A highly mutagenised barley (cv. Golden Promise) TILLING population coupled with strategies for screening-by-sequencing

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.Miriam Schreiber, Abdellah Barakate, Nicola Uzrek, Malcolm Macaulay, Adeline Sourdille, Jenny Morris, Pete E. Hedley, Luke Ramsay and Robbie Waug

Inter-Homolog Crossing-Over and Synapsis in Arabidopsis Meiosis Are Dependent on the Chromosome Axis Protein AtASY3

In this study we have analysed AtASY3, a coiled-coil domain protein that is required for normal meiosis in Arabidopsis. Analysis of an Atasy3-1 mutant reveals that loss of the protein compromises chromosome axis formation and results in reduced numbers of meiotic crossovers (COs). Although the frequency of DNA double-strand breaks (DSBs) appears moderately reduced in Atasy3-1, the main recombination defect is a reduction in the formation of COs. Immunolocalization studies in wild-type meiocytes indicate that the HORMA protein AtASY1, which is related to Hop1 in budding yeast, forms hyper-abundant domains along the chromosomes that are spatially associated with DSBs and early recombination pathway proteins. Loss of AtASY3 disrupts the axial organization of AtASY1. Furthermore we show that the AtASY3 and AtASY1 homologs BoASY3 and BoASY1, from the closely related species Brassica oleracea, are co-immunoprecipitated from meiocyte extracts and that AtASY3 interacts with AtASY1 via residues in its predicted coiled-coil domain. Together our results suggest that AtASY3 is a functional homolog of Red1. Since studies in budding yeast indicate that Red1 and Hop1 play a key role in establishing a bias to favor inter-homolog recombination (IHR), we propose that AtASY3 and AtASY1 may have a similar role in Arabidopsis. Loss of AtASY3 also disrupts synaptonemal complex (SC) formation. In Atasy3-1 the transverse filament protein AtZYP1 forms small patches rather than a continuous SC. The few AtMLH1 foci that remain in Atasy3-1 are found in association with the AtZYP1 patches. This is sufficient to prevent the ectopic recombination observed in the absence of AtZYP1, thus emphasizing that in addition to its structural role the protein is important for CO formation

Arabidopsis PCH2 mediates meiotic chromosome remodeling and maturation of crossovers

Meiotic chromosomes are organized into linear looped chromatin arrays by a protein axis localized along the loop-bases. Programmed remodelling of the axis occurs during prophase I of meiosis. Structured illumination microscopy (SIM) has revealed dynamic changes in the chromosome axis in Arabidopsis thaliana and Brassica oleracea. We show that the axis associated protein ASY1 is depleted during zygotene concomitant with synaptonemal complex (SC) formation. Study of an Atpch2 mutant demonstrates this requires the conserved AAA+ ATPase, PCH2, which localizes to the sites of axis remodelling. Loss of PCH2 leads to a failure to deplete ASY1 from the axes and compromizes SC polymerisation. Immunolocalization of recombination proteins in Atpch2 indicates that recombination initiation and CO designation during early prophase I occur normally. Evidence suggests that CO interference is initially functional in the mutant but there is a defect in CO maturation following designation. This leads to a reduction in COs and a failure to form COs between some homologous chromosome pairs leading to univalent chromosomes at metaphase I. Genetic analysis reveals that CO distribution is also affected in some chromosome regions. Together these data indicate that the axis remodelling defect in Atpch2 disrupts normal patterned formation of COs