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

Jennifer N Bragg,Yong Q Gu,Mara E Guttman,Sean P Gordon,John P Vogel,Jiajie Wu,Roger Thilmony,Gerard R Lazo,Samuel P Hazen

doi:10.1371/journal.pone.0041916

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.

Highlights

Brachypodium distachyon (Brachypodium) is an annual grass native to the Mediterranean and Middle East and is a member of the Pooideae subfamily [1]
Optimization of transformation We performed a series of experiments to improve transformation efficiency, defined as the number of transgenic plantlets regenerated per number of callus pieces co-cultivated with Agrobacterium, and decrease the labor required per transgenic plant produced
By modifying our transformation protocol we were able to significantly increase transformation efficiency and reduce the length of time necessary to generate transgenic plants

Summary

Introduction

Brachypodium distachyon (Brachypodium) is an annual grass native to the Mediterranean and Middle East and is a member of the Pooideae subfamily [1] This group contains cereal and forage grasses including economically important species with complex genomes such as Triticum aestivum (bread wheat), Hordeum vulgare (barley), and Avena sativa (oats). Many aspects of grass biology, such as cell wall composition and architecture [4,5], development, and grain properties, are distinct from dicots In these cases, a grass such as Brachypodium, that possesses all of the biological, physical and genomic attributes required for an experimental system [2,6,7], represents a more relevant model than the dicot model Arabidopsis. Brachypodium is self-fertile and rarely outcrosses [12] which facilitates breeding homozygous lines for applications that require the maintenance of large numbers of independent genotypes, such as mapping experiments, mutant analysis, and studies of natural diversity [13,14,15,16,17]

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