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Microsatellite or simple sequence repeat (SSR) markers are important tools for genetic analyses, especially those targeting diversity. The primary objective of this study was to develop robust oat-based microsatellite markers from newly enriched genomic libraries to expand on a relatively small existing oat SSR toolbox. Microsatellite motifs characterized by (CA/GT), (AAT/TTA), (ATG/TAC) and (CATC/GTAG) repeats were targeted for enrichment. Preliminary screening showed that 90% of clones from the (CA/GT) and 79% of the clones from the (ATG/TAC) libraries contained repeats, while < 11% of the clones from (AAT/TTA) and (CATC/GTAG) libraries contained repeats. Subsequent sequencing of 1536 clones from the (CA/GT) and (ATG/TAC) libraries resulted in 539 and 578 SSRs for which primers were designed, respectively. A total of 246 SSRs were polymorphic across 11 oat lines. One hundred and twenty-five of the markers produced highly reproducible assays that interrogated 369 alleles at 193 loci. Of these, 79 robust assays interrogated 146 codominant alleles. These markers will be useful for a wide range of genetic analyses in oat including assessment of diversity and marker-assisted breeding.

Plant height and spike length and angle are important agronomic traits in the production of barley ( Hordeum vulgare L.) due to strong correlations with lodging and disease. The objective of this study was to use QTL analysis to identify genetic regions associated with each trait in a recombinant inbred line (RIL) mapping population derived from a cross of Falcon by Azhul. Falcon is a spring six-row hulless feed barley with long spikes displaying obtuse angles, while Azhul is a spring dwarf, six-row hulless food barley with short spikes displaying acute angles. The population was genotyped using SNP, DArT and SSR markers and quantitative trait loci (QTL) were detected on chromosomes 2H (102.8 cM, spike length), 3H (89.2 cM, plant height and 38.2, spike angle and length), 4H (19.0 cM, spike length), and 5H (106.7 cM, spike angle). In conclusion, we developed a barley genetic map, which incorporated SNP, DArT and SSR markers, for detection of height and spike length and angle QTL. Three spike angle, one spike length and one plant height QTL were novel and by using comparative genomics we identified possible candidate genes involved in gibberellic acid signaling and auxin- and ethylene-responsive pathways. This knowledge can be used to generate suitable markers for barley breeding improvement.

Selectable markers used in plant transformation, such as the bar gene, have been chosen for selection efficacy and for the absence of pleiotropic effects. Recent research has suggested that expression of bar in Arabidopsis affects the transcriptome, and in at least one barley cultivar, high bar expression has been associated with severe fitness reductions. However, the observed sensitivity in barley was thought to be an isolated case associated with a specific genetic stock. In this study, fitness reductions were observed in some near-isogenic lines in the background of the cultivar Garnet when the bar locus was homozygous. The fitness of the near-isogenic lines was correlated with the levels of bar mRNA in the donor parents. Previous studies had shown heritability of bar expression in F1 barley hybrids, so these results suggested that Garnet barley is intolerant of high phosphinothricin acetyltransferase activity. Phosphinothricin acetyltransferase activities were determined for eight near-isogenic lines that showed a range of phenotypes (apparently normal to abnormal and nonviable) when homozygous for bar. Fitness was clearly and negatively correlated with phosphinothricin acetyltransferase activity. These results suggest genetic variability for sensitivity to bar expression in barley, and the possibility of negative but non-obvious effects in apparently tolerant cultivars cannot be ruled out. Similar problems may be associated, but not yet detected, with other selectable markers. The potential for pleiotropic effects of selectable markers can be avoided by transformation strategies that enable the elimination of selectable markers.

The utility of transgenic plants for both experimental and practical agronomic purposes is highly dependent on stable, predictable, and heritable expression of the introduced genes. This requirement is frequently unfulfilled, and transgenes often are completely silenced. Studies of transgenic loci have shown that rearrangements of transgenes occur during the integration process, some of which are potent cues that induce silencing. Conversely, intact, single-copy transgenes produced via transposon-mediated gene delivery have shown relatively stable expression, at least in early-generation progeny. To examine the long-term expression stability of a bar expression cassette delivered via Dissociation (Ds)-mediated transposition, we examined qualitative and quantitative expression in barley (Hordeum vulgare L.) populations developed for transposon tagging. Qualitative assessments of herbicide resistance among 106 lines showed bar expression to be stable for at least five generations of advance via self-pollination. Similarly, qualitatively stable expression was observed among 31 near-isogenic lines derived from at least seven backcrosses to the cultivar Garnet. Quantitative RT-PCR measurements of bar expression were conducted for eight near-isogenic lines and their donor parents. The expression of bar was highly correlated in parent and progeny near-isogenic lines, showing high heritability of bar expression. These data demonstrate stable, predictable transgene expression following Ds-mediated delivery.

Hexaploid oat ( L., 2 = 6 = 42) is a member of the Poaceae family and has a large genome (∼12.5 Gb) containing 21 chromosome pairs from three ancestral genomes. Physical rearrangements among parental genomes have hindered the development of linkage maps in this species. The objective of this work was to develop a single high-density consensus linkage map that is representative of the majority of commonly grown oat varieties. Data from a cDNA-derived single-nucleotide polymorphism (SNP) array and genotyping-by-sequencing (GBS) were collected from the progeny of 12 biparental recombinant inbred line populations derived from 19 parents representing oat germplasm cultivated primarily in North America. Linkage groups from all mapping populations were compared to identify 21 clusters of conserved collinearity. Linkage groups within each cluster were then merged into 21 consensus chromosomes, generating a framework consensus map of 7202 markers spanning 2843 cM. An additional 9678 markers were placed on this map with a lower degree of certainty. Assignment to physical chromosomes with high confidence was made for nine chromosomes. Comparison of homeologous regions among oat chromosomes and matches to orthologous regions of rice ( L.) reveal that the hexaploid oat genome has been highly rearranged relative to its ancestral diploid genomes as a result of frequent translocations among chromosomes. Heterogeneous chromosome rearrangements among populations were also evident, probably accounting for the failure of some linkage groups to match the consensus. This work contributes to a further understanding of the organization and evolution of hexaploid grass genomes.

Background The INNER NO OUTER (INO) gene is expressed in the outermost cell layer of the outer integument of bitegmic ovules and is essential for this organ’s growth. The role and cross-species functional conservation of INO orthologs were examined in members of the Solanaceae, which have unitegmic ovules. Unitegmy has evolved several times in disparate angiosperm lineages. INO expression has been observed in the outermost cell layers of all examined unitegmic ovules, but the functional role of INO in unitegmic ovules has not previously been evaluated. Results INO orthologs were unambiguously identified in tobacco and tomato by sequence homology. Expression of the tomato INO gene was limited to the outer cell layer of the single integument indicating that this single integument has properties of the outer integument. Expression occurred only after integument initiation, later than observed in ovules of other examined angiosperms. Virus-induced knock-down of expression of the INO ortholog in tobacco inhibited growth of the outer cell layer of the integument leading to a decrease in both integument extension and curvature of the ovule. The altered ovules closely resemble those of the aberrant testa shape (ats) ino mutant combination in Arabidopsis where we see the effect of the ino mutation on a single fused integument produced by the ats mutation. Despite significant sequence identity and similar expression patterns, the tomato INO coding region was not able to complement the Arabidopsis ino mutant. Conclusions The similarity of effects of ino mutations on the unitegmic ovules of tobacco and the fused integuments of the Arabidopsis ats mutant show that: 1) INO orthologs play the same role in promoting integument growth in ovules of tobacco and Arabidopsis; and 2) the unitegmic ovules of tobacco (and hence other solanaceous species) are most likely the result of a congenital fusion of two ancestral integuments. Our results further indicate that INO has a conserved role in growth of the outermost cell layer of integuments. The curvature of solanaceous ovules is driven by unequal growth of the outer layers of the single integument that likely correspond to an ancestral outer integument. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0835-z) contains supplementary material, which is available to authorized users.

The plant hormone ethylene is important to many plant processes from germination through senescence, including responses to in vitro growth and plant regeneration. Knowledge of the number and function of genes that are involved in ethylene biosynthesis and reception is necessary to determine the role of specific genes within gene families known to influence ethylene biosynthesis and other aspects of ethylene function in plants. Our objective was built on previous studies that have established the critical role of ethylene in the in vitro response of barley (Hordeum vulgare L.), and that have identified ethylene-related QTL in the barley genome. In this study, we have identified the locations of genes in the barley 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), ACC oxidase (ACO), and ethylene receptor (ETR) gene families. Specific primers for PCR amplification of each gene were developed and used to map these genes in the Oregon Wolf Barley mapping population. Five ACS, 8 ACO, and 7 ETR genes were identified and mapped to six of the barley chromosomes. Gene locations were syntenous to the orthologs in rice except for two that mapped to chromosome 6H. Gene duplication was evident for ACO genes on chromosomes 5H and 6H. Gene-specific primers will be useful for determining expression of each gene under various environmental conditions, including in vitro environments, to better understand the role of ethylene. Of the six known QTL for green plant regeneration in barley, three were located near the genes mapped in this study.

A polymerase chain reaction (PCR)-based adapter ligation method was developed to determine native barley (Hordeum vulgare L.) sequences flanking Dissociation (Ds) insertions and barley expressed sequence tags (ESTs). This method is simple and efficient, with the majority (∼70%) of queries returning valid sequence information. The success of this method can be traced to experimental protocols that control sample cross contamination and that segregate specific products from nonspecific products. This report describes the protocol in detail, quantifies its efficiency to enable comparisons to future modifications, and discusses specific problems that may be addressed to enable further improvements to this method.

A simple, polymerase chain reaction (PCR) and agarose gel-based system was used to efficiently detect barley (Hordeum vulgare L.) lines with novel transpositions of a modified maize (Zea mays L.) Ds element. One line contained a Ds insertion in a gene critical to barley spike development which appears to be a regulatory component of the ABC model of floral development. This model, elucidated based on studies of dicots, is widely conserved and function of key genes–such as AP2–have been demonstrated in monocots such as rice (Oryza sativa L.) and maize, but significant differences in orthologous gene function exist. This report describes the generation of a Ds-insertional mutant of a putative barley ortholog of miR172. The miR172 family of microRNAs (miRNAs) includes a member shown to control the expression of AP2 and AP2-like orthologs during floral development. The Ds-miR172 mutant contains a 3.6 kb insertion in the mature miRNA sequence, thus abolishing function, and the associated floral abnormalities are consistent with the failure to properly regulate the barley AP2 ortholog. Ds-miR172 mutants show abnormal spikelet development, including the conversion of glumes to partially developed florets in apical regions of spikes. Basal regions of the spike show an abnormal branching phenotype resulting from indeterminate spikelet meristem development, with each branch consisting of multiple, abnormal spikelets and other floral organs in place of a single spikelet. This phenotype is similar to ts4 in maize, the only other known mutation affecting a miR172 ortholog.

1Present address: National Small Grains Germplasm Research Facility, USDA-ARS, Aberdeen, ID 83210, USA SUMMARY Santalales comprise mainly parasitic plants including mistletoes and sandalwoods. Bitegmic ovules similar to those found in most other angiosperms are seen in many members of the order, but other members exhibit evolutionary reductions to the unitegmic and ategmic conditions. In some mistletoes, extreme reduction has resulted in the absence of emergent ovules such that embryo sacs appear to remain embedded in placental tissues. Three santalalean representatives (Comandra, Santalum, and Phoradendron), displaying unitegmic, and ategmic ovules, were studied. Observed ovule morphologies were consistent with published reports, including Phoradendron serotinum, which we interpret as having reduced ategmic ovules, consistent with earlier reports on this species. For further understanding of the nature of the ovule reductions we isolated orthologs of the Arabidopsis genes AINTEGUMENTA (ANT) and BELL1 (BEL1), which are associated with ovule development in this species. We observed ovular expression of ANT and BEL1 in patterns largely resembling those seen in the integumented ovules of Arabidopsis. These genes were found to be expressed in the integument of unitegmic ovules and in the surface layers of ategmic ovules, and in some cases, expression of BEL1 was also observed in the surrounding carpel tissue. We hypothesize that ategmic ovules derive from a fusion of the integuments with the nucellus or that the nucellus has taken on some of the characteristics confined to integuments in ancestral species.

Maize-derived sequences from the transposable elements Activator (Ac) and Dissociation (Ds) have enabled studies of gene function via transposon tagging. The characteristics of modified, transgene-containing Ds elements constructed for some of these studies have demonstrated their ability to resolve complex loci, separate transgenes from marker genes and vector sequences, and to support high and heritable levels of transgene expression. To most efficiently design breeding schemes for developing transgenic populations via Ds-mediated transposition, detailed knowledge of the dynamics and characteristics of transposition in barley is necessary. Examination of a barley transposon tagging population (n = 4,954) derived from crosses of lines containing Ds-bar insertions to lines expressing Ac transposase showed that the frequencies of transposition from eight original Ds-bar loci ranged from 5 to 41 % among F2 individuals. Sequence analysis of Ds-bar terminal sequences and of flanking genomic sequences for 107 F2 and F3 individuals indicated precise integrations. Analysis of 173 flanking sequences derived from these populations and from previously produced populations, primarily using sequence-based methods, enabled the mapping of 159 to a specific chromosome and 136 to specific map locations. Of the 156 DsT loci that could be located to specific contigs, most were located in gene-rich areas and approximately 40 % were either in or near (within 1 kb) expressed sequences or predicted proteins. These data will enable the design of optimal breeding schemes for developing and using Ds-based systems for transposon tagging and for transgene delivery that are specific to barley.

Wnt signaling is an important component in patterning the early embryo and specifically the neural plate. Studies in Xenopus, mouse, and zebrafish have shown that signaling by members of the Wnt family of secreted signaling factors, their Frizzled receptors and several inhibitors (sFRP1, sFRP2, sFRP3/Frzb1, Crescent/Frzb2, Dkk1, and Cerberus) are involved. However, very little is known about the expression of genes in the Wnt signaling pathway during early anterior neural patterning in chick. We have performed an expression analysis at neural plate stages of several Wnts, Frizzled genes, and Wnt signaling pathway inhibitors using in situ hybridization. The gene expression patterns of these markers are extremely dynamic. We have identified two candidate molecules for anterior patterning of the neural plate, Wnt1 and Wnt8b, which are expressed in the rostral ectoderm at these stages. Further functional studies on the roles of these markers are underway.

Little is known about the molecular processes that govern female gametophyte (FG) development and function, and few FG-expressed genes have been identified. We report the identification and phenotypic analysis of 31 new FG mutants in Arabidopsis. These mutants have defects throughout development, indicating that FG-expressed genes govern essentially every step of FG development. To identify genes involved in cell death during FG development, we analyzed this mutant collection for lines with cell death defects. From this analysis, we identified one mutant, gfa2, with a defect in synergid cell death. Additionally, the gfa2 mutant has a defect in fusion of the polar nuclei. We isolated the GFA2 gene and show that it encodes a J-domain-containing protein. Of the J-domain-containing proteins in Saccharomyces cerevisiae (budding yeast), GFA2 is most similar to Mdj1p, which functions as a chaperone in the mitochondrial matrix. GFA2 is targeted to mitochondria in Arabidopsis and partially complements a yeast mdj1 mutant, suggesting that GFA2 is the Arabidopsis ortholog of yeast Mdj1p. These data suggest a role for mitochondria in cell death in plants.

Advances in genomic sequencing technologies in the past decade have revolutionized the field of genomics, resulting in faster and less expensive sequencing. Holding back the potential for innovation, however, is a widespread lack of understanding of genomics and sequencing by the general public. In an attempt to remedy this problem, this paper presents an introduction to the fields of genomics, bioinformatics, and proteomics using the blueberry genome as a model case study of the plant genomics field. The blueberry (Vaccinium sect. Cyanococcus) is often cited as a “super food” in the media due to its nutritional benefits and global economic importance. There have been a number of related genomic publications in the past 20 years; however, a completed genome and a full analysis into the health-related pathways are still needed. As exemplified by this blueberry case study, there are opportunities for future genomic research into numerous beneficial plant species. The solid background presented in this paper provides future researchers the foundation to explore these uncharted areas.

Background The INNER NO OUTER (INO) gene, which encodes a YABBY-type transcription factor, specifies and promotes the growth of the outer integument of the ovule in Arabidopsis. INO expression is limited to the abaxial cell layer of the developing outer integument of the ovule and is regulated by multiple regions of the INO promoter, including POS9, a positive element that when present in quadruplicate can produce low-level expression in the normal INO pattern. Results Significant redundancy in activity between different regions of the INO promoter is demonstrated. For specific regulatory elements, multimerization or the addition of the cauliflower mosaic virus 35S general enhancer was able to activate expression of reporter gene constructs that were otherwise incapable of expression on their own. A new promoter element, POS6, is defined and is shown to include sufficient positive regulatory information to reproduce the endogenous pattern of expression in ovules, but other promoter regions are necessary to fully suppress expression outside of ovules. The full-length INO promoter, but not any of the INO promoter deletions tested, is able to act as an enhancer-blocking insulator to prevent the ectopic activation of expression by the 35S enhancer. Sequence conservation between the promoter regions of Arabidopsis thaliana, Brassica oleracea and Brassica rapa aligns closely with the functional definition of the POS6 and POS9 regions, and with a defined INO minimal promoter. The B. oleracea INO promoter is sufficient to promote a similar pattern and level of reporter gene expression in Arabidopsis to that observed for the Arabidopsis promoter. Conclusions At least two independent regions of the INO promoter contain sufficient regulatory information to direct the specific pattern but not the level of INO gene expression. These regulatory regions act in a partially redundant manner to promote the expression in a specific pattern in the ovule and suppress expression outside of ovules. Establishment of this pattern requires cooperation and competition between multiple positive and negative regulatory elements.