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Translational control of transcription factor ATF4 through paired upstream ORFs (uORFs) plays an important role in eukaryotic gene regulation. While it is typically induced by phosphorylation of eIF2α, ATF4 translation can be also induced by expression of a translational inhibitor protein, eIF5-mimic protein 1 (5MP1, also known as BZW2) in mammals. Here we show that the 5MP gene is maintained in eukaryotes under strong purifying selection, but is uniquely missing in two major phyla, nematoda and ascomycota. The common function of 5MP from protozoa, plants, fungi and insects is to control translation by inhibiting eIF2. The affinity of human 5MP1 to eIF2β was measured as being equivalent to the published value of human eIF5 to eIF2β, in agreement with effective competition of 5MP with eIF5 for the main substrate, eIF2. In the red flour beetle, Tribolium castaneum, RNA interference studies indicate that 5MP facilitates expression of GADD34, a downstream target of ATF4. Furthermore, both 5MP and ATF4 are essential for larval development. Finally, 5MP and the paired uORFs allowing ATF4 control are conserved in the entire metazoa except nematoda. Based on these findings, we discuss the phylogenetic and functional linkage between ATF4 regulation and 5MP expression in this group of eukaryotes.

Summary: Wheat is a globally vital crop, but its limited genetic variation creates a challenge for breeders aiming to maintain or accelerate agricultural improvements over time. Introducing novel genes and alleles from wheat's wild relatives into the wheat breeding pool via introgression lines is an important component of overcoming this low variation but is constrained by poor genomic resolution and limited understanding of the genomic impact of introgression breeding programmes. By sequencing 17 hexaploid wheat/Ambylopyrum muticum introgression lines and the parent lines, we have precisely pinpointed the borders of introgressed segments, most of which occur within genes. We report a genome assembly and annotation of Am. muticum that has facilitated the identification of Am. muticum resistance genes commonly introgressed in lines resistant to stripe rust. Our analysis has identified an abundance of structural disruption and homoeologous pairing across the introgression lines, likely caused by the suppressed Ph1 locus. mRNAseq analysis of six of these introgression lines revealed that novel introgressed genes are rarely expressed and those that directly replace a wheat orthologue have a tendency towards downregulation, with no discernible compensation in the expression of homoeologous copies. This study explores the genomic impact of introgression breeding and provides a schematic that can be followed to characterize introgression lines and identify segments and candidate genes underlying the phenotype. This will facilitate more effective utilization of introgression pre‐breeding material in wheat breeding programmes. [ABSTRACT FROM AUTHOR]

Wheat leaf rust, caused by Puccinia triticina Eriks., infects millions of acres of wheat (Triticum aestivum L.) and causes leaves to senesce prematurely, which in turn decreases yield by reducing the plant's ability to complete kernel fill. Lr17a, a leaf rust resistance gene in wheat, is present in many wheat varieties available today. The objective of this research was to identify molecular markers linked to Lr17a. Microsatellite markers were used on a mapping population of 161 [F.sub.2] lines made from a cross of Chinese Spring and Thatcher-Lr17a. Capillary fragment analysis was performed and eight markers were linked to Lr17a, of which Xgwm614 and Xwmc407 flanked Lr17a at genetic distances of 0.7 and 2.5 cM, respectively. An evaluation of cultivars with and without Lr17a, and grown in the midwestern United States, revealed that multiple alleles were present for all markers and little correspondence between alleles of closely linked markers and Lr17a was observed. Therefore, caution must taken when using them for marker-assisted selection.

Resistance to Fusarium head blight (FHB) (caused mainly by Fusarium graminearum Schw.) is difficult to obtain because of the quantitative nature and the numerous types of resistance involved, cDNA expression arrays have become a rapidly growing area for the identification and characterization of genes involved in complex pathways. A cDNA library was made from wheat (Triticum aestivum L.) spikes of the variety 'Sumai 3', 24 h after inoculation with F. graminearum. Clones were sequenced and putative function assigned to each clone based on BLASTX alignments. Nylon membrane arrays were made from the 580 unigenes present in the library, of which 75 expressed clones were induced in the first 24 h after inoculation. Of these unigenes, 14 are involved in defense response, 9 in gene expression and regulation, 29 in other cell functions, and 24 are without a known function. The induced genes catalyze key steps in the formation of lignin, energy production, and production of phytoalexins suggesting that resistance in wheat to Fusarium is provided by a different pathway than that of the hypersensitive response. Two of the induced genes, BE585589 and BE585627, were physically mapped to regions of chromosomes 3AS and 6BL, respectively, known to contain major QTL for FHB resistance. Abbreviations: DON, deoxynivalenol; EST, expressed sequence tag; FHB, Fusarium head blight; hai, hours after inoculation; OPR, 12-oxophytodienoate reductase.

The Q gene is largely responsible for the widespread cultivation of wheat because it confers the free-threshing character. It also pleiotropically influences many other domestication-related traits such as glume shape and tenacity, rachis fragility, spike length, plant height, and spike emergence time. We isolated the Q gene and verified its identity by analysis of knockout mutants and transformation. The Q gene has a high degree of similarity to members of the AP2 family of transcription factors. The Q allele is more abundantly transcribed than q, and the two alleles differ for a single amino acid. An isoleucine at position 329 in the Q protein leads to an abundance of homodimer formation in yeast cells, whereas a valine in the q protein appears to limit homodimer formation. Ectopic expression analysis allowed us to observe both silencing and overexpression effects of Q. Rachis fragility, glume shape, and glume tenacity mimicked the q phenotype in transgenic plants exhibiting post-transcriptional silencing of the transgene and the endogenous Q gene. Variation in spike compactness and plant height were associated with the level of transgene transcription due to the dosage effects of Q. The q allele is the more primitive, and the mutation that gave rise to Q occurred only once leading to the world's cultivated wheats.

We report the map-based cloning of the leaf rust resistance gene Lr21, previously mapped to a generich region at the distal end of chromosome arm 1DS of bread wheat (Triticum aestivum L.). Molecular cloning of Lr21 was facilitated by diploid/polyploid shuttle mapping strategy. Cloning of Lr21 was confirmed by genetic transformation and by a stably inherited resistance phenotype in transgenic plants. Lr21 spans 4318 bp and encodes a 1080-amino-acid protein containing a conserved nucleotide-binding site (NBS) domain, 13 imperfect leucine-rich repeats (LRRs), and a unique 151-amino-acid sequence missing from known NBS-LRR proteins at the N terminus. Fine-structure genetic analysis at the Lr21 locus detected a noncrossover (recombination without exchange of flanking markers) within a 1415-bp region resulting from either a gene conversion tract of at least 191 bp or a double crossover. The successful map-based cloning approach as demonstrated here now opens the door for cloning of many crop-specific agronomic traits located in the gene-rich regions of bread wheat.

The Q locus played a major role in the domestication of wheat because it confers the free-threshing character and influences many other agronomically important traits. We constructed a physical contig spanning the Q locus using a Triticum monococcum BAC library. Three chromosome walking steps were performed by complete sequencing of BACs and identification of low-copy markers through similarity searches of database sequences. The BAC contig spans a physical distance of ~300 kb corresponding to a genetic distance of 0.9 cM. The physical map of T. monococcum had perfect colinearity with the genetic map of wheat chromosome arm 5AL. Recombination data in conjunction with analysis of fast neutron deletions confirmed that the contig spanned the Q locus. The Q gene was narrowed to a 100-kb segment, which contains an APETALA2 (AP2)-like gene that cosegregates with Q. AP2 is known to play a major role in controlling floral homeotic gene expression and thus is an excellent candidate for Q.

Conserved motifs within resistance genes have been utilized in polymerase chain reaction (PCR)-based strategies to isolate resistance gene analogs (RGAs) and resistance gene-like (RGLs) sequences from many plant species. RGAs have the potential to serve as closely linked markers for marker-assisted breeding or even as resistance gene candidates. The objectives of this study were to clone, sequence, and map RGAs and kinase analogs (KA). Three motifs in nucleotide binding site-leucine-rich repeat (NBS-LRR) resistance genes and two conserved motifs within R-gene kinases were used to design degenerate primers and amplify RGAs and KAs from wheat (Triticum aestivum L.). Eight NBS-LRR and 26 KAs were isolated. The clones were physically mapped to chromosomes by means of wheat nulli-tetrasomic lines. The probes detected 137 fragments that could be assigned to 20 of the 21 chromosomes; nearly half of the fragments mapped in the B genome. None of the fragments mapped to chromosome 4D. Genetic mapping of clones showed simple and complex loci indicating both single and multigene families. The RGAs and KAs will be useful as markers for mapping resistance gene loci., DISEASE RESISTANCE GENES (R-genes) have been cloned from a number of plant species and can be categorized into four classes on the basis of the conserved amino acid sequences of [...]

New technology is allowing marker-assisted selection to fulfill the promise of increasing efficiency of cultivar development. However, these techniques depend upon the ability to extract DNA from large populations of plants. The objective of this project was to develop a high-throughput DNA extraction procedure without the need for greenhouse space or growing wheat (Triticum aestivum L.) plants. A sodium hydroxide rapid DNA extraction was modified for a 96-well format to reduce costs. Seeds were germinated in 8-well tissue culture plates, and 4-d-old seedling tissue was used to extract DNA by means of sodium hydroxide methodology. Approximately 1 µg of genomic DNA per 10 mg of tissue was isolated at a cost of about $0.10. The DNA quality was verified by amplification of microsatellite markers. Results were consistent with either fresh or stored tissue extracts. This technique allows one person to extract nearly 1000 storage-stable DNA samples daily, while keeping costs at a minimum., MARKER-ASSISTED SELECTION in plant breeding programs is limited by the availability of markers, ability to extract DNA from large populations of plants, and, most importantly, high cost. New technologies, such [...]

The homeotic selector genes of the red flour beetle, Tribolium castaneum, are located in a single cluster. We have sequenced the region containing the homeotic selector genes required for proper development of the head and anterior thorax, which is the counterpart of the ANTC in Drosophila. This 280-kb interval contains eight homeodomain-encoding genes, including single orthologs of the Drosophila genes labial, proboscipedia, Deformed, Sex combs reduced, fushi tarazu, and Antennapedia, as well as two orthologs of zerknullt. These genes are all oriented in the same direction, as are the Hox genes of amphioxus, mice, and humans. Although each transcription unit is similar to its Drosophila counterpart in size, the Tribolium genes contain fewer introns (with the exception of the two zerknullt genes), produce shorter mRNAs, and encode smaller proteins. Unlike the ANTC, this region of the Tribolium HOMC contains no additional genes.

The genome-linked protein-proteinase (NIa) genes of three potyviruses (tobacco rein mottling virus[TVMV], tobacco etch virus[TEV], and potato virus Y [PVY]) were introduced into tobacco (Nicotiana tabacum L.) cultivar Burley 21 and transgenic plant lines were evaluated for viral symptoms and virus accumulation. Plant lines that expressed the TVMV NIa gene were resistant to infection by TVMV; inoculated plants did not develop disease symptoms after inoculation, and virus did not accumulate. Most lines that carried the TEV NIa and PVY NIa genes did develop symptoms after inoculation with TEV and PVY, respectively, but these symptoms abated with time (a response termed as recovery). Three genes, each consisting of two NIa coding regions (TEV NIa-PVY NIa, TEV NIa-TVMV NIa, and TVMV NIa-PVY NIa) were also introduced into Burley 21. Transgenic lines that carried either TEV NIa-PVY NIa or TEV NIa-TVMV NIa constructs either did not develop disease symptoms or exhibited the process of recovery after inoculation with TEV. TEV NIa-TVMV NIa and TVMV NIa-PVY NIa lines recovered from an initial infection after inoculation with TVMV. Likewise, TEV NIa-PVY NIa and TVMV NIa-PVY NIa transformed lines recovered from initial infection after inoculation with PVY. These results demonstrate that different potyvirus NIa-proteinase genes can be used for protection against virus infection and that multiple NIa genes may be used to produce plants that are protected against more than one potyvirus., POTYVIRUSES are the largest group of plant viruses and infect a wide range of host plants. Potyviruses are transmitted by aphids and are non-persistent in the aphid vector. Classical breeding [...]

Abstract Background Wheat leaf rust (Puccinia triticina Eriks; Pt) and stem rust fungi (P. graminis f.sp. tritici; Pgt) are significant economic pathogens having similar host ranges and life cycles, but different alternate hosts. The Pt genome, currently estimated at 135 Mb, is significantly larger than Pgt, at 88 Mb, but the reason for the expansion is unknown. Three genomic loci of Pt conserved proteins were characterized to gain insight into gene content, genome complexity and expansion. Results A bacterial artificial chromosome (BAC) library was made from P. triticina race 1, BBBD and probed with Pt homologs of genes encoding two predicted Pgt secreted effectors and a DNA marker mapping to a region of avirulence. Three BACs, 103 Kb, 112 Kb, and 166 Kb, were sequenced, assembled, and open reading frames were identified. Orthologous genes were identified in Pgt and local conservation of gene order (microsynteny) was observed. Pairwise protein identities ranged from 26 to 99%. One Pt BAC, containing a RAD18 ortholog, shares syntenic regions with two Pgt scaffolds, which could represent both haplotypes of Pgt. Gene sequence is diverged between the species as well as within the two haplotypes. In all three BAC clones, gene order is locally conserved, however, gene shuffling has occurred relative to Pgt. These regions are further diverged by differing insertion loci of LTR-retrotransposon, Gypsy, Copia, Mutator, and Harbinger mobile elements. Uncharacterized Pt open reading frames were also found; these proteins are high in lysine and similar to multiple proteins in Pgt. Conclusions The three Pt loci are conserved in gene order, with a range of gene sequence divergence. Conservation of predicted haustoria expressed secreted protein genes between Pt and Pgt is extended to the more distant poplar rust, Melampsora larici-populina. The loci also reveal that genome expansion in Pt is in part due to higher occurrence of repeat-elements in this species.

Abstract Background Rust fungi are biotrophic basidiomycete plant pathogens that cause major diseases on plants and trees world-wide, affecting agriculture and forestry. Their biotrophic nature precludes many established molecular genetic manipulations and lines of research. The generation of genomic resources for these microbes is leading to novel insights into biology such as interactions with the hosts and guiding directions for breakthrough research in plant pathology. Results To support gene discovery and gene model verification in the genome of the wheat leaf rust fungus, Puccinia triticina (Pt), we have generated Expressed Sequence Tags (ESTs) by sampling several life cycle stages. We focused on several spore stages and isolated haustorial structures from infected wheat, generating 17,684 ESTs. We produced sequences from both the sexual (pycniospores, aeciospores and teliospores) and asexual (germinated urediniospores) stages of the life cycle. From pycniospores and aeciospores, produced by infecting the alternate host, meadow rue (Thalictrum speciosissimum), 4,869 and 1,292 reads were generated, respectively. We generated 3,703 ESTs from teliospores produced on the senescent primary wheat host. Finally, we generated 6,817 reads from haustoria isolated from infected wheat as well as 1,003 sequences from germinated urediniospores. Along with 25,558 previously generated ESTs, we compiled a database of 13,328 non-redundant sequences (4,506 singlets and 8,822 contigs). Fungal genes were predicted using the EST version of the self-training GeneMarkS algorithm. To refine the EST database, we compared EST sequences by BLASTN to a set of 454 pyrosequencing-generated contigs and Sanger BAC-end sequences derived both from the Pt genome, and to ESTs and genome reads from wheat. A collection of 6,308 fungal genes was identified and compared to sequences of the cereal rusts, Puccinia graminis f. sp. tritici (Pgt) and stripe rust, P. striiformis f. sp. tritici (Pst), and poplar leaf rust Melampsora species, and the corn smut fungus, Ustilago maydis (Um). While extensive homologies were found, many genes appeared novel and species-specific; over 40% of genes did not match any known sequence in existing databases. Focusing on spore stages, direct comparison to Um identified potential functional homologs, possibly allowing heterologous functional analysis in that model fungus. Many potentially secreted protein genes were identified by similarity searches against genes and proteins of Pgt and Melampsora spp., revealing apparent orthologs. Conclusions The current set of Pt unigenes contributes to gene discovery in this major cereal pathogen and will be invaluable for gene model verification in the genome sequence.

Abstract Background The Hessian fly (Mayetiola destructor) is an important insect pest of wheat. It has tractable genetics, polytene chromosomes, and a small genome (158 Mb). Investigation of the Hessian fly presents excellent opportunities to study plant-insect interactions and the molecular mechanisms underlying genome imprinting and chromosome elimination. A physical map is needed to improve the ability to perform both positional cloning and comparative genomic analyses with the fully sequenced genomes of other dipteran species. Results An FPC-based genome wide physical map of the Hessian fly was constructed and anchored to the insect's polytene chromosomes. Bacterial artificial chromosome (BAC) clones corresponding to 12-fold coverage of the Hessian fly genome were fingerprinted, using high information content fingerprinting (HIFC) methodology, and end-sequenced. Fluorescence in situ hybridization (FISH) co-localized two BAC clones from each of the 196 longest contigs on the polytene chromosomes. An additional 70 contigs were positioned using a single FISH probe. The 266 FISH mapped contigs were evenly distributed and covered 60% of the genome (95,668 kb). The ends of the fingerprinted BACs were then sequenced to develop the capacity to create sequenced tagged site (STS) markers on the BACs in the map. Only 3.64% of the BAC-end sequence was composed of transposable elements, helicases, ribosomal repeats, simple sequence repeats, and sequences of low complexity. A relatively large fraction (14.27%) of the BES was comprised of multi-copy gene sequences. Nearly 1% of the end sequence was composed of simple sequence repeats (SSRs). Conclusion This physical map provides the foundation for high-resolution genetic mapping, map-based cloning, and assembly of complete genome sequencing data. The results indicate that restriction fragment length heterogeneity in BAC libraries used to construct physical maps lower the length and the depth of the contigs, but is not an absolute barrier to the successful application of the technology. This map will serve as a genomic resource for accelerating gene discovery, genome sequencing, and the assembly of BAC sequences. The Hessian fly BAC-clone assembly, and the names and positions of the BAC clones used in the FISH experiments are publically available at http://genome.purdue.edu/WebAGCoL/Hfly/WebFPC/.

Wheat (Triticum aestivum L.) rusts are a worldwide production problem. Plant breeders have used genetic resistance to combat these fungi. However, single‐gene resistance is rapidly overcome as a result of frequent occurrence of new virulent fungal strains. Thus, a supply of new resistance sources is continually needed, and new resistance sources are limited within hexaploid wheat genetic stocks. Wild relatives are able to be a resource for new resistance genes but are hindered because of chromosome incapability with domesticated wheats. Twenty‐eight double‐haploid hexaploid wheat/Amblyopyrum muticum (Boiss.) Eig introgression lines, with introgressions covering the majority of the T genome, were evaluated for resistance to Puccinia triticina Erikss., P. graminis Pers.:Pers. f.sp. tritici Erikss. & E. Henning, and P. striiformis Westend. f.sp. tritici Erikss.. At the seedling level, four lines were resistant to races of P. triticina, six lines were resistant to P. graminis, and 15 lines were resistant to P. striiformis. At the adult stage, 16 lines were resistant to P. triticina. Line 355 had resistance to all three rusts and line 161 had resistance to all tested races of P. triticina. Some of these lines will require further work to reduce the size of the introgressed segment; however, lines 92 and 355 have very small fragments and can be used directly as new resistance donors. [ABSTRACT FROM AUTHOR]