Brachypodium - Bioenergy Crop Plants - page 605

for transferring known DNA sequences into random sites in the host genome. In the resulting
mutant lines, the T-DNA or transposon sequence serves as a tag that can be used to locate the
DNA insertion site within the genome. Large, freely available collections of sequence-indexed,
tagged lines have been an extremely valuable tool in Arabidopsis research, permitting both forward
genetic screens for a particular phenotype within the mutant populations and reverse genetic studies
in which researchers can identify disruptions of specific genes using a simple BLAST search. The
value of a collection of sequence-indexed mutants to facilitate study of candidate genes selected
from the enormous amount of sequence and bioinformatic data currently available is tremendous.
Each insertion event has the potential to cause a knockout phenotype; however, vectors can
also be designed to achieve various research goals, including the identification of promoters and
overexpression of nearby genes. In a gene trap construct, a promoter-less reporter gene (e.g., GUS or
GFP) is placed at the end of the T-DNA sequence that is transferred to the plant genome (An et al.
2005). If the vector DNA integrates downstream of a promoter, reporter gene expression could be
used to infer the expression pattern of the disrupted gene and provide clues about the role of the
disrupted gene. Inclusion of splice acceptor sites adjacent to the reporter genes permits splicing
should the vector DNA fall into an intron. Activation tagging constructs place transcriptional
enhancers within the vector DNA to increase the transcription of genes close to the insertion
site (Weigel et al. 2000; Fits et al. 2001; Nakazawa et al. 2003). Activation tagging is designed to
overexpress nearby genes while still maintaining a wild-type expression pattern, and it is particularly
well suited to studying genes with redundant functions in which knockouts in one family member
fail to produce a phenotype. This strategy can also provide insight into complex processes such as
cell wall biosynthesis because activation tagging can activate global control genes.
A large sequence-indexed Brachypodium mutant population would be a powerful research tool,
and multiple groups have started assembling such a population. The choice of the most efficient
method to produce these collections depends on the efficiency of transformation versus the efficiency
of producing transposon-tagged mutants. Although the transposon approach has the potential
to rapidly generate a large number of insertional mutants, this technique requires optimization
before it will be a productive means of generating Brachypodium mutants. We have transformed
Brachypodium with vectors containing the most commonly used transposons ( Ac / Ds and En / Spm )
and demonstrated that they were active in the Brachypodium genome. However, most transgenic
plants died before setting seed, possibly because the transposons were too active or activated an
endogenous transposon (J. Bragg, unpublished). In contrast, the efficiency of T-DNA tagging has
increased with the optimization of transformation techniques, and one person can easily produce
100 lines per week. Using this approach, at least two substantial populations are in development.
The BrachyTAG project at the John Innes Centre currently lists 4500 T-DNA lines for distribution
to the public. Of these, 1005 have flanking sequence tags, and 61 have nearby genes identified.
Additionally, we have developed over 8000 T-DNA lines that are available for distribution via the
USDA Brachypodium Genome Resources site (see Table 23.2 for links to these resources).
23.3.5 c roSSing B rachypodium
An efficient method of crossing Brachypodium is required for it to serve as a tractable model
genetic system [e.g., to allow positional cloning and mapping of quantitative trait loci (QTLs)].
Brachypodium is primarily an inbreeding species, and flowers rarely open under greenhouse and
growth chamber conditions (Figure 23.1e). In observations of rare open flowers, anthers have already
dehisced on the stigma. This suggests that even open flowers primarily produce self-pollinations,
a notion supported by the highly homozygous nature of wild Brachypodium accessions (Vogel et
al. 2009). Two similar methods, one using a microscope and the other a jeweler's loupe, have been
developed for crossing Brachypodium. The protocols (available at http://brachypodium.pw.usda.
gov/ and http://www.ars.usda.gov/pandp/docs.htm?docid=18531) contain detailed pictures of the
flower stages appropriate for crossing and of each step in the procedure. These protocols are based