Provision of TILLING resources and platforms in wheat
Lead Research Organisation:
John Innes Centre
Department Name: UNLISTED
Abstract
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Technical Summary
Our objective is to make TILLING technology more accessible to the wheat research and breeding community. To enable this we will:
Provide access to existing TILLING libraries from tetraploid and hexaploid wheat
We will generate high quantity and quality DNA from the existing EMS-mutagenised lines in Cadenza and Cham1 to allow the establishment of a long-term community resource. This will involve growing 2000 M2 plants from each population and performing large scale DNA extractions from individual plants. In addition, a diversity set totalling 400 accessions will be developed which can be screened by ecoTILLING.
Provide users with a “TILLING kit”
We will provide users with all the necessary tools and protocols to allow for efficient TILLING. We will develop a “TILLING kit” that will include the individual and pooled DNA as well as additional resources. This will include stocks of celery juice extract, DNA from aneuploid lines to facilitate testing of homoeologue-specific primers for target genes, positive/negative controls to assist users in establishing and troubleshooting their set-up as well as guidance notes
Provide hands-on training for TILLING strategies in polyploid species
We will hold a workshop providing hands-on training in a simple CJE-based method of TILLING, to include genome-specific primer design, PCR, CJE digestion and product detection.
Explore strategies for TILLING by next-generation sequencing
We will examine two strategies for effective TILLING using next-generation sequencing. We will focus on methods to sequence specific genomic fragments and determine pooling depths and the sequencing coverage required to identify mutations. The first method we will explore involves the sequencing of PCR products amplified from the target genes, whereas the second approach involves target enrichment from sheared genomic DNA by solution hybridisation to target-specific oligonucleotides.
Provide access to existing TILLING libraries from tetraploid and hexaploid wheat
We will generate high quantity and quality DNA from the existing EMS-mutagenised lines in Cadenza and Cham1 to allow the establishment of a long-term community resource. This will involve growing 2000 M2 plants from each population and performing large scale DNA extractions from individual plants. In addition, a diversity set totalling 400 accessions will be developed which can be screened by ecoTILLING.
Provide users with a “TILLING kit”
We will provide users with all the necessary tools and protocols to allow for efficient TILLING. We will develop a “TILLING kit” that will include the individual and pooled DNA as well as additional resources. This will include stocks of celery juice extract, DNA from aneuploid lines to facilitate testing of homoeologue-specific primers for target genes, positive/negative controls to assist users in establishing and troubleshooting their set-up as well as guidance notes
Provide hands-on training for TILLING strategies in polyploid species
We will hold a workshop providing hands-on training in a simple CJE-based method of TILLING, to include genome-specific primer design, PCR, CJE digestion and product detection.
Explore strategies for TILLING by next-generation sequencing
We will examine two strategies for effective TILLING using next-generation sequencing. We will focus on methods to sequence specific genomic fragments and determine pooling depths and the sequencing coverage required to identify mutations. The first method we will explore involves the sequencing of PCR products amplified from the target genes, whereas the second approach involves target enrichment from sheared genomic DNA by solution hybridisation to target-specific oligonucleotides.
Planned Impact
unavailable
Organisations
People |
ORCID iD |
| Cristobal Uauy (Principal Investigator) |