A highly mutagenised barley ( cv. Golden Promise) TILLING population coupled with strategies for screening-by-sequencing
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Plant Methods Open Access
METHODOLOGY
A highly mutagenised barley (cv. Golden Promise) TILLING population coupled with strategies for screening‑by‑sequencing Miriam Schreiber1, Abdellah Barakate1, Nicola Uzrek1, Malcolm Macaulay1, Adeline Sourdille2, Jenny Morris1, Pete E. Hedley1, Luke Ramsay1 and Robbie Waugh1,2,3*
Abstract Background: We developed and characterised a highly mutagenised TILLING population of the barley (Hordeum vulgare) cultivar Golden Promise. Golden Promise is the ‘reference’ genotype for barley transformation and a primary objective of using this cultivar was to be able to genetically complement observed mutations directly in order to prove gene function. Importantly, a reference genome assembly of Golden Promise has also recently been developed. As our primary interest was to identify mutations in genes involved in meiosis and recombination, to characterise the population we focused on a set of 46 genes from the literature that are possible meiosis gene candidates. Results: Sequencing 20 plants from the population using whole exome capture revealed that the mutation density in this population is high (one mutation every 154 kb), and consequently even in this small number of plants we identified several interesting mutations. We also recorded some issues with seed availability and germination. We subsequently designed and applied a simple two-dimensional pooling strategy to identify mutations in varying numbers of specific target genes by Illumina short read pooled-amplicon sequencing and subsequent deconvolution. In parallel we assembled a collection of semi-sterile mutants from the population and used a custom exome capture array targeting the 46 candidate meiotic genes to identify potentially causal mutations. Conclusions: We developed a highly mutagenised barley TILLING population in the transformation competent cultivar Golden Promise. We used novel and cost-efficient screening approaches to successfully identify a broad range of potentially deleterious variants that were subsequently validated by Sanger sequencing. These resources combined with a high-quality genome reference sequence opens new possibilities for efficient functional gene validation. Keywords: Barley, Amplicon sequencing, Meiosis, Functional genomics, Exome capture, Recombination Background Barley (2x = 2n = 14) is one of the world’s oldest and most important crops. While most of the harvested grain is used as animal feed, barley also underpins sectors of the food and particularly the drinks industry where it is a mainstay for the production of premium alcoholic beverages including beer and whisky. While high quality grain is needed to produce malted barley for the drinks *Correspondence: [email protected] 1 Cell and Molecular Sciences, The James Hutton Institute, Errol Road, Invergowrie, Dundee DD2 5DA, Scotland, UK Full list of author information is available at the end of the article
industry (i.e. grain subjected to controlled germination then dried), grain failing to meet premium standards along with
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