Fragmentation of pooled PCR products for highly multiplexed TILLING

Improvements to massively parallel sequencing have allowed the routine recovery of natural and induced sequence variants. A broad range of biological disciplines have benefited from this, ranging from plant breeding to cancer research. The need for high sequence coverage to accurately recover single nucleotide variants and small insertions and deletions limits the applicability of whole genome approaches. This is especially true in organisms with a large genome size or for applications requiring the screening of thousands of individuals, such as the reverse-genetic technique known as TILLING. Using PCR to target and sequence chosen genomic regions provides an attractive alternative as the vast reduction in interrogated bases means that sample size can be dramatically increased through amplicon multiplexing and multidimensional sample pooling while maintaining suitable coverage for recovery of small mutations. Direct sequencing of PCR products is limited, however, due to limitations in read lengths of many next generation sequencers. In the present study we show the optimization and use of ultrasonication for the simultaneous fragmentation of multiplexed PCR amplicons for TILLING highly pooled samples. Sequencing performance was evaluated in a total of 32 pooled PCR products produced from 4096 chemically mutagenized Hordeum vulgare DNAs pooled in three dimensions. Evaluation of read coverage and base quality across amplicons suggests this approach is suitable for high-throughput TILLING and other applications employing highly pooled complex sampling schemes. Induced mutations previously identified in a traditional TILLING screen were recovered in this dataset further supporting the efficacy of the approach

Histology and symplasmic tracer distribution during development of barley androgenic embryos

The present study concerns three aspects of barley androgenesis: (1) the morphology and histology of the embryos during their development, (2) the time course of fluorescent symplasmic tracers’ distribution, and (3) the correlation between symplasmic communication and cell differentiation. The results indicate that barley embryos, which are developing via an androgenic pathway, resemble their zygotic counterparts with respect to their developmental stages, morphology and histology. Analysis of the distribution of the symplasmic tracers, HPTS, and uncaged fluorescein indicates the symplasmic isolation of (1) the protodermis from the underlying cells of the late globular stage onwards, and (2) the embryonic organs at the mature stage of development

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

Gene segregation in a barley DH population

A large population of anther culture-derived barley regenerants and their progeny was tested for allele segregation at 1 isozyme and 8 morphological marker loci. The segregation of genetic markers was examined separately for haploid, diploid and polyploid regenerants. All the 9 analysed genes except al (albino lemma) on chromosome 3 segregated according to the expected 1:1 ratio in the microspore-derived barley population. There was no difference in allele distribution between haploid and diploid regenerants. Among the limited number of 34 analysed tetraploids a significant excess of the dominant allele at locus о (orange lemma) of chromosome 6 was also observed. The recombination frequency between linked genes (n - lk2 on chromosome 1 and r - s on chromosome 7) estimated in the DH population did not differ significantly from recombination rates calculated in F₂ progeny or presented in barley chromosome maps. The phenomenon of gametic selection is discussed in relation to the genotype dependency of anther culture response and procedures used for DH production in barley

Cytological and genetic evaluation of anther culture-derived doubled haploids in barley

Gametoclonal variation among anther culture-derived plants of three barley genotypes was estimated on the basis of cytological analysis (DH1, DH2 generation), observation of morphological variants (DH2, DH3) and chlorophyll mutation test (DH2, DH3). Individual head rows were grown in the field to detect possible chimeric structure of regenerants and to assess the number of variants and mutations in each line. Spontaneously doubled plants were the most frequent class (70%) among regenerants and almost 90% of them were completely fertile. There was a difference in proportion of haploids produced by different genotypes, but the highest frequency observed did not exceed 21%. The remaining regenerants were tetraploid, and contained chromosomal mutations or chimeras. In total, there were about 15% of polyploids and plants carrying chromosomal aberrations (translocations, inversions) among DH1 individuals. The changes in chromosome number and structure were the main source of observed variation. The level of gene mutation induced in vitro was relatively low. No more than 1% of microspore-derived plants expressed visible morphological changes in DH2 progeny. Only two morphological variants derived from the Bruce cultivar proved to be homozygous mutants (dwarf type) stable up the to third generation. The frequency of DH plants carrying chlorophyll mutation was 5.8%, but most of them (82%) were chimeric and had only a small mutation sector. The level of gametoclonal variation depended on the donor plant genotype. The highest proportion of variants and mutations was observed among DH plants derived from the Bruce cultivar, while the lowest was recorded among plants regenerated from anther culture of the doubled haploid line H930-36. Mechanisms leading to the observed variation and implications resulting from the presented experiments concerning implementation of anther culture in barley breeding were discussed. It was concluded that this method resulted in a high frequency of spontaneous doubling, a low frequency of genetic changes, and being less time and effort-consuming than the ’Bulbosum’ technique, can be applied to most barley breeding programs