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  • Review Article
  • Published:

Moving forward in reverse: genetic technologies to enable genome-wide phenomic screens in Arabidopsis

Nature Reviews Genetics volume 7, pages 524–536 (2006)Cite this article

Key Points

  • Analysis of the genome sequence of Arabidopsis thaliana, as well as that of all other organisms for which whole-genome sequences are available, has revealed the existence of a surprisingly large number of genes of unknown function. Genomic approaches have been developed to investigate different aspects of gene function, including the location and timing of gene expression, and whether the corresponding protein products interact to form functional complexes. In most cases, however, such information is still insufficient to conclusively assign a gene to a specific biological process.

  • Mutant analysis has been extensively and fruitfully used to determine gene function. Two basic strategies, forward and reverse genetics, are usually used.

  • In forward genetics, large populations of randomly mutagenized individuals are screened for phenotypic alterations in a particular biological process. Although recent DNA array-based mapping approaches are expected to hasten map-based cloning of genes, the effort required to identify the mutation that underlies a particular phenotype of interest can be significant. Additionally, the identification of genes that correspond to large numbers of mutations is more or less a serial process.

  • By contrast, in the reverse genetic approach, a gene or genes of interest are selected on the basis of genotypic information, such as sequence homology to a gene of known function or the particular pattern of expression. Mutations in these genes are obtained simply using DNA sequence-based computer searches of mutation databases, and the phenotypes of the mutant lines are then examined.

  • Various experimental procedures have been developed to generate mutations in the A. thaliana genome. Different mutagens (chemical, physical and biological agents) produce specific types of lesion and vary in their efficiency and degree of randomness of mutations. The relative ease by which transposons and Agrobacterium-mediated T-DNA can be used to create sequence-tagged insertion mutations in plant genomes has made these the mutagens of choice among reverse genetic strategies. Nevertheless, insertional mutagenesis has clear limitations, and complementary gene-directed mutagenesis methodologies are often used in parallel.

  • Several gene-directed mutagenesis strategies have been developed in A. thaliana. Targeted gene replacement by homologous recombination is the 'holy grail' of reverse genetic approaches. So far, the low efficiency of this methodology in plants has limited its use. However, recent studies have shown that substantially increased rates of recombination might be possible.

  • The low success rate of current gene-replacement techniques has prompted the development of alternative strategies such as gene silencing, which relies on the alteration of the gene activity rather than on changes in the gene sequence. Directed genome-sequence alterations that are based on the DNA-recognition properties of engineered zinc-finger proteins could also represent a viable alternative.

  • In the near future, parallel functional analysis of all genes in the A. thaliana genome will become possible with the help of one or more of the methodologies described in this review. Utilization of genome-wide collections of gene-indexed mutants in traditional forward genetic screens promises to accelerate the discovery of gene functions in A. thaliana to unprecedented rates.

Abstract

Genome sequencing, in combination with various computational and empirical approaches to sequence annotation, has made possible the identification of more than 30,000 genes in Arabidopsis thaliana. Increasingly sophisticated genetic tools are being developed with the long-term goal of understanding how the coordinated activity of these genes gives rise to a complex organism. The combination of classical forward genetics with recently developed genome-wide, gene-indexed mutant collections is beginning to revolutionize the way in which gene functions are studied in plants. High-throughput screens using these mutant populations should provide a means to analyse plant gene functions — the phenome — on a genomic scale.

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Figure 1: From genome sequence to gene function.
Figure 2: Generation of genetic diversity.
Figure 3: Systematic reverse genetics.

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Acknowledgements

We thank A. Stepanova for comments on the manuscript. J.R.E is supported by grants from the US National Science Foundation, National Institutes of Health and Department of Energy. J.M.A. is supported by North Carolina State University and grants from the National Science Foundation.

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Authors and Affiliations

  1. Department of Genetics, North Carolina State University, Raleigh, 27695-7614, North Carolina, USA

    Jose M. Alonso

  2. The Salk Institute for Biological Studies, Plant Biology and Genomic Analysis Laboratories, La Jolla, 92037, California, USA

    Joseph R. Ecker

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  1. Jose M. Alonso
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Correspondence to Jose M. Alonso or Joseph R. Ecker.

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FURTHER INFORMATION

Arabidopsis Biological Resource Center Stocks

Arabidopsis 2010

Lehle Seeds

New Wisconsin T-DNA Lines — pDs–Lox Vector web site

Nottingham Arabidopsis Stock Center

SIGnAL — Salk Institute Genomic Analysis Laboratory web site

The Arabidopsis Information Resource homepage

Glossary

Whole-genome tiling microarray

A high-density oligonucleotide array that represents the majority of DNA sequences of an organism's genome.

Massively parallel signature sequencing

A sequencing procedure that allows the reading, in parallel, of short sequence segments, about 17 or 12 nt long, from hundreds of thousands of microbead-attached cDNAs.

Serial analysis of gene expression

A technique that is used to obtain short sequence tags (typically 16 nt long) from large numbers of cDNA clones by cutting, concatemerizing and finally sequencing cDNA fragments.

Shotgun mass spectrometry

A bottom-up proteomics approach that is used in the identification of individual components of a complex protein mixture by the identification of peptide fragments on the basis of their mass.

Mismatch-repair detection on tag arrays

A hybridization-based technique that combines a bacterial selection assay and microarray analysis to rapidly scan large genomic regions for the presence of DNA polymorphisms.

loxP

A sequence that is specifically recognized by the Cre recombinase from the bacteriophage P1. Cre catalyses the recombination between two loxP sequences.

RNA interference

A form of gene silencing in which dsRNA induces the degradation of homologous endogenous mRNA transcripts, thereby mimicking the effect of reduction, or loss, of gene activity.

Co-suppression

Silencing the expression of an endogenous gene, which is caused by the expression of a transgene bearing high levels of sequence identity with the endogenous gene.

Post-transcriptional gene silencing

Refers to the general mechanisms that are involved in gene silencing at the post-transcriptional level independently of the initial silencing agent, that is, double-stranded, antisense or sense RNA.

Small interfering RNA

20–25 nucleotide long RNA molecules that are generated during the post-transcriptional gene-silencing process and act as key determinants of the sequence specificity of the silencing mechanism.

MicroRNA genes

Genome-encoded or artificial genes; the RNAs that are transcribed from them are processed to generate small RNA fragments (20–25 nt long) that target specific mRNAs for degradation or inhibit their translation into proteins.

Saturation mutagenesis

Mutagenesis as a result of which there should be a 100% probability of identifying at least one mutation in any given gene.

Thermal asymmetrical interlaced PCR

A PCR-based strategy that utilizes nested primers, which are complementary to a known DNA sequence, and degenerate primers to amplify the unknown flanking DNA regions.

Adaptor ligation PCR

A PCR-based approach that is used to determine the sequence flanking a DNA region of known sequence. It utilizes two sets of nested primers and a DNA adaptor that, after its ligation to fragmented DNA, facilitates the amplification of the desired DNA region.

Accession

A sample of a plant variety that is collected at a specific location and time. The terms ecotype, wild type and accession are not uniformly used in the Arabidopsis field and often cause confusion. The term accession is probably the most appropriate way to describe the Arabidopsis laboratory lines that are collected initially from the wild.

Virus-induced gene silencing

Gene silencing that is triggered by a viral vector that encodes a dsRNA with high sequence identity to an endogenous gene.

Sequencing by synthesis

A group of new sequencing technologies in which the identity of the bases is determined as they are added to a newly synthesized DNA molecule.

Ultra high-throughput sequencing

A compendium of new sequencing technologies that have a common final aim of accelerating (from years to days or hours) and reducing the cost (from millions to hundreds or thousands of dollars) of genome sequencing.

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Alonso, J., Ecker, J. Moving forward in reverse: genetic technologies to enable genome-wide phenomic screens in Arabidopsis. Nat Rev Genet 7, 524–536 (2006). https://doi.org/10.1038/nrg1893

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