Semi-viviparous embryo development and dehydrin expression in the mangrove Rhizophora mucronata Lam.

Rhizophora mucronata Lam. is a tropical mangrove with semi-viviparous (cotyledon body protrusion before shedding), non-quiescent and non-desiccating (recalcitrant) seeds. As recalcitrance has been thought to relate to the absence of desiccation-related proteins such as dehydrins, we for the first time systematically described and classified embryogenesis in R. mucronata and assessed the presence of dehydrin-like proteins. Embryogenesis largely follows the classic pattern till stage eight, the torpedo stage, with the formation of a cotyledonary body. Ovule and embryo express radical adaptations to semi-vivipary in the saline environment: (1) A large, highly vacuolated and persistent endosperm without noticeable food reserves that envelopes the developing embryo. (2) Absence of vascular tissue connections between embryo and maternal tissue, but, instead, transfer layers in between endosperm and integument and endosperm and embryo. Dehydrin-like proteins (55–65 kDa) were detected by the Western analysis, in the ovules till stage 10 when the integuments are dehisced. An additional 50 kDa band was detected at stages 6–8. Together these results suggest a continuous flow of water with nutrients from the integument via the endosperm to the embryo, circumventing the vascular route and probably suppressing the initially induced dehydrin expression

Genetic diversity at the Dhn3 locus in Turkish Hordeum spontaneum populations with comparative structural analyses

We analysed Hordeum spontaneum accessions from 21 different locations to understand the genetic diversity of HsDhn3 alleles and effects of single base mutations on the intrinsically disordered structure of the resulting polypeptide (HsDHN3). HsDHN3 was found to be YSK2-type with a low-frequency 6-aa deletion in the beginning of Exon 1. There is relatively high diversity in the intron region of HsDhn3 compared to the two exon regions. We have found subtle differences in K segments led to changes in amino acids chemical properties. Predictions for protein interaction profiles suggest the presence of a protein-binding site in HsDHN3 that coincides with the K1 segment. Comparison of DHN3 to closely related cereals showed that all of them contain a nuclear localization signal sequence flanking to the K1 segment and a novel conserved region located between the S and K1 segments [E(D/T)DGMGGR]. We found that H. vulgare, H. spontaneum, and Triticum urartu DHN3s have a greater number of phosphorylation sites for protein kinase C than other cereal species, which may be related to stress adaptation. Our results show that the nature and extent of mutations in the conserved segments of K1 and K2 are likely to be key factors in protection of cells

Isolation and fine mapping of Rps6: An intermediate host resistance gene in barley to wheat stripe rust

A plant may be considered a nonhost of a pathogen if all known genotypes of a plant species are resistant to all known isolates of a pathogen species. However, if a small number of genotypes are susceptible to some known isolates of a pathogen species this plant maybe considered an intermediate host. Barley (Hordeum vulgare) is an intermediate host for Puccinia striiformis f. sp. tritici (Pst), the causal agent of wheat stripe rust. We wanted to understand the genetic architecture underlying resistance to Pst and to determine whether any overlap exists with resistance to the host pathogen, Puccinia striiformis f. sp. hordei (Psh). We mapped Pst resistance to chromosome 7H and show that host and intermediate host resistance is genetically uncoupled. Therefore, we designate this resistance locus Rps6. We used phenotypic and genotypic selection on F2:3 families to isolate Rps6 and fine mapped the locus to a 0.1 cM region. Anchoring of the Rps6 locus to the barley physical map placed the region on two adjacent fingerprinted contigs. Efforts are now underway to sequence the minimal tiling path and to delimit the physical region harbouring Rps6. This will facilitate additional marker development and permit identification of candidate genes in the region

New algorithm improves fine structure of the barley consensus SNP map

<p>Abstract</p> <p>Background</p> <p>The need to integrate information from multiple linkage maps is a long-standing problem in genetics. One way to visualize the complex ordinal relationships is with a directed graph, where each vertex in the graph is a bin of markers. When there are no ordering conflicts between the linkage maps, the result is a directed acyclic graph, or DAG, which can then be linearized to produce a consensus map.</p> <p>Results</p> <p>New algorithms for the simplification and linearization of consensus graphs have been implemented as a package for the R computing environment called DAGGER. The simplified consensus graphs produced by DAGGER exactly capture the ordinal relationships present in a series of linkage maps. Using either linear or quadratic programming, DAGGER generates a consensus map with minimum error relative to the linkage maps while remaining ordinally consistent with them. Both linearization methods produce consensus maps that are compressed relative to the mean of the linkage maps. After rescaling, however, the consensus maps had higher accuracy (and higher marker density) than the individual linkage maps in genetic simulations. When applied to four barley linkage maps genotyped at nearly 3000 SNP markers, DAGGER produced a consensus map with improved fine structure compared to the existing barley consensus SNP map. The root-mean-squared error between the linkage maps and the DAGGER map was 0.82 cM per marker interval compared to 2.28 cM for the existing consensus map. Examination of the barley hardness locus at the 5HS telomere, for which there is a physical map, confirmed that the DAGGER output was more accurate for fine structure analysis.</p> <p>Conclusions</p> <p>The R package DAGGER is an effective, freely available resource for integrating the information from a set of consistent linkage maps.</p

Selection of transformation-efficient barley genotypes based on TFA (transformation amenability) haplotype and higher resolution mapping of the TFA loci

Barley (Hordeum vulgare) cv. ‘Golden Promise’ is one of the most useful and well-studied cultivars for genetic manipulation. In a previous report, we identified several transformation amenability (TFA) loci responsible for Agrobacterium-mediated transformation using the F2 generation of immature embryos, derived from ‘Haruna Nijo’ × ‘Golden Promise,’ as explants. In this report, we describe higher density mapping of these TFA regions with additional SNP markers using the same transgenic plants. To demonstrate the robustness of transformability alleles at the TFA loci, we genotyped 202 doubled haploid progeny from the cross ‘Golden Promise’ × ‘Full Pint.’ Based on SNP genotype, we selected lines having ‘Golden Promise’ alleles at TFA loci and used them for transformation. Of the successfully transformed lines, DH120366 came the closest to achieving a level of transformation efficiency comparable to ‘Golden Promise.’ The results validate that the genetic substitution of TFA alleles from ‘Golden Promise’ can facilitate the development of transformation-efficient lines from recalcitrant barley cultivars

454 sequencing of pooled BAC clones on chromosome 3H of barley

<p>Abstract</p> <p>Background</p> <p>Genome sequencing of barley has been delayed due to its large genome size (ca. 5,000Mbp). Among the fast sequencing systems, 454 liquid phase pyrosequencing provides the longest reads and is the most promising method for BAC clones. Here we report the results of pooled sequencing of BAC clones selected with ESTs genetically mapped to chromosome 3H.</p> <p>Results</p> <p>We sequenced pooled barley BAC clones using a 454 parallel genome sequencer. A PCR screening system based on primer sets derived from genetically mapped ESTs on chromosome 3H was used for clone selection in a BAC library developed from cultivar "Haruna Nijo". The DNA samples of 10 or 20 BAC clones were pooled and used for shotgun library development. The homology between contig sequences generated in each pooled library and mapped EST sequences was studied. The number of contigs assigned on chromosome 3H was 372. Their lengths ranged from 1,230 bp to 58,322 bp with an average 14,891 bp. Of these contigs, 240 showed homology and colinearity with the genome sequence of rice chromosome 1. A contig annotation browser supplemented with query search by unique sequence or genetic map position was developed. The identified contigs can be annotated with barley cDNAs and reference sequences on the browser. Homology analysis of these contigs with rice genes indicated that 1,239 rice genes can be assigned to barley contigs by the simple comparison of sequence lengths in both species. Of these genes, 492 are assigned to rice chromosome 1.</p> <p>Conclusions</p> <p>We demonstrate the efficiency of sequencing gene rich regions from barley chromosome 3H, with special reference to syntenic relationships with rice chromosome 1.</p

The instantaneous helical axis of the subtalar and talocrural joints: a non-invasive in vivo dynamic study

<p>Abstract</p> <p>Background</p> <p>An understanding of rear-foot (talocrural and subtalar joints) kinematics is critical for diagnosing foot pathologies, designing total ankle implants, treating rear-foot injuries and quantifying gait abnormalities. The majority of kinematic data available have been acquired through static cadaver work or passive <it>in vivo </it>studies. The applicability of these data to dynamic <it>in vivo </it>situations remains unknown. Thus, the purpose of this study was to fully quantify subtalar, talocrural and calcaneal-tibial <it>in vivo </it>kinematics in terms of the instantaneous helical axis (IHA) in twenty-five healthy ankles during a volitional activity that simulated single-leg toe-raises with partial-weight support, requiring active muscle control.</p> <p>Methods</p> <p>Subjects were each placed supine in a 1.5 T MRI and asked to repeat this simulated toe-raise while a full sagittal-cine-phase contrast (dynamic) MRI dataset was acquired. From the cine-phase contrast velocity a full kinematic description for each joint was derived.</p> <p>Results</p> <p>Nearly all motion quantified at the calcaneal-tibial joint was attributable to the talocrural joint. The subtalar IHA orientation and position were highly variable; whereas, the talocrural IHA orientation and position were extremely consistent.</p> <p>Conclusion</p> <p>The talocrural was well described by the IHA and could be modeled as a fixed-hinge joint, whereas the subtalar could not be.</p

Genome-Wide Analysis of Müller Glial Differentiation Reveals a Requirement for Notch Signaling in Postmitotic Cells to Maintain the Glial Fate

Previous studies have shown that Müller glia are closely related to retinal progenitors; these two cell types express many of the same genes and after damage to the retina, Müller glia can serve as a source for new neurons, particularly in non-mammalian vertebrates. We investigated the period of postnatal retinal development when progenitors are differentiating into Müller glia to better understand this transition. FACS purified retinal progenitors and Müller glia from various ages of Hes5-GFP mice were analyzed by Affymetrix cDNA microarrays. We found that genes known to be enriched/expressed by Müller glia steadily increase over the first three postnatal weeks, while genes associated with the mitotic cell cycle are rapidly downregulated from P0 to P7. Interestingly, progenitor genes not directly associated with the mitotic cell cycle, like the proneural genes Ascl1 and Neurog2, decline more slowly over the first 10–14 days of postnatal development, and there is a peak in Notch signaling several days after the presumptive Müller glia have been generated. To confirm that Notch signaling continues in the postmitotic Müller glia, we performed in situ hybridization, immunolocalization for the active form of Notch, and immunofluorescence for BrdU. Using genetic and pharmacological approaches, we found that sustained Notch signaling in the postmitotic Müller glia is necessary for their maturation and the stabilization of the glial identity for almost a week after the cells have exited the mitotic cell cycle

Ml proteins from Mesorhizobium loti and MucR from Brucella abortus: an AT-rich core DNA-target site and oligomerization ability

Mesorhizobium loti contains ten genes coding for proteins sharing high amino acid sequence identity with members of the Ros/MucR transcription factor family. Five of these Ros/MucR family members from Mesorhizobium loti (Ml proteins) have been recently structurally and functionally characterized demonstrating that Ml proteins are DNA-binding proteins. However, the DNA-binding studies were performed using the Ros DNA-binding site with the Ml proteins. Currently, there is no evidence as to when the Ml proteins are expressed during the Mesorhizobium loti life cycle as well as no information concerning their natural DNA-binding site. In this study, we examine the ml genes expression profile in Mesorhizobium loti and show that ml1, ml2, ml3 and ml5 are expressed during planktonic growth and in biofilms. DNA-binding experiments show that the Ml proteins studied bind a conserved AT-rich site in the promoter region of the exoY gene from Mesorhizobium loti and that the proteins make important contacts with the minor groove of DNA. Moreover, we demonstrate that the Ml proteins studied form higher-order oligomers through their N-terminal region and that the same AT-rich site is recognized by MucR from Brucella abortus using a similar mechanism involving contacts with the minor groove of DNA and oligomerization