A protocol for TILLING and Ecotilling in plants and animals

Till, Bradley J.; Zerr, Troy; Comai, Luca; Henikoff, Steven

doi:10.1038/nprot.2006.329

Cited by 193 publications

(179 citation statements)

References 22 publications

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“…For some deficiencies (Df(1)mal10/Binsn, Df(1)16-3-22/ FM6, Df(1)17-257/Binsn, and Df(1)ED7664/FM7h) we used TILLING (Till et al 2006) following the protocols of the Transgenomic SURVEYOR Mutation Detection kit (Transgenomic). This approach involves a DNA endonuclease that cleaves mismatches in heteroduplex DNA.…”

Section: Determination Of Molecular Breakpoints Of Chromosomal Deficimentioning

confidence: 99%

Incompatibility Between X Chromosome Factor and Pericentric Heterochromatic Region Causes Lethality in Hybrids Between Drosophila melanogaster and Its Sibling Species

Cattani

Presgraves

2012

Genetics

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The Dobzhansky-Muller model posits that postzygotic reproductive isolation results from the evolution of incompatible epistatic interactions between species: alleles that function in the genetic background of one species can cause sterility or lethality in the genetic background of another species. Progress in identifying and characterizing factors involved in postzygotic isolation in Drosophila has remained slow, mainly because Drosophila melanogaster, with all of its genetic tools, forms dead or sterile hybrids when crossed to its sister species, D. simulans, D. sechellia, and D. mauritiana. To circumvent this problem, we used chromosome deletions and duplications from D. melanogaster to map two hybrid incompatibility loci in F 1 hybrids with its sister species. We mapped a recessive factor to the pericentromeric heterochromatin of the X chromosome in D. simulans and D. mauritiana, which we call heterochromatin hybrid lethal (hhl), which causes lethality in F 1 hybrid females with D. melanogaster. As F 1 hybrid males hemizygous for a D. mauritiana (or D. simulans) X chromosome are viable, the lethality of deficiency hybrid females implies that a dominant incompatible partner locus exists on the D. melanogaster X. Using small segments of the D. melanogaster X chromosome duplicated onto the Y chromosome, we mapped a dominant factor that causes hybrid lethality to a small 24-gene region of the D. melanogaster X. We provide evidence suggesting that it interacts with hhl mau . The location of hhl is consistent with the emerging theme that hybrid incompatibilities in Drosophila involve heterochromatic regions and factors that interact with the heterochromatin. M ORE than 70 years ago, Dobzhansky (1937) and Muller (1942) proposed a simple two-locus model in which postzygotic isolation arises as a byproduct of divergence between effectively allopatric populations. Genes that function well in one species' genetic background might not be functional in a hybrid genetic background, rendering hybrids dead or sterile. Despite early acceptance of the Dobzhansky-Muller model, progress in identifying and characterizing factors involved in hybrid incompatibilities has remained slow. One of the reasons for the slow progress is that Drosophila melanogaster, with its many genetic tools, forms largely dead or sterile hybrids when crossed to its sister species D. simulans, D. sechellia, and D. mauritiana (Sturtevant 1920(Sturtevant , 1929Lachaise et al. 1986;Provine 1991;Barbash 2010).In general, three methods have been used to circumvent this problem. First, alleles that suppress postzygotic isolation, so-called hybrid rescue mutations, have been used to characterize hybrid inviability between D. melanogaster and its three sister species in the D. simulans complex. When D. melanogaster females are crossed to sibling species males, only hybrid females are produced while males die as larvae (Sturtevant 1920;Lachaise et al. 1986). These males are rescued by the D. melanogaster mutant allele of Hybrid male rescue (Hmr) (Hutter...

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Section: Determination Of Molecular Breakpoints Of Chromosomal Deficimentioning

confidence: 99%

Incompatibility Between X Chromosome Factor and Pericentric Heterochromatic Region Causes Lethality in Hybrids Between Drosophila melanogaster and Its Sibling Species

Cattani

Presgraves

2012

Genetics

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“…However, these methods are generally slow, costly, and labor intensive. Application of NGS has been shown to be a cost-effective mutation detection system by re-sequencing the gene of interest in mutagenized plants [90,91]. The availability of genome sequence enables the use of reverse genetic approaches to identify mutations in specific target genes, thereby accelerating the generation of novel phenotypes.…”

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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“…Typically, a PCR amplicon yield of 10 ng/μl is sufficient. The amount of genomic DNA needed to produce this amount can be roughly estimated by size of the genome (Till et al 2006b). The yield of PCR product should be sufficiently high to produce cleavage fragments visible by agarose gel electrophoresis (see Figs.…”

Section: Agarose Gel Electrophoresis and Data Analysismentioning

confidence: 99%

A Protocol for Validation of Doubled Haploid Plants by Enzymatic Mismatch Cleavage

Till

Hofinger

Şen

et al. 2016

Biotechnologies for Plant Mutation Breeding

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Doubled haploidy is an important tool for plant breeders. It provides a rapid means of developing recombinant populations consisting of individuals that are homozygous and therefore genetically fixed. Homozygosity is also important in plant mutation breeding where many induced mutations are predicted to be recessive and mutant alleles need to be in a homozygous state before new traits are expressed. While production of doubled haploids has been described for many plant species, efficient means to validate that produced materials are indeed homozygous are needed. Polymorphism discovery methods utilizing enzymatic mismatch cleavage are ideally suited for validation of doubled haploid plants. We describe here a low-cost protocol that utilizes self-extracted single-strand-specific nucleases, standard PCR reactions and agarose gel electrophoresis that can be applied to most plant species.

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A protocol for TILLING and Ecotilling in plants and animals

Cited by 193 publications

References 22 publications

Incompatibility Between X Chromosome Factor and Pericentric Heterochromatic Region Causes Lethality in Hybrids Between Drosophila melanogaster and Its Sibling Species

Incompatibility Between X Chromosome Factor and Pericentric Heterochromatic Region Causes Lethality in Hybrids Between Drosophila melanogaster and Its Sibling Species

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

A Protocol for Validation of Doubled Haploid Plants by Enzymatic Mismatch Cleavage

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