Barley Stripe Rust Resistance QTL: Development and Validation of SNP Markers for Resistance to Puccinia striiformis f. sp. hordei.

Quantitative trait loci (QTL) for barley stripe rust resistance were mapped in recombinant inbred lines (RIL) from a 'Lenetah' × 'Grannelose Zweizeilige' (GZ) cross. GZ is known for a major seedling resistance QTL on chromosome 4H but linked markers suitable for marker-assisted selection have not been developed. This study identified the 4H QTL (log of the likelihood [LOD] = 15.94 at 97.19 centimorgans [cM]), and additional QTL on chromosomes 4H and 6H (LOD = 5.39 at 72.7 cM and 4.24 at 34.46 cM, respectively). A QTL on chromosome 7H (LOD = 2.04 at 81.07 cM) was suggested. All resistance alleles were derived from GZ. Evaluations of adult plant response in Corvallis, OR in 2013 and 2015 provided evidence of QTL at the same positions. However, the minor QTL on 4H was not statistically significant in either location/year, while the 7H QTL was significant in both. The single-nucleotide polymorphism markers flanking the resistance QTL were validated in RIL from a '95SR316A' × GZ cross for their ability to predict seedling resistance. In 95SR316A × GZ, 91 to 92% of RIL with GZ alleles at the major 4H QTL and at least one other were resistant to moderate in reaction. In these populations, at least two QTL were required to transfer the barley stripe rust resistance from GZ.

Mapping Ds insertions in barley using a sequence-based approach.

A transposon tagging system, based upon maize Ac/Ds elements, was developed in barley (Hordeum vulgaresubsp. vulgare). The long-term objective of this project is to identify a set of lines with Ds insertions dispersed throughout the genome as a comprehensive tool for gene discovery and reverse genetics. AcTPase and Ds-bar elements were introduced into immature embryos of Golden Promise by biolistic transformation. Subsequent transposition and segregation of Ds away from AcTPase and the original site of integration resulted in new lines, each containing a stabilized Ds element in a new location. The sequence of the genomic DNA flanking the Ds elements was obtained by inverse PCR and TAIL-PCR. Using a sequence-based mapping strategy, we determined the genome locations of the Ds insertions in 19 independent lines using primarily restriction digest-based assays of PCR-amplified single nucleotide polymorphisms and PCR-based assays of insertions or deletions. The principal strategy was to identify and map sequence polymorphisms in the regions corresponding to the flanking DNA using the Oregon Wolfe Barley mapping population. The mapping results obtained by the sequence-based approach were confirmed by RFLP analyses in four of the lines. In addition, cloned DNA sequences corresponding to the flanking DNA were used to assign map locations to Morex-derived genomic BAC library inserts, thus integrating genetic and physical maps of barley. BLAST search results indicate that the majority of the transposed Ds elements are found within predicted or known coding sequences. Transposon tagging in barley using Ac/Ds thus promises to provide a useful tool for studies on the functional genomics of the Triticeae.

Somaclonal variation in the progeny of transgenic barley.

Somaclonal variation (SCV) in transgenic plants may slow the incorporation of introduced genes into commercially competitive cultivars. Somaclonal variation in transgenic barley (Hordeum vulgare L.) was assessed in one experiment by comparing the agronomic characteristics of 44 segregating transgenic lines in the T2 generation to their non-transformed parent ('Golden Promise'). A second experiment examined the agronomic characteristics of seven transgenic-derived, null (non-transgenic) segregant lines in the T2 and T4 generations. Compared to their uncultured parent, Golden Promise, most of these lines were shorter, lower yielding, and had smaller seed, and the variability among individual plants was higher. The frequency and severity of the observed SCV was unexpectedly high, and the transformation procedure appeared to induce greater SCV than tissue culture in the absence of transformation. Attempts to understand the sources of SCV, and to modify transformation procedures to reduce the generation of SCV, should be made.

Enhancement of plant regeneration from embryogenic callus of commercial barley cultivars.

Genotypic restrictions on plant regeneration from cultured cells have hindered the genetic transformation of most barley cultivars. Optimizing culturing protocols for specific cultivars of commercial interest may facilitate their genetic transformation. Plant regeneration from embryogenic callus of `Harrington', `Morex', and `Hector' as affected by certain protocol modifications was examined in replicated experiments. Regeneration was improved for all cultivars by separately autoclaving certain components of the culture media and by reducing the amount of embryogenic callus cultured per petri dish. Regeneration improvements in response to various concentrations of copper and 2,4-dichlorophenoxyacetic acid were more genotype specific. This study suggests that the development and use of genotype-specific protocols can enhance plant regeneration. Enhancements in plant regeneration are expected to facilitate the transformation of commercial barley germplasm.

Callus formation and plant regeneration from somatic embryos of oat (Avena sativa L.).

Globular-stage somatic embryos were isolated by vortexing friable, embryogenic callus of oat (Avena sativa L.) followed by fractionation based on size. Somatic embryos were most frequently found in the 300-380 µm size fraction. Friable, embryogenic callus was reinitiated from 55% of isolated somatic embryos. Fertile plants were regenerated from 22% of isolated somatic embryos. Reinitiation of callus from somatic embryos and growth of friable, embryogenic callus was inhibited by the selective agents G418 and methotrexate. These results suggest that somatic embryos isolated from friable, embryogenic callus of oat may be useful totipotent targets for particle acceleration-mediated transformation.