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Application of TILLING and EcoTILLING as Reverse Genetic Approaches to Elucidate the Function of Genes in Plants and Animals.

Barkley NA, Wang ML - Curr. Genomics (2008)

Bottom Line: Ultimately, it can provide an allelic series of silent, missense, nonsense, and splice site mutations to examine the effect of various mutations in a gene.TILLING has proven to be a practical, efficient, and an effective approach for functional genomic studies in numerous plant and animal species.EcoTILLING, which is a variant of TILLING, examines natural genetic variation in populations and has been successfully utilized in animals and plants to discover SNPs including rare ones.

View Article: PubMed Central - PubMed

Affiliation: USDA-ARS, Plant Genetic Resources Conservation Unit (PGRCU), 1109 Experiment Street, Griffin, GA 30223, USA.

ABSTRACT
With the fairly recent advent of inexpensive, rapid sequencing technologies that continue to improve sequencing efficiency and accuracy, many species of animals, plants, and microbes have annotated genomic information publicly available. The focus on genomics has thus been shifting from the collection of whole sequenced genomes to the study of functional genomics. Reverse genetic approaches have been used for many years to advance from sequence data to the resulting phenotype in an effort to deduce the function of a gene in the species of interest. Many of the currently used approaches (RNAi, gene knockout, site-directed mutagenesis, transposon tagging) rely on the creation of transgenic material, the development of which is not always feasible for many plant or animal species. TILLING is a non-transgenic reverse genetics approach that is applicable to all animal and plant species which can be mutagenized, regardless of its mating / pollinating system, ploidy level, or genome size. This approach requires prior DNA sequence information and takes advantage of a mismatch endonuclease to locate and detect induced mutations. Ultimately, it can provide an allelic series of silent, missense, nonsense, and splice site mutations to examine the effect of various mutations in a gene. TILLING has proven to be a practical, efficient, and an effective approach for functional genomic studies in numerous plant and animal species. EcoTILLING, which is a variant of TILLING, examines natural genetic variation in populations and has been successfully utilized in animals and plants to discover SNPs including rare ones. In this review, TILLING and EcoTILLING techniques, beneficial applications and limitations from plant and animal studies are discussed.

No MeSH data available.


EcoTILLING images produced from a collection of Vigna radiata. Polymorphic sites are marked with boxes and were subsequently confirmed by sequencing. A size standard (50-700 bp) was included to estimate the size of the cleaved fragments and the target gene.
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Figure 2: EcoTILLING images produced from a collection of Vigna radiata. Polymorphic sites are marked with boxes and were subsequently confirmed by sequencing. A size standard (50-700 bp) was included to estimate the size of the cleaved fragments and the target gene.

Mentions: EcoTILLING was employed to identify polymorphisms in a germplasm collection of mung bean (Vigna radiata (L.) R. Wilczek), which previously showed limited diversity (Barkley et al. submitted data). Vigna radiata, which is classified in the family Fabaceae, is an important economic crop and a dietary staple in many developing countries [70]. The species radiata can be further subdivided into botanical varieties such as radiata and sublobata, of which sublobata is currently acknowledged as the putative progenitor of radiata [71, 72]. Polymorphic sites were abundant when comparing V. radiata var. sublobata to V. radiata var. radiata; however, when accessions of V. radiata var. radiata were pooled together relatively few polymorphisms were identified. This suggests that accessions classified as V. radiata var. radiata could have a narrow genetic base (Fig. 2). Morphological data taken from accessions of V. radiata var. radiata also demonstrated limited diversity in the flowers and pod descriptors. The majority of polymorphisms detected between sublobata and radiata were found in putative introns. The banding patterns varied from simple to complex as the number of DNA polymorphisms between two pooled samples increased. Overall, this modification of the TILLING method has proven to identify natural genetic variation in a gene of interest and to mine for SNPs in plants. Furthermore, it can be effectively used as an efficient, rapid technique to identify DNA polymorphisms in populations with high genetic identity and to mine for SNPs in collections of plant germplasm (Barkley et al. submitted data).


Application of TILLING and EcoTILLING as Reverse Genetic Approaches to Elucidate the Function of Genes in Plants and Animals.

Barkley NA, Wang ML - Curr. Genomics (2008)

EcoTILLING images produced from a collection of Vigna radiata. Polymorphic sites are marked with boxes and were subsequently confirmed by sequencing. A size standard (50-700 bp) was included to estimate the size of the cleaved fragments and the target gene.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2682938&req=5

Figure 2: EcoTILLING images produced from a collection of Vigna radiata. Polymorphic sites are marked with boxes and were subsequently confirmed by sequencing. A size standard (50-700 bp) was included to estimate the size of the cleaved fragments and the target gene.
Mentions: EcoTILLING was employed to identify polymorphisms in a germplasm collection of mung bean (Vigna radiata (L.) R. Wilczek), which previously showed limited diversity (Barkley et al. submitted data). Vigna radiata, which is classified in the family Fabaceae, is an important economic crop and a dietary staple in many developing countries [70]. The species radiata can be further subdivided into botanical varieties such as radiata and sublobata, of which sublobata is currently acknowledged as the putative progenitor of radiata [71, 72]. Polymorphic sites were abundant when comparing V. radiata var. sublobata to V. radiata var. radiata; however, when accessions of V. radiata var. radiata were pooled together relatively few polymorphisms were identified. This suggests that accessions classified as V. radiata var. radiata could have a narrow genetic base (Fig. 2). Morphological data taken from accessions of V. radiata var. radiata also demonstrated limited diversity in the flowers and pod descriptors. The majority of polymorphisms detected between sublobata and radiata were found in putative introns. The banding patterns varied from simple to complex as the number of DNA polymorphisms between two pooled samples increased. Overall, this modification of the TILLING method has proven to identify natural genetic variation in a gene of interest and to mine for SNPs in plants. Furthermore, it can be effectively used as an efficient, rapid technique to identify DNA polymorphisms in populations with high genetic identity and to mine for SNPs in collections of plant germplasm (Barkley et al. submitted data).

Bottom Line: Ultimately, it can provide an allelic series of silent, missense, nonsense, and splice site mutations to examine the effect of various mutations in a gene.TILLING has proven to be a practical, efficient, and an effective approach for functional genomic studies in numerous plant and animal species.EcoTILLING, which is a variant of TILLING, examines natural genetic variation in populations and has been successfully utilized in animals and plants to discover SNPs including rare ones.

View Article: PubMed Central - PubMed

Affiliation: USDA-ARS, Plant Genetic Resources Conservation Unit (PGRCU), 1109 Experiment Street, Griffin, GA 30223, USA.

ABSTRACT
With the fairly recent advent of inexpensive, rapid sequencing technologies that continue to improve sequencing efficiency and accuracy, many species of animals, plants, and microbes have annotated genomic information publicly available. The focus on genomics has thus been shifting from the collection of whole sequenced genomes to the study of functional genomics. Reverse genetic approaches have been used for many years to advance from sequence data to the resulting phenotype in an effort to deduce the function of a gene in the species of interest. Many of the currently used approaches (RNAi, gene knockout, site-directed mutagenesis, transposon tagging) rely on the creation of transgenic material, the development of which is not always feasible for many plant or animal species. TILLING is a non-transgenic reverse genetics approach that is applicable to all animal and plant species which can be mutagenized, regardless of its mating / pollinating system, ploidy level, or genome size. This approach requires prior DNA sequence information and takes advantage of a mismatch endonuclease to locate and detect induced mutations. Ultimately, it can provide an allelic series of silent, missense, nonsense, and splice site mutations to examine the effect of various mutations in a gene. TILLING has proven to be a practical, efficient, and an effective approach for functional genomic studies in numerous plant and animal species. EcoTILLING, which is a variant of TILLING, examines natural genetic variation in populations and has been successfully utilized in animals and plants to discover SNPs including rare ones. In this review, TILLING and EcoTILLING techniques, beneficial applications and limitations from plant and animal studies are discussed.

No MeSH data available.