Discovery of Rare Mutations in Populations: TILLING by Sequencing

Tsai, Helen; Howell, T. A.; Nitcher, Rebecca; Missirian, Victor; Watson, Brian; Ngo, Kathie J.; Lieberman, Meric; Fass, Joseph; Uauy, Cristóbal; Tran, Robert K.; Khan, Asif Ali; Filkov, Vladimir; Tai, Thomas H.; Dubcovsky, Jorge; Comai, Luca

doi:10.1104/pp.110.169748

Cited by 236 publications

(260 citation statements)

References 26 publications

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“…For example, the TILLING by sequencing protocol described in Chap. 20 provides a three-dimensional pooling strategy for 768 individuals and simultaneous mutation discovery in many gene targets (Tsai et al 2011). The use of advanced tools is not limited to reversegenetics.…”

Section: Higher-throughput Genotyping and Mutation Discovery Methodsmentioning

confidence: 99%

Mutagenesis for Crop Breeding and Functional Genomics

Jankowicz-Cieślak

Chikelu

Till

2016

Biotechnologies for Plant Mutation Breeding

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Genetic variation is a source of phenotypic diversity and is a major driver of evolutionary diversification. Heritable variation was observed and used thousands of years ago in the domestication of plants and animals. The mechanisms that govern the inheritance of traits were later described by Mendel. In the early decades of the twentieth century, scientists showed that the relatively slow rate of natural mutation could be increased by several orders of magnitude by treating Drosophila and cereals with X-rays. What is striking about these achievements is that they came in advance of experimental evidence that DNA is the heritable material. This highlights one major advantage of induced mutations for crop breeding: prior knowledge of genes or gene function is not required to successfully create plants with improved traits and to release new varieties. Indeed, mutation induction has been an important tool for crop breeding since the release of the first mutant variety of tobacco in the 1930s. In addition to plant mutation breeding, induced mutations have been used extensively for functional genomics in model organisms and crops. Novel reverse-genetic strategies, such as Targeting Induced Local Lesions IN Genomes (TILLING), are being used for the production of stable genetic stocks of mutant plant populations such as Arabidopsis, barley, soybean, tomato and wheat. These can be kept for many years and screened repeatedly for different traits. Robust and efficient methods are required for the seamless integration of induced mutations in breeding and functional genomics studies. This chapter provides an overview of the principles and methodologies that underpin the set of protocols and guidelines for the use of induced mutations to improve crops.

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Section: Higher-throughput Genotyping and Mutation Discovery Methodsmentioning

confidence: 99%

Mutagenesis for Crop Breeding and Functional Genomics

Jankowicz-Cieślak

Chikelu

Till

2016

Biotechnologies for Plant Mutation Breeding

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“…The use of multidimensional pooling of DNA samples enables screening of DNA pools for multiple independent mutations in any target gene using NGS, which provides a cost-effective assay. This has led to the discovery of rare mutations in rice and wheat, termed TILLING by sequencing [92], tef [93], and in animals [94]. Different sample pooling schemes for NGS, which further enhance the power of NGS in processing multiple samples in parallel have been developed [95].…”

Section: Targeting Induced Local Lesions In Genomes (Tilling) and Ngsmentioning

confidence: 99%

Perspectives on the Application of Next-generation Sequencing to the Improvement of Africa’s Staple Food Crops

Gedil¹,

Ferguson²,

Girma³

et al. 2016

Next Generation Sequencing - Advances, Applications and Challenges

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The persistent challenge of insufficient food, unbalanced nutrition, and deteriorating natural resources in the most vulnerable nations, characterized by fast population growth, calls for utilization of innovative technologies to curb constraints of crop production. Enhancing genetic gain by using a multipronged approach that combines conventional and genomic technologies for the development of stress-tolerant varieties with high yield and nutritional quality is necessary. The advent of nextgeneration sequencing (NGS) technologies holds the potential to dramatically impact the crop improvement process. NGS enables whole-genome sequencing (WGS) and re-sequencing, transcriptome sequencing, metagenomics, as well as high-throughput genotyping, which can be applied for genome selection (GS). It can also be applied to diversity analysis, genetic and epigenetic characterization of germplasm and pathogen detection, identification, and elimination. High-throughput phenotyping, integrated data management, and decision support tools form the necessary supporting environment for effective utilization of genome sequence information. It is important that these opportunities for mainstreaming innovative breeding strategies, enabled by cutting-edge "Omics" technologies, are seized in Africa; however, several constraints must be addressed before the benefit of NGS can be fully realized. African breeding programs must have access to high-throughput genotyping facilities, capacity in the application of genome selection and marker-assisted breeding must be built and supported by capacity in genomic analysis and bioinformatics. This chapter demonstrates how interventions with NGS-enabled innovative strategies can be applied to increase genetic gain with insights from the Consortium of International

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“…Such an approach requires sufficient sequencing coverage to allow the discrimination of real mutations from changes introduced through sequencing errors. While the technology exists to detect singlenucleotide polymorphisms by whole-genome resequencing (Ossowski et al 2010), the application of this approach to large mutant populations is still too costly for most species (Tsai et al 2011). Instead, mutation discovery using massive parallel sequencing approaches have been focused on the gene space, where mutations are most likely to affect function.…”

Section: Introductionmentioning

confidence: 99%

“…This approach has been adapted for use by laboratories with limited resources (Jankowicz-Cieslak et al 2012). However, discovery of mutations in pools of more than eight individuals is challenging, and once a putative mutation has been detected, Sanger sequencing of all individuals in the pool is needed to verify the mutation (McCallum et al 2000;Bentley et al 2000;Comai and Henikoff 2006;Jankowicz-Cieslak et al 2012;Tsai et al 2011). Because Sanger sequencing requires a large amount of template and is prone to sequencing errors, traditional TILLING can be slow and cost prohibitive.…”

Section: Introductionmentioning

confidence: 99%

“…Because Sanger sequencing requires a large amount of template and is prone to sequencing errors, traditional TILLING can be slow and cost prohibitive. The advent of next-generation DNA sequencing (NGS), which is cheaper and faster than the classic Sanger method, provides new opportunities for mutant identification in TILLING populations (Tsai et al 2011).…”

Section: Introductionmentioning

confidence: 99%

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Next-Generation Sequencing for Targeted Discovery of Rare Mutations in Rice

Burkart‐Waco

Tsai

Ngo

et al. 2016

Biotechnologies for Plant Mutation Breeding

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Advances in DNA sequencing (i.e., next-generation sequencing, NGS) have greatly increased the power and efficiency of detecting rare mutations in large mutant populations. Targeting Induced Local Lesions in Genomes (TILLING) is a reverse genetics approach for identifying gene mutations resulting from chemical mutagenesis. In traditional TILLING, mutation discovery is accomplished through mismatch cleavage of mutant and wild-type DNA heteroduplexes using endonucleases. This is followed by Sanger sequencing to determine the specific sequence changes. TILLING by sequencing (TBS) uses NGS to facilitate the concurrent detection and sequence characterization of mutations, which allows researchers to prioritize mutants for further analyses. NGS increases the sensitivity of mutation detection and thus improves screening efficiency by allowing the pooling of more DNAs. Here we describe a protocol for TBS using rice as an example. First, DNA from a mutant population is quantified and combined in an overlapping pool design. Then, target genes are amplified from DNA pools and amplicons are combined to maximize throughput and increase likelihood of mutation detection during sequencing. Once sequence data is obtained, mutations are called using statistical approaches that weigh likelihood of rare mutations versus the probability of PCR and sequencing error.

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Discovery of Rare Mutations in Populations: TILLING by Sequencing

Cited by 236 publications

References 26 publications

Mutagenesis for Crop Breeding and Functional Genomics

Mutagenesis for Crop Breeding and Functional Genomics

Perspectives on the Application of Next-generation Sequencing to the Improvement of Africa’s Staple Food Crops

Next-Generation Sequencing for Targeted Discovery of Rare Mutations in Rice

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