Overexpression of an ethylene-forming ACC oxidase (ACO) gene precedes the Minute Hilum seed coat phenotype in Glycine max.

Background: To elucidate features of seed development, we investigated the transcriptome of a soybean isoline from the germplasm collection that contained an introgressed allele known as minute hilum (mi) which confers a smaller hilum region where the seed attaches to the pod and also results in seed coat cracking surrounding the hilum region.

Results: RNAs were extracted from immature seed from an extended hilum region (i.e.

An embryo lethal transgenic line manifests global expression changes and elevated protein/oil ratios in heterozygous soybean plants.

Understanding the molecular processes of seed development is important especially in agronomic crops that produce large amounts of nutrient reserves. Because soybean is a vital source of vegetable protein worldwide, producers are concerned about increasing the total amount of protein in the seed without substantially lowering the amount of oil, another economically important product. Here we describe a transgenic soybean line with increased protein and protein/oil ratio, containing an average of 42.

Nonallelic homologous recombination events responsible for copy number variation within an RNA silencing locus.

The structure of chalcone synthase () gene repeats in different alleles of the (inhibitor) locus in soybean spawns endogenous RNA interference (RNAi) that leads to phenotypic change in seed coat color of this major agronomic crop. Here, we examined gene copy number by digital PCR and single nucleotide polymorphisms (SNPs) through whole genome resequencing of 15 cultivars that varied in alleles of the locus (, , , and ) that control the pattern distribution of pigments in the seed coats. Lines homozygous for the allele had the highest copy number followed by the and cultivars which were more related to each other than to the lines with alleles.

Host-derived gene silencing of parasite fitness genes improves resistance to soybean cyst nematodes in stable transgenic soybean.

Soybean expressing small interfering RNA of SCN improved plant resistance to SCN consistently, and small RNA-seq analysis revealed a threshold of siRNA expression required for resistance ability. Soybean cyst nematode (SCN), Heterodera glycines, is one of the most destructive pests limiting soybean production worldwide, with estimated losses of $1 billion dollars annually in the USA alone. RNA interference (RNAi) has become a powerful tool for silencing gene expression.

Ribosome profiling reveals changes in translational status of soybean transcripts during immature cotyledon development.

To understand translational capacity on a genome-wide scale across three developmental stages of immature soybean seed cotyledons, ribosome profiling was performed in combination with RNA sequencing and cluster analysis. Transcripts representing 216 unique genes demonstrated a higher level of translational activity in at least one stage by exhibiting higher translational efficiencies (TEs) in which there were relatively more ribosome footprint sequence reads mapping to the transcript than were present in the control total RNA sample. The majority of these transcripts were more translationally active at the early stage of seed development and included 12 unique serine or cysteine proteases and 16 2S albumin and low molecular weight cysteine-rich proteins that may serve as substrates for turnover and mobilization early in seed development.

Mutations in Illuminate Epistatic Interactions of the and Loci Leading to Saddle Seed Color Patterns in .

The soybean () seed coat has distinctive, genetically programmed patterns of pigmentation, and the recessive mutation can epistatically overcome the dominant and alleles, which inhibit seed color by producing small interfering RNAs (siRNAs) targeting () mRNAs. Small RNA sequencing of dissected regions of immature seed coats demonstrated that siRNA levels cause the patterns produced by the and alleles of the locus, which restrict pigment to the hilum or saddle region of the seed coat, respectively. To identify the locus, we compared RNA-seq data from dissected regions of two Clark isolines having similar saddle phenotypes mediated by siRNAs but different genotypes (homozygous versus homozygous ).

Simple gene silencing using the trans-acting siRNA pathway.

In plants, particular micro-RNAs (miRNAs) induce the production of a class of small interfering RNAs (siRNA) called trans-acting siRNA (ta-siRNA) that lead to gene silencing. A single miRNA target is sufficient for the production of ta-siRNAs, which target can be incorporated into a vector to induce the production of siRNAs, and ultimately gene silencing. The term miRNA-induced gene silencing (MIGS) has been used to describe such vector systems in Arabidopsis.

Direct detection of transcription factors in cotyledons during seedling development using sensitive silicon-substrate photonic crystal protein arrays.

Transcription factors control important gene networks, altering the expression of a wide variety of genes, including those of agronomic importance, despite often being expressed at low levels. Detecting transcription factor proteins is difficult, because current high-throughput methods may not be sensitive enough. One-dimensional, silicon-substrate photonic crystal (PC) arrays provide an alternative substrate for printing multiplexed protein microarrays that have greater sensitivity through an increased signal-to-noise ratio of the fluorescent signal compared with performing the same assay upon a traditional aminosilanized glass surface.

Methylation affects transposition and splicing of a large CACTA transposon from a MYB transcription factor regulating anthocyanin synthase genes in soybean seed coats.

We determined the molecular basis of three soybean lines that vary in seed coat color at the R locus which is thought to encode a MYB transcription factor. RM55-r(m) is homozygous for a mutable allele (r(m)) that specifies black and brown striped seeds; RM30-R* is a stable black revertant isoline derived from the mutable line; and RM38-r has brown seed coats due to a recessive r allele shown to translate a truncated MYB protein. Using long range PCR, 454 sequencing of amplicons, and whole genome re-sequencing, we determined that the variegated RM55-r(m) line had a 13 kb CACTA subfamily transposon insertion (designated TgmR*) at a position 110 bp from the beginning of Intron2 of the R locus, Glyma09g36983.

Transcription factors and glyoxylate cycle genes prominent in the transition of soybean cotyledons to the first functional leaves of the seedling.

During early seedling growth, the cotyledons transition from a storage tissue to become effectively the first leaf-like structures of the plant. In this programmed developmental process, they likely undergo a massive change in gene expression to redirect their metabolism and physiological processes. To define the developmental shifts in gene expression and begin to understand the gene regulatory networks that set this transition in motion, we carried out high-throughput RNA sequencing of cotyledons from seven developmental stages of soybean seedlings.

RNA-Seq profiling of a defective seed coat mutation in Glycine max reveals differential expression of proline-rich and other cell wall protein transcripts.

The plant cell wall performs a number of essential functions including providing shape to many different cell types and serving as a defense against potential pathogens. The net pattern mutation creates breaks in the seed coat of soybean (Glycine max) because of ruptured cell walls. Using RNA-Seq, we examined the seed coat transcriptome from three stages of immature seed development in two pairs of isolines with normal or defective seed coat phenotypes due to the net pattern.

The transition from primary siRNAs to amplified secondary siRNAs that regulate chalcone synthase during development of Glycine max seed coats.

The I locus is a 27-kb inverted repeat cluster of chalcone synthase genes CHS1-3-4 that mediates siRNA down-regulation of CHS7 and CHS8 target mRNAs during seed development leading to yellow seed coats lacking anthocyanin pigments. Here, we report small RNA sequencing of ten stages of seed development from a few days post fertilization through maturity, revealing the amplification from primary to secondary short interfering RNAs (siRNAs) occurring during development. The young seed populations had a higher proportion of siRNAs representing the CHS1-3-4 gene family members, consistent with this region as the origin of the primary siRNAs.

Genome-wide identification of binding sites for NAC and YABBY transcription factors and co-regulated genes during soybean seedling development by ChIP-Seq and RNA-Seq.

Background: Two plant-specific transcription factors, NAC and YABBY, are involved in important plant developmental processes. However their molecular mechanisms, especially DNA binding sites and co-regulated genes, are largely unknown during soybean seedling development.

Results: In order to identify genome-wide binding sites of specific members of the NAC and YABBY transcription factors and co-regulated genes, we performed Chromatin Immunoprecipitation Sequencing (ChIP-Seq) and RNA Sequencing (RNA-Seq) using cotyledons from soybean seedling developmental stages.

Using RNA-Seq to profile soybean seed development from fertilization to maturity.

To understand gene expression networks leading to functional properties and compositional traits of the soybean seed, we have undertaken a detailed examination of soybean seed development from a few days post-fertilization to the mature seed using Illumina high-throughput transcriptome sequencing (RNA-Seq). RNA was sequenced from seven different stages of seed development, yielding between 12 million and 78 million sequenced transcripts. These have been aligned to the 79,000 gene models predicted from the soybean genome recently sequenced by the Department of Energy Joint Genome Institute.

Divergent patterns of endogenous small RNA populations from seed and vegetative tissues of Glycine max.

Background: Small non-coding RNAs (smRNAs) are known to have major roles in gene regulation in eukaryotes. In plants, knowledge of the biogenesis and mechanisms of action of smRNA classes including microRNAs (miRNAs), short interfering RNAs (siRNAs), and trans-acting siRNAs (tasiRNAs) has been gained mostly through studies with Arabidopsis. In recent years, high throughput sequencing of smRNA populations has enabled extension of knowledge from model systems to plants with larger, more complex genomes.

Identification of soybean seed developmental stage-specific and tissue-specific miRNA targets by degradome sequencing.

Background: MicroRNAs (miRNAs) regulate the expression of target genes by mediating gene silencing in both plants and animals. The miRNA targets have been extensively investigated in Arabidopsis and rice using computational prediction, experimental validation by overexpression in transgenic plants, and by degradome or PARE (parallel analysis of RNA ends) sequencing. However, miRNA targets mostly remain unknown in soybean (Glycine max).

The interaction of the novel 30C02 cyst nematode effector protein with a plant β-1,3-endoglucanase may suppress host defence to promote parasitism.

Phytoparasitic nematodes secrete an array of effector proteins to modify selected recipient plant cells into elaborate and essential feeding sites. The biological function of the novel 30C02 effector protein of the soybean cyst nematode, Heterodera glycines, was studied using Arabidopsis thaliana as host and the beet cyst nematode, Heterodera schachtii, which contains a homologue of the 30C02 gene. Expression of Hg30C02 in Arabidopsis did not affect plant growth and development but increased plant susceptibility to infection by H.

Using photonic crystal enhanced fluorescence on quartz substrates to improve the sensitivity of DNA microarrays.

Gene expression analysis of low abundance genes remains difficult when DNA microarrays are performed on standard glass substrates. However, we have shown that by using photonic crystals (PC) made on quartz substrates, the fluorescence intensity of Cyanine-5 (Cy5) labeled microarray spots is greatly enhanced. In a 1-color microarray experiment studying gene expression of soybean cotyledon tissue, an average signal enhancement factor of 17.

Transcript profiling reveals expression differences in wild-type and glabrous soybean lines.

Background: Trichome hairs affect diverse agronomic characters such as seed weight and yield, prevent insect damage and reduce loss of water but their molecular control has not been extensively studied in soybean. Several detailed models for trichome development have been proposed for Arabidopsis thaliana, but their applicability to important crops such as cotton and soybean is not fully known.

Results: Two high throughput transcript sequencing methods, Digital Gene Expression (DGE) Tag Profiling and RNA-Seq, were used to compare the transcriptional profiles in wild-type (cv.

Improved sensitivity of DNA microarrays using photonic crystal enhanced fluorescence.

DNA microarrays are used to profile changes in gene expression between samples in a high-throughput manner, but measurements of genes with low expression levels can be problematic with standard microarray substrates. In this work, we expand the detection capabilities of a standard microarray experiment using a photonic crystal (PC) surface that enhances fluorescence observed from microarray spots. This PC is inexpensively and uniformly fabricated using a nanoreplica molding technique, with very little variation in its optical properties within- and between-devices.

Flux of transcript patterns during soybean seed development.

Background: To understand gene expression networks leading to functional properties of the soybean seed, we have undertaken a detailed examination of soybean seed development during the stages of major accumulation of oils, proteins, and starches, as well as the desiccating and mature stages, using microarrays consisting of up to 27,000 soybean cDNAs. A subset of these genes on a highly-repetitive 70-mer oligonucleotide microarray was also used to support the results.

Results: It was discovered that genes related to cell growth and maintenance processes, as well as energy processes like photosynthesis, decreased in expression levels as the cotyledons approached the mature, dry stage.

Optimizing the spatial resolution of photonic crystal label-free imaging.

A theory is derived to describe the relationship between photonic crystal (PC) label-free imaging resolution and PC resonance spectral linewidth and location. PCs are fabricated and patterned with a resolution standard photomask in order to verify this relationship experimentally. Two distinct linear resolutions of <1 microm and 3.

Endogenous, tissue-specific short interfering RNAs silence the chalcone synthase gene family in glycine max seed coats.

Two dominant alleles of the I locus in Glycine max silence nine chalcone synthase (CHS) genes to inhibit function of the flavonoid pathway in the seed coat. We describe here the intricacies of this naturally occurring silencing mechanism based on results from small RNA gel blots and high-throughput sequencing of small RNA populations. The two dominant alleles of the I locus encompass a 27-kb region containing two perfectly repeated and inverted clusters of three chalcone synthase genes (CHS1, CHS3, and CHS4).

A detection instrument for enhanced-fluorescence and label-free imaging on photonic crystal surfaces.

We report on the design and demonstration of an optical imaging system capable of exciting surface-bound fluorophores within the resonant evanescent electric field of a photonic crystal surface and gathering fluorescence emission that is directed toward the imaging objective by the photonic crystal. The system also has the ability to quantify shifts in the local resonance angle induced by the adsorption of biomolecules on the photonic crystal surface for label-free biomolecular imaging. With these two capabilities combined within a single detection system, we demonstrate label-free images self-registered to enhanced fluorescence images with 328x more sensitive fluorescence detection relative to a glass surface.

A putative autonomous 20.5 kb-CACTA transposon insertion in an F3'H allele identifies a new CACTA transposon subfamily in Glycine max.

Background: The molecular organization of very few genetically defined CACTA transposon systems have been characterized thoroughly as those of Spm/En in maize, Tam1 of Antirrhinum majus Candystripe1 (Cs1) from Sorghum bicolor and CAC1 from Arabidopsis thaliana, for example. To date, only defective deletion derivatives of CACTA elements have been described for soybean, an economically important plant species whose genome sequence will be completed in 2008.

Results: We identified a 20.