Location: Crop Improvement and Genetics ResearchTitle: Sequencing and functional validation of the JGI Brachypodium distachyon T-DNA collection Author
|Hsia, Mon - University Of California|
|O'malley, Ronan - Salk Institute|
|Cartwright, Amy - Joint Genome Institute|
|Nieu, Rita - University Of California|
|Gordon, Sean - Joint Genome Institute|
|Kelly, Sandra - Agriculture Canada|
|Wood, Delilah - De|
|Zhao, Yunjun - National Center For Agriculture And Forestry Technologies (CENTA)|
|Jordan, Mark - Agriculture Canada|
|Pauly, Markus - University Of California|
|Ecker, Joseph - Salk Institute|
|Vogel, John - Department Of Energy Joint Genome|
Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/18/2017
Publication Date: 6/21/2017
Citation: Hsia, M., O'Malley, R., Cartwright, A., Nieu, R., Gordon, S., Kelly, S., Williams, T.G., Wood, D.F., Zhao, Y., Bragg, J.N., Jordan, M., Pauly, M., Ecker, J., Gu, Y.Q., Vogel, J.P. 2017. Sequencing and functional validation of the JGI Brachypodium distachyon T-DNA collection. Plant Journal. doi: 10.1111/tpj.13582.
Interpretive Summary: Methods for creating and sequencing T-DNA lines are very time-intensive and costly. We optimized the methods to gain significant time, cost and labor savings without compromising the accuracy of the data. The largest gain was attained by using our method to sequence the DNA flanking the insertion sites and pooling leaf samples from 10,000 lines into 40 for DNA extraction. This approach can be applied to other sequencing applications where the sequence adjacent to a known sequence is desired from a large number of organisms. The sequence indexed T-DNA lines produced by this project are valuable resources for the plant science community that is already heavily used.
Technical Abstract: Brachypodium distachyon is a powerful experimental model for the grasses with a large and growing collection of genomic and experimental resources. We have added to these resources by greatly expanding the number of sequence-indexed T-DNA lines. We sequenced 21,165 T-DNA lines, 15,569 of which were produced in this study, using a high throughput approach called TDNA-Seq. This method uses four dimensional pooling and Illumina sequencing to greatly decrease the labor and cost of sequencing DNA flanking insertion sites. This increased the number of unique insertion sites in the T-DNA collection by 21,078 bringing the overall total to 26,112. Thirty seven percent (9,754) of these insertion sites are within genes and 28% (7,217) are within 500 bp of a gene. Approximately 31% of the genes in the v2.1 annotation have been tagged in this population. To demonstrate the utility of this collection, we phenotypically characterized five T-DNA lines with insertions in genes previously shown in other systems to be involved in cellulose biosynthesis, hemicellulose biosynthesis, secondary cell wall development, DNA damage repair, wax biosynthesis, and chloroplast synthesis. In all cases, the phenotypes observed supported previous studies demonstrating the power of this collection for plant functional genomics. The Brachypodium T-DNA Resource can be accessed online at http://jgi.doe.gov/our-science/science-programs/plant-genomics/brachypodium/.