Functional Genomics in Plants

Abstract

The first complete plant genome sequence to be determined was that of the model plant Arabidopsis thaliana, which was published in 2000. This was followed by the first drafts of the rice genome sequence in 2002. Since the publication of these landmark genomes, the development of high throughput sequencing technologies has reduced the time and cost of obtaining genome sequences to a fraction of that required a decade ago. This has enabled the sequencing of many plant genomes and thus a vast amount of genome sequence information is available to the researcher. The goal of functional genomics is to determine how these genome sequences generate plant phenotypes. The focus of most functional genomics research is to determine the function of all the genes involved in a particular process. To achieve this, functional genomics uses techniques and analyses that can survey the entire complement of genes in a genome. This chapter discusses the most commonly used functional genomics methods.

Key Concepts:

  • Advances in sequencing technologies are making entire genome sequences relatively easy to obtain.

  • The goal of plant functional genomics is to understand how the genome generates the phenotype of the plant.

  • Comparison to other gene sequences can often identify a likely function for a gene.

  • Array and sequencing based methods can be used to show where and when all the genes in an organism are expressed.

  • Gene function can be determined by knocking out or reducing gene function and can be achieved by insertion, chemical mutagenesis or RNAi.

  • High throughput methods using recombination‐based cloning enable many tests of gene function to be applied on a genome‐wide scale.

Keywords: expression arrays; transcriptomics; reverse genetics; sequence annotations; insertion mutants; RNAi

Figure 1.

Array technologies for comprehensive assessment of gene expression. Microarrays are made by amplification of individual inserts from plasmid libraries containing different cDNAs, genes or gene fragments by polymerase chain reaction (PCR). The amplified fragments are individually dotted onto a glass slide by a gridding robot (top left of figure). DNA chips are synthesised by printing or deposition of oligonucleotides designed from genome or cDNA sequence data (bottom left of figure). Sample mRNAs are labelled with fluorescently labelled nucleotides, for example by reverse transcription with an oligo dT primer to start the labelling reaction. By labelling two samples with different fluorescent dyes a comparison of mRNA abundance between the two samples can be made. The two labelled samples are mixed and hybridised to the same array or chip. The fluorescence signal at each element on the array is visualised by scanning with a sensitive microfluorimeter. After normalisation of the signals from the two dyes, yellow indicates equal expression while red or green signals indicate that the gene was more highly expressed in sample 1 or 2 respectively. DNA chips are often hybridised with one sample at once and the signal intensities for the oligonucleotides for each gene are then used to quantify mRNA expression and these values can then be compared between chips.

Figure 2.

Commonly used approaches to obtaining loss‐of‐gene function or reduced gene function in plants. (a) Large populations of insertion mutants are produced using either T‐DNA or transposable elements. DNA is prepared from individuals in the population and then pooled for screening by PCR. Typically, PCR is carried out using a primer in the insertion and another in the gene of interest; multiple primer sets can be used to cover a whole gene and allow for both orientations of the inserted elements. Further rounds of PCR are then carried out on DNA from individual plants from positive pools to identify the plant with the insertion in the gene of interest. (b) Databases of known T‐DNA and transposon insertion sites exist for many model species. These can be used to identify and order insertion mutants in a candidate gene. Small genes, such as those encoding miRNAs, often have no insertion and hence other methods need to be used to assess their function. (c) The mRNA of a candidate gene can be targeted by either hairpin RNAi where an inverted repeat of about 300–500 nucleotides of the target mRNA is expressed in a plant or by an artificial miRNA construct where the precursor of a plant miRNA is engineered to produce a miRNA that targets the candidate gene. (d) TILLING identifies plants carrying point mutations in a candidate gene. To identify plants with point mutations in the gene of interest, PCR is carried out across a region of the candidate gene in pools of DNA from plant lines carrying point mutations. The PCR products are melted and re‐annealed giving rise to a base mismatch where a mutant and wild type sequence are annealed. The mismatch is digested by Cel I; cleavage is usually detected by electrophoresis methods.

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Further Reading

Kahl G and Meksem K (2008) The Handbook of Plant Functional Genomics: Concepts and Protocols. Weinheim: Wiley‐VCH.

Morot‐Gaudry JF, Lea P and Briat JF (2007) Functional Plant Genomics. Enfield, NH: Science Publishers.

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Helliwell, Chris(May 2012) Functional Genomics in Plants. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002023.pub2]