Dataset Information

Mlo-based powdery mildew resistance in hexaploid bread wheat generated by a non-transgenic TILLING approach.

ABSTRACT: Wheat is one of the most widely grown cereal crops in the world and is an important food grain source for humans. However, wheat yields can be reduced by many abiotic and biotic stress factors, including powdery mildew disease caused by Blumeria graminis f.sp. tritici (Bgt). Generating resistant varieties is thus a major effort in plant breeding. Here, we took advantage of the non-transgenic Targeting Induced Lesions IN Genomes (TILLING) technology to select partial loss-of-function alleles of TaMlo, the orthologue of the barley Mlo (Mildew resistance locus o) gene. Natural and induced loss-of-function alleles (mlo) of barley Mlo are known to confer durable broad-spectrum powdery mildew resistance, typically at the expense of pleiotropic phenotypes such as premature leaf senescence. We identified 16 missense mutations in the three wheat TaMlo homoeologues, TaMlo-A1, TaMlo-B1 and TaMlo-D1 that each lead to single amino acid exchanges. Using transient gene expression assays in barley single cells, we functionally analysed the different missense mutants and identified the most promising candidates affecting powdery mildew susceptibility. By stacking of selected mutant alleles we generated four independent lines with non-conservative mutations in each of the three TaMlo homoeologues. Homozygous triple mutant lines and surprisingly also some of the homozygous double mutant lines showed enhanced, yet incomplete, Bgt resistance without the occurrence of discernible pleiotropic phenotypes. These lines thus represent an important step towards the production of commercial non-transgenic, powdery mildew-resistant bread wheat varieties.

SUBMITTER: Acevedo-Garcia J

PROVIDER: S-EPMC5316926 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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mlo-based powdery mildew resistance in hexaploid bread wheat generated by a non-transgenic TILLING approach.

Acevedo-Garcia Johanna J Spencer David D Thieron Hannah H Reinstädler Anja A Hammond-Kosack Kim K Phillips Andrew L AL Panstruga Ralph R

Plant biotechnology journal 20160925 3

Wheat is one of the most widely grown cereal crops in the world and is an important food grain source for humans. However, wheat yields can be reduced by many abiotic and biotic stress factors, including powdery mildew disease caused by Blumeria graminis f.sp. tritici (Bgt). Generating resistant varieties is thus a major effort in plant breeding. Here, we took advantage of the non-transgenic Targeting Induced Lesions IN Genomes (TILLING) technology to select partial loss-of-function alleles of T ...[more]

PMID: 27565953

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Project description:Genetically modified (GM) plants offer an ideal model system to study the influence of single genes that confer constitutive resistance to pathogens on the ecological behaviour of plants. We used phytometers to study competitive interactions between GM lines of spring wheat Triticum aestivum carrying such genes and control lines. We hypothesized that competitive performance of GM lines would be reduced due to enhanced transgene expression under pathogen levels typically encountered in the field. The transgenes pm3b from wheat (resistance against powdery mildew Blumeria graminis) or chitinase and glucanase genes from barley (resistance against fungi in general) were introduced with the ubiquitin promoter from maize (pm3b and chitinase genes) or the actin promoter from rice (glucanase gene). Phytometers of 15 transgenic and non-transgenic wheat lines were transplanted as seedlings into plots sown with the same 15 lines as competitive environments and subject to two soil nutrient levels. Pm3b lines had reduced mildew incidence compared with control lines. Chitinase and chitinase/glucanase lines showed the same high resistance to mildew as their control in low-nutrient treatment and slightly lower mildew rates than the control in high-nutrient environment. Pm3b lines were weaker competitors than control lines. This resulted in reduced yield and seed number. The Pm3b line with the highest transgene expression had 53.2% lower yield than the control whereas the Pm3b line which segregated in resistance and had higher mildew rates showed only minor costs under competition. The line expressing both chitinase and glucanase genes also showed reduced yield and seed number under competition compared with its control. Our results suggest that single transgenes conferring constitutive resistance to pathogens can have ecological costs and can weaken plant competitiveness even in the presence of the pathogen. The magnitude of these costs appears related to the degree of expression of the transgenes.

Project description:Wheat (Triticum ssp.) is an important food source for humans in many regions around the world. However, the ability to understand and modify gene function for crop improvement is hindered by the lack of available genomic resources. TILLING is a powerful reverse genetics approach that combines chemical mutagenesis with a high-throughput screen for mutations. Wheat is specially well-suited for TILLING due to the high mutation densities tolerated by polyploids, which allow for very efficient screens. Despite this, few TILLING populations are currently available. In addition, current TILLING screening protocols require high-throughput genotyping platforms, limiting their use.We developed mutant populations of pasta and common wheat and organized them for TILLING. To simplify and decrease costs, we developed a non-denaturing polyacrylamide gel set-up that uses ethidium bromide to detect fragments generated by crude celery juice extract digestion of heteroduplexes. This detection method had similar sensitivity as traditional LI-COR screens, suggesting that it represents a valid alternative. We developed genome-specific primers to circumvent the presence of multiple homoeologous copies of our target genes. Each mutant library was characterized by TILLING multiple genes, revealing high mutation densities in both the hexaploid (~1/38 kb) and tetraploid (~1/51 kb) populations for 50% GC targets. These mutation frequencies predict that screening 1,536 lines for an effective target region of 1.3 kb with 50% GC content will result in ~52 hexaploid and ~39 tetraploid mutant alleles. This implies a high probability of obtaining knock-out alleles (P = 0.91 for hexaploid, P = 0.84 for tetraploid), in addition to multiple missense mutations. In total, we identified over 275 novel alleles in eleven targeted gene/genome combinations in hexaploid and tetraploid wheat and have validated the presence of a subset of them in our seed stock.We have generated reverse genetics TILLING resources for pasta and bread wheat and achieved a high mutation density in both populations. We also developed a modified screening method that will lower barriers to adopt this promising technology. We hope that the use of this reverse genetics resource will enable more researchers to pursue wheat functional genomics and provide novel allelic diversity for wheat improvement.

Dataset Information

Mlo-based powdery mildew resistance in hexaploid bread wheat generated by a non-transgenic TILLING approach.

Publications

mlo-based powdery mildew resistance in hexaploid bread wheat generated by a non-transgenic TILLING approach.

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