Developing expressed sequence tag libraries and the discovery of simple sequence repeat markers for two species of raspberry (\u3cem\u3eRubus\u3c/em\u3e L.)

Background Due to a relatively high level of codominant inheritance and transferability within and among taxonomic groups, simple sequence repeat (SSR) markers are important elements in comparative mapping and delineation of genomic regions associated with traits of economic importance. Expressed sequence tags (ESTs) are a source of SSRs that can be used to develop markers to facilitate plant breeding and for more basic research across genera and higher plant orders. Methods Leaf and meristem tissue from ‘Heritage’ red raspberry (Rubus idaeus) and ‘Bristol’ black raspberry (R. occidentalis) were utilized for RNA extraction. After conversion to cDNA and library construction, ESTs were sequenced, quality verified, assembled and scanned for SSRs. Primers flanking the SSRs were designed and a subset tested for amplification, polymorphism and transferability across species. ESTs containing SSRs were functionally annotated using the GenBank non-redundant (nr) database and further classified using the gene ontology database. Results To accelerate development of EST-SSRs in the genus Rubus (Rosaceae), 1149 and 2358 cDNA sequences were generated from red raspberry and black raspberry, respectively. The cDNA sequences were screened using rigorous filtering criteria which resulted in the identification of 121 and 257 SSR loci for red and black raspberry, respectively. Primers were designed from the surrounding sequences resulting in 131 and 288 primer pairs, respectively, as some sequences contained more than one SSR locus. Sequence analysis revealed that the SSR-containing genes span a diversity of functions and share more sequence identity with strawberry genes than with other Rosaceous species. Conclusion This resource of Rubus-specific, gene-derived markers will facilitate the construction of linkage maps composed of transferable markers for studying and manipulating important traits in this economically important genus

Folian-cv1 Is a Member of a Highly Acidic Phosphoprotein Class Derived From the Foliated Layer of the Eastern Oyster (\u3ci\u3eCrassostrea virginica\u3c/i\u3e) Shell and Identified in Hemocytes and Mantle

The proteins derived from the foliated shell layer of the oyster, Crassostrea virginica, are unusually acidic and highly phosphorylated. Here we report the identification of a gene encoding a member of this class of phosphoproteins that we collectively refer to as folian. Using an in silico approach, a virtual probe was constructed from an N-terminal sequence (DEADAGD) determined for a 48 kDa folian phosphoprotein and used to screen an oyster EST databank. A sequence that matched the N-terminus of the 48 kDa protein was found and used to identify the full-length gene from a C. virginica BAC library. The molecular weight of the deduced gene product is 32 kDa and was named folian-cv1. Genomic Southern analysis revealed two variants of the gene. The mature protein is composed of 43.3% Asp, 32.6% Ser, and 9.1% Glu with 37.5% of the amino acids of the protein potentially phosphorylated. The primary sequence of folian-cv1 is organized in blocks, with a short relatively hydrophobic block at the N-terminus and with the remainder containing low complexity regions largely dominated by aspartic acid and serine. Overall, the protein is predicted to be highly disordered. PCR and sequence analyses identified folian-cv1 expression in the mantle and hemocytes. Immuno-histochemical staining of mantle tissue reveals that cells of the shell-facing epithelium and in the periostracal groove secrete a continuous layer of folian-positive material and that folian-positive hemocytes move through the mantle epithelium. The function in shell formation of folian proteins including folian-cv1 is not known. However, based on the complexity of this class of proteins and the two methods of their delivery to the region of shell formation, it is possible they are involved in diverse ways in this process

Characterization of extrachromosomal circular DNA (eccDNA) structure, function, and dynamics in a CHO K-1 clone

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Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1

Meiosis depends on homologous recombination (HR) in most sexually reproducing organisms. Efficient meiotic HR requires the activity of the meiosis-specific recombinase, Dmc1. Previous work shows Dmc1 is expressed in Entamoeba histolytica, a eukaryotic parasite responsible for amoebiasis throughout the world, suggesting this organism undergoes meiosis. Here, we demonstrate Dmc1 protein is expressed in E. histolytica. We show that purified ehDmc1 forms presynaptic filaments and catalyzes ATP-dependent homologous DNA pairing and DNA strand exchange over at least several thousand base pairs. The DNA pairing and strand exchange activities are enhanced by the presence of calcium and the meiosis-specific recombination accessory factor, Hop2-Mnd1. In combination, calcium and Hop2-Mnd1 dramatically increase the rate of DNA strand exchange activity of ehDmc1. The biochemical system described herein provides a basis on which to better understand the role of ehDmc1 and other HR proteins in E. histolytica

Transcriptomics reveal the genetic coordination of early defense to Armillaria root rot (ARR) in Prunus spp

Armillaria root rot (ARR) poses a significant threat to the long-term productivity of stone-fruit and nut crops in the predominant production area of the United States. To mitigate this issue, the development of ARR-resistant and horticulturally-acceptable rootstocks is a crucial step towards the maintenance of production sustainability. To date, genetic resistance to ARR has been found in exotic plum germplasm and a peach/plum hybrid rootstock, ’MP-29‘. However, the widely-used peach rootstock Guardian® is susceptible to the pathogen. To understand the molecular defense mechanisms involved in ARR resistance in Prunus rootstocks, transcriptomic analyses of one susceptible and two resistant Prunus spp. were performed using two causal agents of ARR, including Armillaria mellea and Desarmillaria tabescens. The results of in vitro co-culture experiments revealed that the two resistant genotypes showed different temporal response dynamics and fungus-specific responses, as seen in the genetic response. Gene expression analysis over time indicated an enrichment of defense-related ontologies, including glucosyltransferase activity, monooxygenase activity, glutathione transferase activity, and peroxidase activity. Differential gene expression and co-expression network analysis highlighted key hub genes involved in the sensing and enzymatic degradation of chitin, GSTs, oxidoreductases, transcription factors, and biochemical pathways likely involved in Armillaria resistance. These data provide valuable resources for the improvement of ARR resistance in Prunus rootstocks through breeding

A new reference genome for Sorghum bicolor reveals high levels of sequence similarity between sweet and grain genotypes: implications for the genetics of sugar metabolism.

BackgroundThe process of crop domestication often consists of two stages: initial domestication, where the wild species is first cultivated by humans, followed by diversification, when the domesticated species are subsequently adapted to more environments and specialized uses. Selective pressure to increase sugar accumulation in certain varieties of the cereal crop Sorghum bicolor is an excellent example of the latter; this has resulted in pronounced phenotypic divergence between sweet and grain-type sorghums, but the genetic mechanisms underlying these differences remain poorly understood.ResultsHere we present a new reference genome based on an archetypal sweet sorghum line and compare it to the current grain sorghum reference, revealing a high rate of nonsynonymous and potential loss of function mutations, but few changes in gene content or overall genome structure. We also use comparative transcriptomics to highlight changes in gene expression correlated with high stalk sugar content and show that changes in the activity and possibly localization of transporters, along with the timing of sugar metabolism play a critical role in the sweet phenotype.ConclusionsThe high level of genomic similarity between sweet and grain sorghum reflects their historical relatedness, rather than their current phenotypic differences, but we find key changes in signaling molecules and transcriptional regulators that represent new candidates for understanding and improving sugar metabolism in this important crop

New genomic resources for switchgrass: a BAC library and comparative analysis of homoeologous genomic regions harboring bioenergy traits

<p>Abstract</p> <p>Background</p> <p>Switchgrass, a C4 species and a warm-season grass native to the prairies of North America, has been targeted for development into an herbaceous biomass fuel crop. Genetic improvement of switchgrass feedstock traits through marker-assisted breeding and biotechnology approaches calls for genomic tools development. Establishment of integrated physical and genetic maps for switchgrass will accelerate mapping of value added traits useful to breeding programs and to isolate important target genes using map based cloning. The reported polyploidy series in switchgrass ranges from diploid (2X = 18) to duodecaploid (12X = 108). Like in other large, repeat-rich plant genomes, this genomic complexity will hinder whole genome sequencing efforts. An extensive physical map providing enough information to resolve the homoeologous genomes would provide the necessary framework for accurate assembly of the switchgrass genome.</p> <p>Results</p> <p>A switchgrass BAC library constructed by partial digestion of nuclear DNA with <it>Eco</it>RI contains 147,456 clones covering the effective genome approximately 10 times based on a genome size of 3.2 Gigabases (~1.6 Gb effective). Restriction digestion and PFGE analysis of 234 randomly chosen BACs indicated that 95% of the clones contained inserts, ranging from 60 to 180 kb with an average of 120 kb. Comparative sequence analysis of two homoeologous genomic regions harboring orthologs of the rice <it>OsBRI1 </it>locus, a low-copy gene encoding a putative protein kinase and associated with biomass, revealed that orthologous clones from homoeologous chromosomes can be unambiguously distinguished from each other and correctly assembled to respective fingerprint contigs. Thus, the data obtained not only provide genomic resources for further analysis of switchgrass genome, but also improve efforts for an accurate genome sequencing strategy.</p> <p>Conclusions</p> <p>The construction of the first switchgrass BAC library and comparative analysis of homoeologous harboring <it>OsBRI1 </it>orthologs present a glimpse into the switchgrass genome structure and complexity. Data obtained demonstrate the feasibility of using HICF fingerprinting to resolve the homoeologous chromosomes of the two distinct genomes in switchgrass, providing a robust and accurate BAC-based physical platform for this species. The genomic resources and sequence data generated will lay the foundation for deciphering the switchgrass genome and lead the way for an accurate genome sequencing strategy.</p

A genetically anchored physical framework for Theobroma cacao cv. Matina 1-6

<p>Abstract</p> <p>Background</p> <p>The fermented dried seeds of <it>Theobroma cacao </it>(cacao tree) are the main ingredient in chocolate. World cocoa production was estimated to be 3 million tons in 2010 with an annual estimated average growth rate of 2.2%. The cacao bean production industry is currently under threat from a rise in fungal diseases including black pod, frosty pod, and witches' broom. In order to address these issues, genome-sequencing efforts have been initiated recently to facilitate identification of genetic markers and genes that could be utilized to accelerate the release of robust <it>T. cacao </it>cultivars. However, problems inherent with assembly and resolution of distal regions of complex eukaryotic genomes, such as gaps, chimeric joins, and unresolvable repeat-induced compressions, have been unavoidably encountered with the sequencing strategies selected.</p> <p>Results</p> <p>Here, we describe the construction of a BAC-based integrated genetic-physical map of the <it>T. cacao </it>cultivar Matina 1-6 which is designed to augment and enhance these sequencing efforts. Three BAC libraries, each comprised of 10× coverage, were constructed and fingerprinted. 230 genetic markers from a high-resolution genetic recombination map and 96 Arabidopsis-derived conserved ortholog set (COS) II markers were anchored using pooled overgo hybridization. A dense tile path consisting of 29,383 BACs was selected and end-sequenced. The physical map consists of 154 contigs and 4,268 singletons. Forty-nine contigs are genetically anchored and ordered to chromosomes for a total span of 307.2 Mbp. The unanchored contigs (105) span 67.4 Mbp and therefore the estimated genome size of <it>T. cacao </it>is 374.6 Mbp. A comparative analysis with <it>A. thaliana, V. vinifera</it>, and <it>P. trichocarpa </it>suggests that comparisons of the genome assemblies of these distantly related species could provide insights into genome structure, evolutionary history, conservation of functional sites, and improvements in physical map assembly. A comparison between the two <it>T. cacao </it>cultivars Matina 1-6 and Criollo indicates a high degree of collinearity in their genomes, yet rearrangements were also observed.</p> <p>Conclusions</p> <p>The results presented in this study are a stand-alone resource for functional exploitation and enhancement of <it>Theobroma cacao </it>but are also expected to complement and augment ongoing genome-sequencing efforts. This resource will serve as a template for refinement of the <it>T. cacao </it>genome through gap-filling, targeted re-sequencing, and resolution of repetitive DNA arrays.</p

Genomic tools development for Aquilegia: construction of a BAC-based physical map

<p>Abstract</p> <p>Background</p> <p>The genus <it>Aquilegia</it>, consisting of approximately 70 taxa, is a member of the basal eudicot lineage, Ranuculales, which is evolutionarily intermediate between monocots and core eudicots, and represents a relatively unstudied clade in the angiosperm phylogenetic tree that bridges the gap between these two major plant groups. <it>Aquilegia </it>species are closely related and their distribution covers highly diverse habitats. These provide rich resources to better understand the genetic basis of adaptation to different pollinators and habitats that in turn leads to rapid speciation. To gain insights into the genome structure and facilitate gene identification, comparative genomics and whole-genome shotgun sequencing assembly, BAC-based genomics resources are of crucial importance.</p> <p>Results</p> <p>BAC-based genomic resources, including two BAC libraries, a physical map with anchored markers and BAC end sequences, were established from <it>A. formosa</it>. The physical map was composed of a total of 50,155 BAC clones in 832 contigs and 3939 singletons, covering 21X genome equivalents. These contigs spanned a physical length of 689.8 Mb (~2.3X of the genome) suggesting the complex heterozygosity of the genome. A set of 197 markers was developed from ESTs induced by drought-stress, or involved in anthocyanin biosynthesis or floral development, and was integrated into the physical map. Among these were 87 genetically mapped markers that anchored 54 contigs, spanning 76.4 Mb (25.5%) across the genome. Analysis of a selection of 12,086 BAC end sequences (BESs) from the minimal tiling path (MTP) allowed a preview of the <it>Aquilegia </it>genome organization, including identification of transposable elements, simple sequence repeats and gene content. Common repetitive elements previously reported in both monocots and core eudicots were identified in <it>Aquilegia </it>suggesting the value of this genome in connecting the two major plant clades. Comparison with sequenced plant genomes indicated a higher similarity to grapevine (<it>Vitis vinifera</it>) than to rice and <it>Arabidopsis </it>in the transcriptomes.</p> <p>Conclusions</p> <p>The <it>A. formosa </it>BAC-based genomic resources provide valuable tools to study <it>Aquilegia </it>genome. Further integration of other existing genomics resources, such as ESTs, into the physical map should enable better understanding of the molecular mechanisms underlying adaptive radiation and elaboration of floral morphology.</p

Adventures in the Enormous: A 1.8 Million Clone BAC Library for the 21.7 Gb Genome of Loblolly Pine

Loblolly pine (LP; Pinus taeda L.) is the most economically important tree in the U.S. and a cornerstone species in southeastern forests. However, genomics research on LP and other conifers has lagged behind studies on flowering plants due, in part, to the large size of conifer genomes. As a means to accelerate conifer genome research, we constructed a BAC library for the LP genotype 7-56. The LP BAC library consists of 1,824,768 individually-archived clones making it the largest single BAC library constructed to date, has a mean insert size of 96 kb, and affords 7.6X coverage of the 21.7 Gb LP genome. To demonstrate the efficacy of the library in gene isolation, we screened macroarrays with overgos designed from a pine EST anchored on LP chromosome 10. A positive BAC was sequenced and found to contain the expected full-length target gene, several gene-like regions, and both known and novel repeats. Macroarray analysis using the retrotransposon IFG-7 (the most abundant repeat in the sequenced BAC) as a probe indicates that IFG-7 is found in roughly 210,557 copies and constitutes about 5.8% or 1.26 Gb of LP nuclear DNA; this DNA quantity is eight times the Arabidopsis genome. In addition to its use in genome characterization and gene isolation as demonstrated herein, the BAC library should hasten whole genome sequencing of LP via next-generation sequencing strategies/technologies and facilitate improvement of trees through molecular breeding and genetic engineering. The library and associated products are distributed by the Clemson University Genomics Institute (www.genome.clemson.edu)