BackgroundA high-throughput genotyping platform is needed to enable marker-assisted breeding in the allo-octoploid cultivated strawberry Fragaria × ananassa. Short-read sequences from one diploid and 19 octoploid accessions were aligned to the diploid Fragaria vesca ‘Hawaii 4’ reference genome to identify single nucleotide polymorphisms (SNPs) and indels for incorporation into a 90 K Affymetrix® Axiom® array. We report the development and preliminary evaluation of this array.ResultsAbout 36 million sequence variants were identified in a 19 member, octoploid germplasm panel. Strategies and filtering pipelines were developed to identify and incorporate markers of several types: di-allelic SNPs (66.6%), multi-allelic SNPs (1.8%), indels (10.1%), and ploidy-reducing “haploSNPs” (11.7%). The remaining SNPs included those discovered in the diploid progenitor F. iinumae (3.9%), and speculative “codon-based” SNPs (5.9%). In genotyping 306 octoploid accessions, SNPs were assigned to six classes with Affymetrix’s “SNPolisher” R package. The highest quality classes, PolyHigh Resolution (PHR), No Minor Homozygote (NMH), and Off-Target Variant (OTV) comprised 25%, 38%, and 1% of array markers, respectively. These markers were suitable for genetic studies as demonstrated in the full-sib family ‘Holiday’ × ‘Korona’ with the generation of a genetic linkage map consisting of 6,594 PHR SNPs evenly distributed across 28 chromosomes with an average density of approximately one marker per 0.5 cM, thus exceeding our goal of one marker per cM.ConclusionsThe Affymetrix IStraw90 Axiom array is the first high-throughput genotyping platform for cultivated strawberry and is commercially available to the worldwide scientific community. The array’s high success rate is likely driven by the presence of naturally occurring variation in ploidy level within the nominally octoploid genome, and by effectiveness of the employed array design and ploidy-reducing strategies. This array enables genetic analyses including generation of high-density linkage maps, identification of quantitative trait loci for economically important traits, and genome-wide association studies, thus providing a basis for marker-assisted breeding in this high value crop.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1310-1) contains supplementary material, which is available to authorized users.

Ribozymes are catalytic RNA molecules that can be designed to cleave specific RNA sequences. To investigate the potential use of synthetic stabilized ribozymes for the treatment of chronic hepatitis C virus (HCV) infection, we designed and synthesized hammerhead ribozymes targeting 15 conserved sites in the 5Ј untranslated region (UTR) of HCV RNA. This region forms an internal ribosome entry site that allows for efficient translation of the HCV polyprotein. The 15 synthetic ribozymes contained modified nucleotides and linkages that stabilize the molecules against nuclease degradation. All 15 ribozymes were tested for their ability to reduce expression in an HCV 5Ј UTR/ luciferase reporter system and for their ability to inhibit replication of an HCV-poliovirus (HCV-PV) chimera. Treatment with several ribozymes resulted in significant downregulation of HCV 5Ј UTR/luciferase reporter expression (range 40% to 80% inhibition, P F .05). Moreover, several ribozymes showed significant inhibition (G90%, Chronic infection with hepatitis C virus (HCV) can lead to cirrhosis, liver failure and/or hepatocellular carcinoma over a period of 10 to 20 years. 1,2 The Centers for Disease Control recently reported the number of chronically infected Americans to be approximately 4.5 million; thus, HCV infection is over four times as prevalent as human immunodeficiency virus infection. 3 Worldwide the prevalence of chronic HCV is similar to that found in the United States. 3 Thus, chronic HCV infection represents an important public health problem throughout the world.PTreatment of chronic HCV infection with interferon alfa leads to sustained viral clearance in only approximately 12% of patients. 4 Newer therapeutic regimes, such as the combination of interferon alfa and ribavirin, can lead to 38% to 43% of patients having a sustained virological response. 5,6 However, even with current combination regimes, approximately 60% of patients have no sustained virological benefit. Additionally, treatment with interferon alfa and ribavirin leads to significant toxicities. 5,6 Therefore, there still remains a great need for improved therapeutic modalities.HCV is a 9.5-kb, plus-strand, RNA virus that is a member of the human flavivirus family. 7,8 Although the sequence of the HCV-RNA genome is highly variable among clinical isolates, the 5Ј untranslated region (UTR) of the genome is highly conserved with respect to RNA sequence identity. 9,10 The conserved sequence/structure of the 5Ј UTR of HCV RNA contains an internal ribosome entry site (IRES) to mediate translation independent of a 5Ј-cap structure. 10,11 The HCV IRES does not require any viral protein for initiation of translation 12 and IRES elements also occur in cellular messenger RNAs. 13,14 Because the components of IRES-mediated translation are shared between cellular and HCV messenger RNAs, targeting the mechanism of HCV-IRES-mediated translation can be problematic. However, because the HCV-IRES sequence is highly conserved among viral genotypes, it is an excellent target for ribo...

RNAs 33 nucleotides in length can direct accurate initiation of subgenomic RNA synthesis by the brome mosaic virus RNA-dependent RNA polymerase (RdRp), provided that the native sequences are maintained at five positions: ؊17, ؊14, ؊13, ؊11, and the ؉1 initiation site. The functional groups in the bases of these essential nucleotides required to interact with RdRp were examined by using chemically synthesized RNAs containing base analogs at each of the five positions. Analysis using a template competition assay revealed that the mode of recognition for the initiation nucleotide (؉1) is distinct from that of the other essential nucleotides in the promoter. Competition experiments also determined that three template nucleotides are sufficient for stable interaction with RdRp. These results identify base moieties in the brome mosaic virus subgenomic promoter required for efficient RNA synthesis and support the hypothesis that the recognition of a RNA promoter by a viral RdRp is analogous to the recognition of DNA promoters by DNAdependent RNA polymerases.Replication of viral RNA genomes requires specific recognition of RNA features by proteins. The viral and cellular proteins that comprise the RNA-dependent RNA polymerase (RdRp) complex are responsible for directing viral RNA synthesis from the infecting RNA templates. Unlike other polymerases, RdRps can initiate RNA synthesis from either the end of the RNA template or from an internal promoter (1). The recognition elements that determine template specificity, ensuring amplification of the appropriate viral RNA species, are not well characterized. An in-depth biochemical knowledge of the features that determine specificity of viral RNA replication is directly relevant to viral pathogenesis and is important for elucidating the principles for protein-RNA interaction.To investigate the mechanism of RNA-directed RNA synthesis, we studied brome mosaic virus (BMV), the type member of the bromovirus group of plant viruses in the alphaviruslike superfamily of (ϩ)-strand RNA viruses (2). Three RNAs designated 1, 2, and 3, and a subgenomic RNA4 that is initiated from (Ϫ)-strand RNA3 comprise the BMV genome. In a highly specific manner, enriched BMV RdRp preparations from infected barley can synthesize (Ϫ)-strand RNA from (ϩ)-strand templates and subgenomic (ϩ)-strand products from (Ϫ)-strand templates (3-5).We have developed a system to study BMV subgenomic RNA initiation from minimal templates, designated proscripts, because they contain both the promoter and template for (ϩ)-strand RNA synthesis. The 20 nts 3Ј of the subgenomic initiation nucleotide are recognized as the subgenomic core promoter (6, 7). In vitro, these 20 nts efficiently and accurately direct (ϩ)-strand RNA synthesis (5, 8). By using this functional assay, we demonstrated that nucleotides Ϫ17, Ϫ14, Ϫ13, and Ϫ11 relative to the subgenomic initiation site must be maintained for RNA synthesis and are recognized in a sequencespecific fashion (8). RdRp interaction with the BMV core promoter represents an...

A novel metal-binding site has been identified in the hammerhead ribozyme by 31 P NMR. The metal-binding site is associated with the A 13 phosphate in the catalytic core of the hammerhead ribozyme and is distinct from any previously identified metal-binding sites. 31 P NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250-570 mM at 25 8C for binding of a single Mg 21 ion. The NMR data also show evidence of a structural change at this site upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the hammerhead ribozyme. These NMR data were combined with the X-ray structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB. 1994. Nature 372:68-74) to model RNA ligands involved in binding the metal at this A 13 site. In this model, the A 13 metal-binding site is structurally similar to the previously identified A 9 metal-binding site and illustrates the symmetrical nature of the tandem G•A base pairs in domain 2 of the hammerhead ribozyme. These results demonstrate that 31 P NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids.

4'-Thio-beta-D-oligoribonucleotides (12 mer and 16 mer) containing a mixed base sequence were synthesized via the phosphoramidite solid support approach. These RNA analogs showed very good nuclease resistance as compared with wild-type RNA. Furthermore, 4'-thio-beta-D-oligoribonucleotides were shown to hybridize with a complementary DNA or RNA strand to form a duplex or with a DNA hairpin to form a triple helix. 4'-Thio-RNA binds more tightly to its complementary RNA strand than to its complementary DNA strand. A 4'-thio-RNA:RNA duplex is as stable as a 2'-O-methyl-RNA:RNA duplex. 4'-Thio-RNA, however, forms a 4'-thio-RNA:DNA:DNA triplex with a stability similar to the corresponding triplex with all wild-type DNA.