Spectrum and Density of Gamma and X-ray Induced Mutations in a Non-Model Rice Cultivar.

Physical mutagens are a powerful tool used for genetic research and breeding for over eight decades. Yet, when compared to chemical mutagens, data sets on the effect of different mutagens and dosages on the spectrum and density of induced mutations remain lacking. To address this, we investigated the landscape of mutations induced by gamma and X-ray radiation in the most widely cultivated crop species: rice. A mutant population of a tropical upland rice, Oryza sativa L., was generated and propagated via self-fertilization for seven generations. Five dosages ranging from 75 Gy to 600 Gy in both X-ray and gamma-irradiated material were applied. In the process of a forward genetic screens, 11 unique rice mutant lines showing phenotypic variation were selected for mutation analysis via whole-genome sequencing. Thousands of candidate mutations were recovered in each mutant with single base substitutions being the most common, followed by small indels and structural variants. Higher dosages resulted in a higher accumulation of mutations in gamma-irradiated material, but not in X-ray-treated plants. The in vivo role of all annotated rice genes is yet to be directly investigated. The ability to induce a high density of single nucleotide and structural variants through mutagenesis will likely remain an important approach for functional genomics and breeding.

sRNA Profiling Combined With Gene Function Analysis Reveals a Lack of Evidence for Cross-Kingdom RNAi in the Wheat - Zymoseptoria tritici Pathosystem.

Cross-kingdom small RNA (sRNA) silencing has recently emerged as a mechanism facilitating fungal colonization and disease development. Here we characterized RNAi pathways in Zymoseptoria tritici, a major fungal pathogen of wheat, and assessed their contribution to pathogenesis. Computational analysis of fungal sRNA and host mRNA sequencing datasets was used to define the global sRNA populations in Z. tritici and predict their mRNA targets in wheat. 389 in planta-induced sRNA loci were identified. sRNAs generated from some of these loci were predicted to target wheat mRNAs including those potentially involved in pathogen defense. However, molecular approaches failed to validate targeting of selected wheat mRNAs by fungal sRNAs. Mutant strains of Z. tritici carrying deletions of genes encoding key components of RNAi such as Dicer-like (DCL) and Argonaute (AGO) proteins were generated, and virulence bioassays suggested that these are dispensable for full infection of wheat. Nonetheless, our results did suggest the existence of non-canonical DCL-independent pathway(s) for sRNA biogenesis in Z. tritici. dsRNA targeting essential fungal genes applied in vitro or generated from an RNA virus vector in planta in a procedure known as HIGS (Host-Induced Gene Silencing) was ineffective in preventing Z. tritici growth or disease. We also demonstrated that Z. tritici is incapable of dsRNA uptake. Collectively, our data suggest that RNAi approaches for gene function analyses in this fungal species and potentially also as a control measure may not be as effective as has been demonstrated for some other plant pathogenic fungi.

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 multi-dimensional 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.

Sequence diversification in recessive alleles of two host factor genes suggests adaptive selection for bymovirus resistance in cultivated barley from East Asia.

KEY MESSAGE: Two distinct patterns of sequence diversity for the recessive alleles of two host factors HvPDIL5 - 1 and HvEIF4E indicated the adaptive selection for bymovirus resistance in cultivated barley from East Asia. Plant pathogens are constantly challenging plant fitness and driving resistance gene evolution in host species. Little is known about the evolution of sequence diversity in host recessive resistance genes that interact with plant viruses. Here, by combining previously published and newly generated targeted re-sequencing information, we systematically analyzed natural variation in a broad collection of wild (Hordeum spontaneum; Hs) and domesticated barleys (Hordeum vulgare; Hv) using the full-length coding sequence of the two host factor genes, HvPDIL5-1 and HvEIF4E, conferring recessive resistance to the agriculturally important Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV). Interestingly, two types of gene evolution conferred by sequence variation in domesticated barley, but not in wild barley were observed. Whereas resistance-conferring alleles of HvEIF4E exclusively contained non-synonymous amino acid substitutions (including in-frame sequence deletions and insertions), loss-of-function alleles were predominantly responsible for the HvPDIL5-1 conferred bymovirus resistance. A strong correlation between the geographic origin and the frequency of barley accessions carrying resistance-conferring alleles was evident for each of the two host factor genes, indicating adaptive selection for bymovirus resistance in cultivated barley from East Asia.

Validation of doubled haploid plants by enzymatic mismatch cleavage.

BACKGROUND: Doubled haploidy is a fundamental tool in plant breeding as it provides the fastest way to generate populations of meiotic recombinants in a genetically fixed state. A wide range of methods has been developed to produce doubled haploid (DH) plants and recent advances promise efficient DH production in otherwise recalcitrant species. Since the cellular origin of the plants produced is not always certain, rapid screening techniques are needed to validate that the produced individuals are indeed homozygous and genetically distinct from each other. Ideal methods are easily implemented across species and in crops where whole genome sequence and marker resources are limited. RESULTS: We have adapted enzymatic mismatch cleavage techniques commonly used for TILLING (Targeting Induced Local Lesions IN Genomes) for the evaluation of heterozygosity in parental, F1 and putative DH plants. We used barley as a model crop and tested 26 amplicons previously developed for TILLING. Experiments were performed using self-extracted single-strand-specific nuclease and standard native agarose gels. Eleven of the twenty-six tested primers allowed unambiguous assignment of heterozygosity in material from F1 crosses and loss of heterozygosity in the DH plants. Through parallel testing of previously developed Simple Sequence Repeat (SSR) markers, we show that 3/32 SSR markers were suitable for screening. This suggests that enzymatic mismatch cleavage approaches can be more efficient than SSR based screening, even in species with well-developed markers. CONCLUSIONS: Enzymatic mismatch cleavage has been applied for mutation discovery in many plant species, including those with little or no available genomic DNA sequence information. Here, we show that the same methods provide an efficient system to screen for the production of DH material without the need of specialized equipment. This gene target based approach further allows discovery of novel nucleotide polymorphisms in candidate genes in the parental lines.

An exceptionally high nucleotide and haplotype diversity and a signature of positive selection for the eIF4E resistance gene in barley are revealed by allele mining and phylogenetic analyses of natural populations.

In barley, the eukaryotic translation initiation factor 4E (eIF4E) gene situated on chromosome 3H is recognized as an important source of resistance to the bymoviruses Barley yellow mosaic virus and Barley mild mosaic virus. In modern barley cultivars, two recessive eIF4E alleles, rym4 and rym5, confer different isolate-specific resistances. In this study, the sequence of eIF4E was analysed in 1090 barley landraces and noncurrent cultivars originating from 84 countries. An exceptionally high nucleotide diversity was evident in the coding sequence of eIF4E but not in either the adjacent MCT-1 gene or the sequence-related eIF(iso)4E gene situated on chromosome 1H. Surprisingly, all nucleotide polymorphisms detected in the coding sequence of eIF4E resulted in amino acid changes. A total of 47 eIF4E haplotypes were identified, and phylogenetic analysis using maximum likelihood provided evidence of strong positive selection acting on this barley gene. The majority of eIF4E haplotypes were found to be specific to distinct geographic regions. Furthermore, the eI4FE haplotype diversity (uh) was found to be considerably higher in East Asia, whereas SNP genotyping identified a comparatively low degree of genome-wide genetic diversity in 16 of 17 tested accessions (each carrying a different eIF4E haplotype) from this same region. In addition, selection statistic calculations using coalescent simulations showed evidence of non-neutral variation for eIF4E in several geographic regions, including East Asia, the region with a long history of the bymovirus-induced yellow mosaic disease. Together these findings suggest that eIF4E may play a role in barley adaptation to local habitats.

High-resolution melting analysis of cDNA-derived PCR amplicons for rapid and cost-effective identification of novel alleles in barley.

An original method has been established for the identification of novel alleles of eukaryotic translation initiation factor 4E (eIF4E) gene, which is required for resistance to agronomically important bymoviruses, in barley germplasm. This method involves scanning for sequence variations in cDNA-derived PCR amplicons using High-resolution melting (HRM) followed by direct Sanger sequencing of only those amplicons which were predicted to carry nucleotide changes. HRM is a simple, cost-effective, rapid and high-throughput assay, which so far has only been widely used in clinical pathology for molecular diagnostic of diseases and patient genotyping. Application of HRM allowed significant reduction in the amount of expensive Sanger sequencing required for allele mining in plants. The method described here involved an investigation of total cDNA rather than genomic DNA, thus permitting the analyses of shorter (up to 300-bp) and fewer overlapping amplicons to cover the coding sequence. This strategy further reduced the allele mining costs. The sensitivity and accuracy of HRM for predicting genotypes carrying a wide range of nucleotide polymorphisms in eIF4E approached 100%. Results of the current study are promising and suggest that this method could also potentially be applied to the discovery of superior alleles controlling other important traits in barley as well in other model and crop plant species.