Genomics-assisted dissection of barley morphology and development– BARCODE
We used Illumina Oligo Pool Assay (OPA) technology to genotype 979 Bowman backcross derived nearly isogenic mutant lines at 3072 mapped genetic loci (6144 alleles) (Druka et al., 2010). We used simple pair-wise comparison between the mutant lines with Bowman, their recurrent parent, to define the boundaries of the segments containing mutated alleles and compile a detailed comparative putative gene content map for each locus based on the barley, rice and emerging Brachypodium and maize genome sequences. While OPA genotyping will narrow the location of each mutant locus to a small genetic interval, identifying the mutated gene from all potential candidates will likely still require a forward genetics approach. To facilitate this, and all similar studies in the future, we contributed to an established international strategy for the staged development of a genetically anchored physical map of the barley genome by end sequencing BACs that are currently undergoing High Information Content Fingerprint analysis and Overgo hybridisation to >12,000 unigenes. This will provide up to 400 Mbp of barley genomic DNA sequence information and will assist in anchoring the physical map to the barley gene map (~5-6000 mapped genes) and rice genome sequence. To demonstrate the utility of the developed information, resources and approach, each partner lab will identify a minimum BAC tiling path spanning a morphological /developmental mutant locus, identify candidate genes and initiate their functional characterisation by a combination of comparative allele sequencing (WT vs. Mutant) and the use of functional genomics tools available to the group (TILLING, transgenics, VIGS). All information will be made publicly available to the community for broader exploitation through existing web-based informatics resources. Over 300,000 Paired BAC End Sequences have been derived and are being incorporated into the barley physical map being constructed out with this project. Additionally 27 F2 populations of >10,000 individuals have been developed for a range of mutant phenotypes and distributed to IPK (9), Udine (9) and SCRI (9) for use in future fine mapping projects.
You can download the BOPA1 genotypic data in Flapjack format for the Bowman isolines here. This enable you to identify regions of the Bowman isolines that differ from Bowman. Every cell coloured green is the same as Bowman and every red cell is different. Thus the two lines differ from Bowman at similar regions of the segment of the barley genome displayed in the Figure.
Several morphological mutant loci were fine mapped using the homozygous recessive individuals of large F2 populations. These included dense spike-ar (dsp-ar) (Shahinnia et al., 2012). For two of these loci this approach helped us to identify and characterise the gene underlying the trait, Third outer glume (Trd1) (Houston et al., 2012) and Zeocriton (AP2) (Houston et al., 2013).
This EU funded project (2007- 2011) was a collaboration between The James Hutton Institute in the UK, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) in Germany, and University of Udine, Italy.
Shahinnia, F., Druka, A., Franckowiak, J., Morgante, M., Waugh, R., & Stein, N. (2012). High resolution mapping of Dense spike-ar (dsp. ar) to the genetic centromere of barley chromosome 7H. Theoretical and Applied Genetics, 124(2), 373-384.
Houston, K., Druka, A., Bonar, N., Macaulay, M., Lundqvist, U., Franckowiak, J., … & Waugh, R. (2012). Analysis of the barley bract suppression gene Trd1. Theoretical and Applied Genetics, 125(1), 33-45.
Houston, K., McKim, S. M., Comadran, J., Bonar, N., Druka, I., Uzrek, N., … & Waugh, R. (2013). Variation in the interaction between alleles of HvAPETALA2 and microRNA172 determines the density of grains on the barley inflorescence. Proceedings of the National Academy of Sciences, 110(41), 16675-16680.
For further information on this project please contact Kelly Houston (firstname.lastname@example.org) from the James Hutton Institute.