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United States Department of Agriculture

Agricultural Research Service

Research Project: Genetic Foundations for Bioenergy Feedstocks

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Title: Genome diversity in Brachypodium distachyon: deep sequencing of highly diverse inbred lines)

item Gordon, Sean
item Priest, Henry
item Des marais, David
item Schackwitz, Wendy
item Figueroa, Melania
item Martin, Joel
item Bragg, Jennifer
item Tyler, Ludmila
item Lee, Cheng-ruel
item Bryant, Doug
item Wang, Wenqin
item Messing, Joachin
item Manzaneda, Antonio
item Barry, Kerrie
item Garvin, David
item Budak, Hikmet
item Tuna, Metin
item Mitchell-olds, Thomas
item Pfender, William
item Juenger, Thomas
item Mockler, Todd
item Vogel, John

Submitted to: Plant Journal
Publication Type: Peer reviewed journal
Publication Acceptance Date: 5/23/2014
Publication Date: 5/31/2014
Citation: Gordon, S.P., Priest, H., Des Marais, D., Schackwitz, W., Figueroa, M., Martin, J., Bragg, J., Tyler, L., Lee, C., Bryant, D., Wang, W., Messing, J., Manzaneda, A., Barry, K., Garvin, D.F., Budak, H., Tuna, M., Mitchell-Olds, T., Pfender, W.F., Juenger, T., Mockler, T., Vogel, J.P. 2014. Genome diversity in Brachypodium distachyon: deep sequencing of highly diverse inbred lines. Plant Journal. 79:361-374.

Interpretive Summary: Considerable natural variation in many agriculturally relevant traits (e.g. drought tolerance, biomass yield, height, flowering time, etc.) is present in existing germplasm collections of the model grass Brachypodium distachyon. This variation can be used as a tool to understand the molecular mechanisms underlying the biological processes controlling these traits. To facilitate the study of natural variation in B. distachyon we sequenced the genomes of six diverse natural accessions to high coverage using the Illumina sequencing platform. We observed considerable genomic variation including 3,803,592 unique single nucleotide polymorphisms. We maximized the number and type of polymorphisms identified by using multiple analysis methods. To ensure the quality of the final datasets, we compared our output to independent data sets. In addition, we sequenced the transcriptome of the most divergent accession and conducted a drought response experiment to identify genes up or down regulated in response to drought in all the lines. Our data is available through and will be invaluable to the research community for future investigations of the genetic basis of natural phenotypic variation and as a source of markers for mapping mutations.

Technical Abstract: Natural variation provides a powerful opportunity to study the genetic basis of biological traits. Brachypodium distachyon is a broadly distributed diploid model grass with a small genome and a large collection of diverse inbred lines. As a step towards understanding the genetic basis of the natural variation in B. distachyon, we sequenced the reference line, Bd21, and six divergent lines to 34- to 58-fold coverage with paired-end Illumina reads. We identified 3,803,592 unique single nucleotide polymorphisms (SNPs) relative to the reference genome and generated a subset of 2,485,097 high-confidence non-redundant SNPs by comparing the output of two SNP calling programs. The high-confidence SNP set contained 96.6% of the SNPs previously used to produce a genetic linkage map. We further identified more than 1,000,000 small and large indels that account for a non-redundant 11.3Mb of sequence inserted or deleted among the lines relative to the reference. We generated assemblies and gene annotations for each line, and we used deep mRNA sequencing (mRNA-Seq) to produce a de novo transcriptome for Bd1-1, the line most divergent from Bd21, revealing more than 2,000 transcripts absent from the reference annotation. We integrated mRNA-Seq data with genomic variant predictions from the six divergent lines and Bd21 to validate the effect of sequence variants on transcript abundance and structure. In addition, we conducted quantitative mRNA-Seq experiments and showed significant differences in expression responses to water deficit in all lines. The genome sequence data, available for download and visualization at, will be a powerful resource for examining the genetic and molecular control of natural trait variation in B. distachyon.

Last Modified: 8/24/2016
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