Globular somatic embryos can be induced from immature cotyledons of soybean (Glycine max L. Merr. cv Jack) placed on high levels of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos develop from the adaxial side of the cotyledon, whereas the abaxial side evolves into a callus. Using a 9,280-cDNA clone array, we have compared steady-state RNA from the adaxial side from which embryos develop and from the abaxial callus at five time points over the course of the 4 weeks necessary for the development of globular embryos. In a second set of experiments, we have profiled the expression of each clone in the adaxial side during the same period. A total of 495 genes differentially expressed in at least one of these experiments were grouped according to the similarity of their expression profiles using a nonhierarchical clustering algorithm. Our results indicate that the appearance of somatic embryos is preceded by dedifferentiation of the cotyledon during the first 2 weeks on auxin. Changes in mRNA abundance of genes characteristic of oxidative stress and genes indicative of cell division in the adaxial side of the cotyledons suggest that the arrangement of the new cells into organized structures might depend on a genetically controlled balance between cell proliferation and cell death. Our data also suggest that the formation of somatic globular embryos is accompanied by the transcription of storage proteins and the synthesis of gibberellic acid.Due to their ability to regenerate into full plants, somatic embryos are the tissue of choice for transformation by particle bombardment in several crop species including soybean (Glycine max L. Merr. cv Jack; Finer and McMullen, 1991). In soybean, somatic embryos are obtained by induction, or culturing, of immature cotyledons on high concentration of auxin. Globular somatic embryos originate from epidermal and subepidermal cells of the adaxial side of the cotyledon (Finer, 1988), whereas the abaxial side evolves into a callus. The adaxial side is the "flat" side of the cotyledons, closest to the axis of the embryo, whereas the abaxial side is the side of the cotyledon in contact with the endosperm and the seed coat. However, the response to tissue culture is highly genotype dependent (Meurer et al., 2001), and the ability to transform a wider range of cultivars could accelerate the production of transgenic plants.Somatic and zygotic embryos follow the same general pattern of development (Zimmerman, 1993;Goldberg et al., 1994). However, large quantities of somatic embryos can be produced in vitro, making them more amenable to experimentation than their zygotic counterparts, which are protected by fruit structures and less accessible. Therefore, somatic embryos constitute a model system to study basic aspects of embryogenesis, as well as a tool for efficient transformation.Little is known of the genes expressed in early globular stage embryos (Zimmerman, 1993). Choi evaluated that only 10% of the proteins visible on a two-dimensional gel are embryo specific (Choi ...

Whole-genome sequencing is fundamental to understanding the genetic composition of an organism. Given the size and complexity of the soybean genome, an alternative approach is targeted random-gene sequencing, which provides an immediate and productive method of gene discovery. In this study, more than 120000 soybean expressed sequence tags (ESTs) generated from more than 50 cDNA libraries were evaluated. These ESTs coalesced into 16928 contigs and 17336 singletons. On average, each contig was composed of 6 ESTs and spanned 788 bases. The average sequence length submitted to dbEST was 414 bases. Using only those libraries generating more than 800 ESTs each and only those contigs with 10 or more ESTs each, correlated patterns of gene expression among libraries and genes were discerned. Two-dimensional qualitative representations of contig and library similarities were generated based on expression profiles. Genes with similar expression patterns and, potentially, similar functions were identified. These studies provide a rich source of publicly available gene sequences as well as valuable insight into the structure, function, and evolution of a model crop legume genome.

BackgroundTransposome-based technologies have enabled the streamlined production of sequencer-ready DNA libraries; however, current methods are highly sensitive to the amount and quality of input nucleic acid.ResultsWe describe a new library preparation technology (Nextera DNA Flex) that utilizes a known concentration of transposomes conjugated directly to beads to bind a fixed amount of DNA, and enables direct input of blood and saliva using an integrated extraction protocol. We further report results from libraries generated outside the standard parameters of the workflow, highlighting novel applications for Nextera DNA Flex, including human genome builds and variant calling from below 1 ng DNA input, customization of insert size, and preparation of libraries from short fragments and severely degraded FFPE samples. Using this bead-linked library preparation method, library yield saturation was observed at an input amount of 100 ng. Preparation of libraries from a range of species with varying GC levels demonstrated uniform coverage of small genomes. For large and complex genomes, coverage across the genome, including difficult regions, was improved compared with other library preparation methods. Libraries were successfully generated from amplicons of varying sizes (from 50 bp to 11 kb), however, a decrease in efficiency was observed for amplicons smaller than 250 bp. This library preparation method was also compatible with poor-quality DNA samples, with sequenceable libraries prepared from formalin-fixed paraffin-embedded samples with varying levels of degradation.ConclusionsIn contrast to solution-based library preparation, this bead-based technology produces a normalized, sequencing-ready library for a wide range of DNA input types and amounts, largely obviating the need for DNA quantitation. The robustness of this bead-based library preparation kit and flexibility of input DNA facilitates application across a wide range of fields.

Background: Microarrays are an important tool with which to examine coordinated gene expression. Soybean (Glycine max) is one of the most economically valuable crop species in the world food supply. In order to accelerate both gene discovery as well as hypothesis-driven research in soybean, global expression resources needed to be developed. The applications of microarray for determining patterns of expression in different tissues or during conditional treatments by dual labeling of the mRNAs are unlimited. In addition, discovery of the molecular basis of traits through

Expressed sequence tags (ESTs) exhibiting homology to a BURP domain containing gene family were identified from the Glycine max (L.) Merr. EST database. These ESTs were assembled into 16 contigs of variable sizes and lengths. Consistent with the structure of known BURP domain containing proteins, the translation products exhibit a modular structure consisting of a C-terminal BURP domain, an N-terminal signal sequence, and a variable internal region. The soybean family members exhibit 35-98% similarity in a -100-amino-acid C-terminal region, and a phylogenetic tree constructed using this region shows that some soybean family members group together in closely related pairs, triplets, and quartets, whereas others remain as singletons. The structure of these groups suggests that multiple gene duplication events occurred during the evolutionary history of this family. The depth and diversity of G. max EST libraries allowed tissue-specific expression patterns of the putative soybean BURPs to be examined. Consistent with known BURP proteins, the newly identified soybean BURPs have diverse expression patterns. Furthermore, putative paralogs can have both spatially and quantitatively distinct expression patterns. We discuss the functional and evolutionary implications of these findings, as well as the utility of EST-based analyses for identifying and characterizing gene families.

Epigenomics is increasingly becoming an important field of research, and the ability to detect and quantify DNA methylation accurately is now critical for numerous fields of study, including disease biology and gene expression. The differential reactivities of methylated and nonmethylated cytosines in DNA with sodium bisulfite forms the basis for their identification in the genome by sequencing. Current whole-genome bisulfite sequencing methods require substantial amounts of starting DNA to compensate for the loss due to bisulfite-mediated DNA degradation. To address issues with sample preparation, we have developed the EpiGnome™ Methyl-Seq Kit, which utilizes a novel pre-library bisulfite conversion scheme to prepare whole-genome bisulfite sequencing libraries with no sample loss and with only 50 ng of input genomic DNA.DNA methylation is not evenly distributed in the mammalian genome.In human somatic cells approximately 60%-80% of all CpGs (~1% of total DNA bases) are methylated. Numerous methods are available to investigate methylation patterns, including those that are enrichment based (e.g., as methylated DNA immunoprecipitation sequencing (MeDIP-seq)), restriction enzyme based (e.g., methylation-sensitive restriction enzyme sequencing (MRE-seq)) and bisulfite based (e.g., reduced-representation bisulfite sequencing (RRBS) and wholegenome bisulfite sequencing (WGBS)). Of these, the only method that captures the complete methylome of a given sample is WGBS, as only through this approach-in which unmethylated cytosine residues are converted to uracil-can genome-wide analysis of 5-methylcytosines be achieved. However, a major challenge in WGBS is the degradation of DNA that occurs during bisulfite conversion under the conditions required for complete conversion. Typically, ~90% of input DNA is degraded, something that is especially problematic when only limited starting amounts are available. Additionally, regions that are rich in unmethylated cytosines are more sensitive to strand breaks. As a consequence, a majority of DNA fragments contained in di-tagged NGS DNA libraries treated with bisulfite "post-library construction" can be rendered inactive due to strand breaks in the DNA sequence flanked by the adapter sequences. These mono-tagged templates are then excluded during library enrichment, resulting in incomplete coverage and bias when performing whole-genome bisulfite sequencing.Here, we describe a novel library construction method, called Method overviewWith the EpiGnome™ Methyl-Seq Kit, bisulfite-treated single-stranded DNA (ssDNA) is randomly primed using a polymerase able to read uracil nucleotides to synthesize DNA strands containing a specific sequence tag (Fig. 1). The 3′ ends of the newly synthesized DNA strands are then selectively tagged with a second specific sequence tag using a patented procedure, resulting in di-tagged DNA molecules with known sequence tags at their 5′ and 3′ ends. The di-tagged DNA is enriched in PCR, resulting in double-stranded DNA (dsDNA) with the appropriate sequences...

Compacting plasmid DNA (pDNA) into a small size is a fundamental necessity for the efficient in vivo transfer of nucleic acids to somatic cells. An approach for accomplishing this is to condense pDNA using cationic detergents with sulfhydryl groups, near their critical micelle concentration. In this study, a model surfactant was used to study how the rate of disulfide bond formation relates to environmental factors. It was shown that the thiol detergent had the ability to form a disulfide bond when oxidized and the presence of polyanions was significantly increased. The addition of a reducing agent disrupted the disulfide bonds initially, but this was followed by disulfide bond reformation in a short time period.

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