Goldberg Lab Research

NSF Single Cell Transcriptome Profiling Project in Soybean & Arabidopsis

We used laser capture microdissection (LCM) to isolate specific compartments, regions, tissues, and cell types from soybean and Arabidopsis seeds at different stages of development – including the seed coat, endosperm, and embryo regions and organ systems. Microarray, Next-generation sequencing, and qRT-PCR experiments were carried out in order to determine (1) the spectrum of genes that are active in different parts of seed during development and (2) transcription factors and biological processes that partitioned within a seed playing important roles in seed differentiation and/or function. Functional studies were also carried out in order to determine what role compartment and tissue-specific transcription factors play in soybean seed development.

We have profiled the mRNA sets present in 71 soybean and Arabidopsis seed compartments from shortly after fertilization through the early maturation stage of development. All GeneChip data generated for this NSF-sponsored project have been deposited in GEO and on our interactive relational seed database, which is built under this project website - seedgenenetwork.net. In the project website, we created the built-in analysis tools to allow the user to not only browse the database by probe identification, gene ontology, and gene function, but also compare gene activity in different seed compartments during development. We also took efforts to annotate the Affymetrix soybean array and the Arabidopsis ATH1 array.

Furthermore, we used next generation sequencing technology, RNA-Seq, to profile mRNAs and non-coding small RNAs on a whole-genome basis during soybean seed development, including (a) whole seeds from all stages of seed development from fertilization to dormancy, (b) isolated cotyledons from maturation-stage seeds and germinating seedlings, (c) isolated axis and seed coat at early-maturation stage, (d) 40 different LCM-captured seed compartments from globular to early-maturation stage, (e) post-germination seedlings, and (f) mature plant leaves, roots, stems, and flowers. To date, we have generated ~ 7 billion reads, or 400 gigabytes of sequences. All generated RNA-Seq and small RNA-Seq data for this NSF-sponsored project have been deposited in GEO.

Click here to download the datasets submitted to GEO.

Click here to download the publication of genomic profiling of Arabidopsis seed regions and subregions.

Using Methylome Profiling To Reveal The Role Of DNA Methylation In Seed Development

We used laser capture microdissection to isolate the seed compartment, tissue, and regions in soybean seeds and then apply NextGen Sequencing (Bisulfite (BS)-DNA-Seq) to characterize methylomes of soybean seeds throughout development on a genome-wide basis - both early in development when differentiation occurs (globular through cotyledon stages) and late in development when maturation and dormancy begin (early-maturation stage to dry seeds). These experiments will provide the first genome-wide characterization of the methylomes of a seed throughout development. By means of studying these unique resources of temporal- and spatial- specific methylome datasets, we will have a better understanding the epigenetic aspects of seed development.

We have developed perfect technology to perform BS-DNA-Seq for whole-genome methylome analysis on small amounts of DNA (~10 ng) from specific seed tissues captured using LCM. We have developed bioinformatics tools, computer algorithms, and pipelines to analyze BS DNA-Seq. To date, we have profiled the methylome sets present in 36 soybean seed, compartments, tissues and regions from differentiation phase seeds (globular stage and cotyledon stage), to maturation phase seeds (early maturation, middle maturation, late maturation), and dormancy seeds (dry seeds) to post-germination phase (seedlings and cotyledon from seedlings) and generated ~ 10 billion reads, or 960 gigabytes of sequences. All generated BS-DNA-Seq data have been deposited in GEO.

We observed dynamic methylome changes during seed development, as well as different DNA methylation levels in seed compartments, tissues and regions. The significance of this project is that important new information will be obtained on the epigenetic mechanisms underlying soybean seed development.

Click here to download the datasets submitted to GEO.

Click here to browse the project website - seedgenenetwork.net

Genomic Profiling of Arabidopsis Plant Life Cycle Using Affymetrix GeneChips

Utilizing a genomics approach, we profiled mRNAs active during Arabidopsis seed development using whole-genome Affymetrix GeneChips ATH1-121501. Specifically, we profiled mRNAs active during seed development at 24 hours post-fertilization, 7-8 days after pollination (DAP), 13-14 DAP, and 18-19 DAP. These gene sets were compared with those active in pre-fertilization ovules, seedlings 3 days after imbibition, leaves, roots, stems, and floral buds of the mature plant. Collectively, we have carried out whole-genome analysis of genes active during the entire Arabidopsis life cycle.

Click here to browse our Arabidopsis GeneChip Database.

Click here to download the publication of this project.

Scarlet Runner Bean Embryo EST Project

Our lab has utilized the SRB as a model system to identify genes and regulatory networks programming early embryo developmental events using a genomic approach. Our long-term goal is to understand the region-specific differentiation processes that occur during early embryo development and how genes are activated specifically in the suspensor and embryo proper. The SRB is unique in this regard because of its giant seed and large embryo, permitting hand dissection of the embryo-proper and suspensor regions at early developmental stages. Before the high-throughput sequencing era, total RNAs isolated from the hand-dissected suspensor and embryo proper were used to construct cDNA libraries. The 5' ends of individual cDNA clones from each library were sequenced using the Sanger sequencing method whereas random cDNA fragments were sequenced using the 454 sequencing technology. ~390,000 ESTs were identified and representing messages active 6-7 days after fertilization in different embryo regions of the early embryo. We've also created a database where you can query your sequences against our Scarlet Runner Bean ESTs using local BLAST tools. All of our EST sequences have been submitted to GenBank. Their accession numbers are: CA896559 to CA916678 and GD289845 to GD660862. You can access all of these sequences at NCBI.

Furthermore, we used laser capture microdissection to isolate the SRB,common bean suspensor, and embryo proper 6-7 days after fertilization and then apply NextGen Sequencing, RNA-Seq to characterize transcriptome in different embryo regions of the early embryo. The common bean is very close to the SRB and its genome has been sequences and published recently. The SRB and common bean have similar early embryo morphology. Eventually we could understand how the embryo proper and suspensor regions are specified from the apical and basal cell of a two-cell embryo by comparing the transcriptome profiles from different species.

Click Here to Visit The Scarlet Runner Bean Embryo EST Project Website

Click Here to BLAST Your Sequences Against our Scarlet Runner Bean EST Sequences.

Molecular and Genetic Dissection of Plant Development

My laboratory has been investigating the molecular processes controlling the development of specialized cells in higher plants.  The major questions that I have addressed in my research are ....Read more

Seed Institute

The Seed Institute was established in order to carry out basic research in seed biology with the objective of uncovering the genes and processes required to "make a seed."  The long-range goal of the Seed Institute, in partnership with Ceres, is to use new insights obtained on the mechanisms that control seed formation to improve seeds in major crop plants....Read More

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