Generation and Characterization of the Western Regional Research Center Brachypodium T-DNA Insertional Mutant Collection

Abstract

The model grass Brachypodium distachyon (Brachypodium) is an excellent system for studying the basic biology underlying traits relevant to the use of grasses as food, forage and energy crops. To add to the growing collection of Brachypodium resources available to plant scientists, we further optimized our Agrobacterium tumefaciens-mediated high-efficiency transformation method and generated 8,491 Brachypodium T-DNA lines. We used inverse PCR to sequence the DNA flanking the insertion sites in the mutants. Using these flanking sequence tags (FSTs) we were able to assign 7,389 FSTs from 4,402 T-DNA mutants to 5,285 specific insertion sites (ISs) in the Brachypodium genome. More than 29% of the assigned ISs are supported by multiple FSTs. T-DNA insertions span the entire genome with an average of 19.3 insertions/Mb. The distribution of T-DNA insertions is non-uniform with a larger number of insertions at the distal ends compared to the centromeric regions of the chromosomes. Insertions are correlated with genic regions, but are biased toward UTRs and non-coding regions within 1 kb of genes over exons and intron regions. More than 1,300 unique genes have been tagged in this population. Information about the Western Regional Research Center Brachypodium insertional mutant population is available on a searchable website (http://brachypodium.pw.usda.gov) designed to provide researchers with a means to order T-DNA lines with mutations in genes of interest.

LACCASE5 Is Required for Lignification of the Brachypodium distachyon Culm

“…leaf) or in nonlignified tissue (endosperm). This coexpression study (Supplemental Table S3) revealed that both laccases are coexpressed with genes involved in monolignol biosynthesis, such as O-methyltransferase (Bradi3g16530; Bragg et al, 2012;Dalmais et al, 2013), cinnamoyl-CoA reductase (Bradi3g36887), or Phe ammonia lyase (Bradi3g49250 and Bradi3g49260). They are also coexpressed with three cellulose synthase genes specific to the secondary cell wall, CESA4 (Bradi3g28350), CESA7 (Bradi4g30540), and CESA8 (Bradi2g49912; Handakumbura et al, 2013).…”

Plant Immune Responses Against Viruses: How Does a Virus Cause Disease?

“…Its fully sequenced genome is highly syntenic to the genomes of important monocot crops such as wheat, rice, barley, millet, and maize, thus allowing functional and comparative genomic studies with potential for direct translation to field crops (International Brachypodium Initiative, 2010). Many genomic and functional resources have been developed around Brachypodium, including microarrays, yeast two-hybrid libraries, ;200 inbred accessions, EMS/TILLING lines, and annotated T-DNA knockout lines (Filiz et al, 2009;Fox et al, 2010;International Brachypodium Initiative, 2010;Brkljacic et al, 2011;Cao et al, 2011;Bragg et al, 2012). Brachypodium has become an accepted model plant for research related to plant-pathogen interactions, as evidenced by the growing list of publications (Routledge et al, 2004;Parker et al, 2008;Peraldi et al, 2011;Cui et al, 2012;Lee et al, 2012;Mandadi and Scholthof, 2012).…”

Brachypodium as an emerging model for cereal–pathogen interactions

“…A substantial collection of brachypodium T-DNA mutants has been created (Thole et al, 2010;Bragg et al, 2012), and efforts are underway to develop homozygous lines for insertions in or near genes. Since publication of the initial description of the WRRC brachypodium T-DNA collection (Bragg et al, 2012), the resource has expanded to contain approx. 22 000 mutants from which >25 000 insertion sites have been identified (http://brachypodium.pw.usda.gov/TDNA/).…”

“…However, genomic brachypodium resources are rapidly accumulating (Brkljacic et al, 2011); key resources include a sizable collection of T-DNA mutants (Bragg et al, 2012) and a publicly available TILLING resource (Dalmais et al, 2013), as noted above. Increasingly powerful sequencing technologies are facilitating high-throughput characterization of mutant collections (Polko et al, 2012) and rapid identification of causal mutations within TILLING populations (Nordstrom et al, 2013).…”