A TILLING Reverse Genetics Tool and a Web-Accessible Collection of Mutants of the Legume <i>Lotus japonicus</i>

Perry, Jillian; Wang, Trevor L.; Welham, Tracey; Gardner, Sarah; Pike, Jodie; Yoshida, Satoko; Parniske, Martin

doi:10.1104/pp.102.017384

Cited by 303 publications

(219 citation statements)

References 13 publications

(16 reference statements)

Supporting

Mentioning

212

Contrasting

Unclassified

Order By: Relevance

“…For example, TILLING organisms with large introns, such as zebrafish, requires short exon targets, leading to the development of alternative protocols for zebrafish TILLING 12,13 . In addition, TILLING can be performed using alternative readout platforms including slab gel and capillary systems 7 (http://www.jicgenomelab.co.uk/services/ mutation-detection/development.html). Although many groups use an enzymatic mismatch cleavage approach similar to that described in this protocol, other mutation discovery methods can potentially be substituted including denaturing HPLC and resequencing (e.g., see refs.…”

Section: Overview Of Tillingmentioning

confidence: 99%

“…TIL-LING is a reverse genetic strategy that combines traditional mutagenesis with high-throughput discovery of single-nucleotide changes 2,3 . The method is general and, following its original application to the model plant Arabidopsis thaliana, has been applied to a variety of plant and animal species including maize, lotus, barley, wheat, Drosophila and zebrafish [4][5][6][7][8][9] .…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A protocol for TILLING and Ecotilling in plants and animals

et al. 2006

View full text Add to dashboard Cite

We describe Targeting-Induced Local Lesions IN Genomes (TILLING), a reverse-genetic strategy for the discovery and mapping of induced mutations. TILLING is suitable for essentially any organism that can be mutagenized. The TILLING procedure has also been adapted for the discovery and cataloguing of natural polymorphisms, a method called Ecotilling. To discover nucleotide changes within a particular gene, PCR is performed with gene-specific primers that are end-labeled with fluorescent molecules. After PCR, samples are denatured and annealed to form heteroduplexes between polymorphic DNA strands. Mismatched base pairs in these heteroduplexes are cleaved by digestion with a single-strand specific nuclease. The resulting products are size-fractionated using denaturing polyacrylamide gel electrophoresis and visualized by fluorescence detection. The migration of cleaved products indicates the approximate location of nucleotide polymorphisms. Throughput is increased and costs are reduced by sample pooling, multi-well liquid handling and automated gel band mapping. Once genomic DNA samples have been obtained, pooled and arrayed, thousands of samples can be screened daily.

show abstract

Section: Overview Of Tillingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A protocol for TILLING and Ecotilling in plants and animals

et al. 2006

View full text Add to dashboard Cite

show abstract

“…Recently, there are several reports of EMC using different kinds of enzymes [56][57][58][59][60][61][62][63]. Among them CEL nuclease is potentially the most effective enzyme for EMC reported recently [64][65][66][67][68][69][70] and is now commercially available as SURVEYOR Nuclease S from Transgenomic (Omaha, NE, USA) [71][72][73].…”

Section: Introductionmentioning

confidence: 99%

Development of a simple and highly sensitive mutation screening system by enzyme mismatch cleavage with optimized conditions for standard laboratories

Tsuji

Niida

2008

Electrophoresis

View full text Add to dashboard Cite

Efficient screening of unknown DNA variations is one of the substantive matters of molecular biology even today. Historically, single-strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) are the most commonly used methods for detecting DNA variations everywhere in the world because of their simplicity. However, the sensitivity of these methods is not satisfactory for screening purpose. Recently, several new PCR based mutation screening methods have been developed, but most of them require special instruments and adjustment of conditions for each DNA sequence to attain the maximum sensitivity, eventually becoming as inconvenient as old methods. Enzyme mismatch cleavage (EMC) is potentially an ideal screening method. With high performance nucleases and once experimental conditions are optimized, it requires only conventional staff and conditions remain the same for each PCR product. In this study we tested four commercially available endonucleases for EMC and optimized the electrophoresis and developing conditions. We prepared 25 known DNA variations consisting of 18 single base substitutions (8 transitions and 10 transversions, including all possible sets of mismatches) and 7 small deletions or insertions. The combination of CEL nuclease, 12% polyacrylamide gel electrophoresis and rapid silver staining can detect all types of mutations and achieved 100% sensitivity.

show abstract

“…Lotus japonicus [8], a model plant in legumes, is an amenable system to study the nodulation and other development processes [9][10][11]. The whole-genome sequencing project has been carried out [12], and several L. japonicus Chr.…”

Section: Introductionmentioning

confidence: 99%

Wrinkled petals and stamens 1, is required for the morphogenesis of petals and stamens in Lotus japonicus

et al. 2006

View full text Add to dashboard Cite

Although much progress has been made in understanding how floral organ identity is determined during the floral development, less is known about how floral organ is elaborated in the late floral developmental stages. Here we describe a novel floral mutant, wrinkled petals and stamens1 (wps1), which shows defects in the development of petals and stamens. Genetic analysis indicates that wps1 mutant is corresponding to a single recessive locus at the long arm of chromosome 3. The early development of floral organs in wps1 mutant is similar to that in wild type, and the malfunction of the mutant commences in late developmental stages, displaying a defect on the appearance of petals and stamens. In the mature flower, petals and stamen filaments in the mutant are wrinkled or folded, and the cellular morphology under L1 layer of petals and stamen filaments is abnormal. It is found that the expression patterns of floral organ identity genes are not affected in wps1 mutants compared with that of wild type, consistent with the unaltered development of all floral organs. Furthermore, the identities of epidermal cells in different type of petals are maintained. The histological analysis shows that in wps1 flowers all petals are irregularly folded, and there are knotted structures in the petals, while the shape and arrangement of inner cells are malformed and unorganized. Based on these results, we propose that Wps1 acts downstream to the class B floral organ identity genes, and functions to modulate the cellular differentiation during the late flower developmental stages.

show abstract

A TILLING Reverse Genetics Tool and a Web-Accessible Collection of Mutants of the Legume Lotus japonicus

Cited by 303 publications

References 13 publications

A protocol for TILLING and Ecotilling in plants and animals

A protocol for TILLING and Ecotilling in plants and animals

Development of a simple and highly sensitive mutation screening system by enzyme mismatch cleavage with optimized conditions for standard laboratories

Wrinkled petals and stamens 1, is required for the morphogenesis of petals and stamens in Lotus japonicus

Contact Info

Product

Resources

About