The alleles from seven varieties with the highest resistance and widest resistance spectra were selected for transformation into the susceptible variety J23B to construct near-isogenic lines (NILs). These NILs showed resistance in a field test in Enshi and Yichang, indicating that the seven novel rice blast-resistance tandem-repeat regions at the Pi2/Pi9 locus of chromosome 6 could potentially serve as a genetic resource for molecular breeding of resistance to rice blast.ConclusionsThe thirteen novel Pi9 alleles identified in this study expand the list of available of blast-resistance alleles. Seven tandem-repeat regions of the Pi2/Pi9 locus from different donors were characterized as broad-spectrum rice blast-resistance fragments; these donors enrich the genetic resources available for rice blast-resistance breeding programs.

Project description:Over the last few decades, the use of molecular markers has played an increasing role in rice breeding and genetics. Of the different types of molecular markers, microsatellites have been utilized most extensively, because they can be readily amplified by PCR and the large amount of allelic variation at each locus. Microsatellites are also known as simple sequence repeats (SSR), and they are typically composed of 1-6 nucleotide repeats. These markers are abundant, distributed throughout the genome and are highly polymorphic compared with other genetic markers, as well as being species-specific and co-dominant. For these reasons, they have become increasingly important genetic markers in rice breeding programs. The evolution of new biotypes of pests and diseases as well as the pressures of climate change pose serious challenges to rice breeders, who would like to increase rice production by introducing resistance to multiple biotic and abiotic stresses. Recent advances in rice genomics have now made it possible to identify and map a number of genes through linkage to existing DNA markers. Among the more noteworthy examples of genes that have been tightly linked to molecular markers in rice are those that confer resistance or tolerance to blast. Therefore, in combination with conventional breeding approaches, marker-assisted selection (MAS) can be used to monitor the presence or lack of these genes in breeding populations. For example, marker-assisted backcross breeding has been used to integrate important genes with significant biological effects into a number of commonly grown rice varieties. The use of cost-effective, finely mapped microsatellite markers and MAS strategies should provide opportunities for breeders to develop high-yield, blast resistance rice cultivars. The aim of this review is to summarize the current knowledge concerning the linkage of microsatellite markers to rice blast resistance genes, as well as to explore the use of MAS in rice breeding programs aimed at improving blast resistance in this species. We also discuss the various advantages, disadvantages and uses of microsatellite markers relative to other molecular marker types.

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Identification of Rice Blast Loss-of-Function Mutant Alleles in the Wheat Genome as a New Strategy for Wheat Blast Resistance Breeding.

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