Progress 08/01/06 to 07/31/10
Outputs
OUTPUTS: The goal of this proposal was to use novel methods for the isolation and characterization of small RNA molecules (21 to 24 nucleotides) from the model legume Medicago truncatula. We also generated a dataset of small RNAs from soybean (Glycine max) and common bean (Phaseolus vulgaris), peanut, and chickpea. We generated data from 21 libraries. The libraries represented diverse wild-type M. truncatula tissues, tissues of plants interacting with symbiotic and pathogenic microbes, and similar tissues from soybean, common bean, chickpea, and peanut. To facilitate public use and access to the legume small RNA data, we created a publicly available, user-friendly website (http://mpss.udel.edu/legume). PARTICIPANTS: D Janine Sherrier, PD; Pamela J Green, co-PD; Blake C Meyers, co-PD; Sherry L Kitto, group leader, lead tissue culture effort; Jixian Zhai, PhD Student, computational studies; Sean Fidance, PhD Student, data validations, Northern Blots; Madhavi Avadani, postdoctoral researcher, molecular biology, plant analysis; Dong Hoon Jeong, postdoctoral researcher, library generation; Sunhee Park, research associate, library generation; Monica Accerbi, research associate, library generation; Kathryn Guhl, research associate, generation of biological material; Heather Danysh, research associate, generation of biological material; Javier Mendiola, research associate, molecular biology, plant analysis; Angel Huang, Lab manager, library generation, data validation; Alvaro J Gonzalez, MS student, computational studies; TARGET AUDIENCES: Target audience, international plant science community; Effort, National Scientific meetings, International Scientific meetings, public website PROJECT MODIFICATIONS: We have expanded to number of species and tissues presented in this work, due to cost efficiency.
Impacts
Small RNAs have specific biological activities in plants, usually as regulators of gene activity. However, prior to the initiation of this work most of these small RNA molecules have never been identified or measured in legumes or legumes interacting with microbes. We have developed a new technology for the identification and measurement of these molecules and this project will be the first large-scale application of the method in legumes. Small RNAs are a relatively-newly described class of molecules that have specific biological activities in many eukaryotes and usually function to regulate the abundance or function of RNA. These molecules can regulate how the plant genome is expressed gene by several modes of action. These effects of small RNAs can produce a specific phenotype. In plants, flowering is controlled by several known small RNAs. Because genomics technologies have yet to be applied on a large scale to the analysis of small RNAs from legumes, there is much that is not yet understood about the complexity, abundance, targeting and function of these important regulatory molecules in this important group of crop plants. This research will identify novel small RNAs that may be involved in plant development or plant-microbe interactions in legumes. The data generated in this study will be made widely available to scientific community through a user friendly database. To date we have generated 21 libraries and sequenced all of them. Each sequencing run yielded 2-3 million sequences representing individual small RNA molecules. The data has been released to the public on our web site. We have generated the first cross-species small RNA database. We have expanded to number of species and tissues presented in this work, due to cost efficiency.
Publications
- Trupti Joshi, Zhe Yan, Marc Libault, Dong-Hoon Jeong, Sunhee Park, Pamela J Green, D Janine Sherrier, Andrew Farmer, Greg May, Blake C Meyers, Dong Xu and Gary Stacey (2010) Prediction of novel miRNAs and associated target genes in Glycine max. BMC Bioinformatics 2010, 11(Suppl 1):S
- NEWStacey A. Simon, Blake C. Meyers, and D. Janine Sherrier (2009) MicroRNAs in the Rhizobia Legume Symbiosis. Plant Physiol. 151: 1002-1008
- Accerbi, M., Schmidt, S.A., De Paoli, E., Park, S., Jeong, D-H., and Green, P.J. Methods for Isolation of total RNA to Recover miRNAs and Other Small RNAs from Diverse Species. In Meyers, B.C. and Green, P.J. (Eds.) Plant microRNA protocols. Humana Press, New York, NY (2010). EPub 2009 Oct 3.
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Progress 08/01/08 to 07/31/09
Outputs
OUTPUTS: Small RNAs in Legume Symbioses - Invited presentation by DJ Sherrier at 9th International Plant Molecular Biology (IPMB) Congress in St Louis, MO PARTICIPANTS: Javier Mendiolia - RNA extraction, characterization of rdr2 mutant, generation of small RNA libraries Angel Huang - sample preparation Sean Fidance - sample preparation, phenotype analysis TARGET AUDIENCES: IMPB 2010 Scientific audience, exchange of information PROJECT MODIFICATIONS: We have created a separate web based database to query the soybean small RNA data generated in this project. With the publication of the soybean genome, we were able to generate this system to allow better interpretation of our data. Previously our soy bean was queried against the Medicago truncatula database.
Impacts
We have extended the project to include an assembled version of the soybean genome on which the soybean small RNA data can be queried. The Medicago and Legume Small RNA database updated to query Medicago truncatula genome version 3.0
Publications
- Trupti Joshi, Zhe Yan, Marc Libault, Dong-Hoon Jeong, Sunhee Park, Pamela J Green, D Janine Sherrier, Andrew Farmer, Greg May, Blake C Meyers, Dong Xu and Gary Stacey (2010) Prediction of novel miRNAs and associated target genes in Glycine max. Bioinformatics 2010, 11(Suppl 1):S
- SA Simon, BC Meyers, Sherrier DJ (2009)MicroRNAs in the Rhizobia Legume Symbiosis. Plant Physiol. 151: 1002-1008
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Progress 08/01/07 to 07/31/08
Outputs
OUTPUTS: The goal of this proposal is to use novel methods for the isolation and characterization of small RNA molecules (21 to 24 nucleotides) from the model legume Medicago truncatula. We will also generate a less extensive dataset of small RNAs from soybean (Glycine max) and common bean (Phaseolus vulgaris), peanut, and chickpea. These molecules will be sequenced using a method called 454. We will generate data from 21 libraries. The libraries will represent diverse wild-type M. truncatula tissues, tissues of plants interacting with symbiotic and pathogenic microbes, and similar tissues from soybean, common bean, chickpea, and peanut. To facilitate public use and access to the legume small RNA data, we will create a publicly available, user-friendly website (http://mpss.udel.edu/legume). In the current reporting period, we have completed the synthesis and sequencing of 21 small RNA libraries from a variety of legume tissues. We have added the 1.0 version of Medicago truncatula genome sequence to our database, and the small RNAs are matched to these sequences. The database indicates the conserved small RNAs identified in the legume tissues. We have identified putative miRNAs, siRNAs, and ta-siRNAs, based on phasing within the genome. PARTICIPANTS: Blake Meyers,Jixian Zhai, Alvaro Gonzalez,Mayumi Nakano,Deepti Kulkarni, Sireesha Guntapalli, Manoj Pillay have worked on database generation. Janine Sherrier, Madhavi Avadhani, Sherry Kitto, Michael Grusak, Maria Harrison, Steve Knapp, and Gary Stacey contributed to tissue generation. Pam Green, Dong-Hoon Jeong, Sunhee Park have worked to libarary generation. TARGET AUDIENCES: Scientific community PROJECT MODIFICATIONS: We have generated more libaries and sequences than anticipated, due to changes in sequencing technology. The generation of stable Medicago truncatula lines been slower than anticipated, although we now have multiple lines.a We plan to extend this project by one year.
Impacts
Our legume small RNA database includes 7.8 Million, 15 Million Soy, 10 Million Common Bean, and 21 Million Medicago truncatula sequences. We have identified new, legume specific small RNAs.
Publications
- No publications reported this period
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Progress 08/01/06 to 07/31/07
Outputs
OUTPUTS: The goal of this proposal is to use novel methods for the isolation and characterization of small RNA molecules (21 to 24 nucleotides) from the model legume Medicago truncatula. We will also generate a less extensive dataset of small RNAs from soybean (Glycine max) and common bean (Phaseolus vulgaris), peanut, and chickpea. These molecules will be sequenced using a method called 454. We will generate data from 21 libraries. The libraries will represent diverse wild-type M. truncatula tissues, tissues of plants interacting with symbiotic and pathogenic microbes, and similar tissues from soybean, common bean, chickpea, and peanut. To facilitate public use and access to the legume small RNA data, we will create a publicly available, user-friendly website (http://mpss.udel.edu/legume). We will analyze a subset of these small RNAs using RACE and RNA gel blots to verify the small RNA abundance and identify source transcripts. To date we have generated 17 libraries and sequenced 16 of them. Each sequencing run yielded 2-3 million sequences representing individual small RNA molecules. Currently we are performing quality control experiments on this massive data set, and are generating the 3 remaining libraries. We expect to release the first sets of data to the public in early fall of 2008. PARTICIPANTS: Dr. Janine Sherrier is responsible for coordinating the growth and collection of biological material and functional analysis of small RNA products. Dr. Pamela Green oversees the postdoctoral associtate in the collection of small RNSs and Functional analysis of small RNA products. Dr. Blake Meyers oversees generation of sequence data, gnereatio of the database, and data analysis. Madhavi S Avadhani Post Doctoral Researcher received 3 months pay. This person assisted with the production of small RNA libraries. Heather Elizabeth Danysh Post Doctoral Research received one months pay. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: We expect to request a budget revision and an extension within the year.
Impacts
We have generated the first cross-species small RNA database. We have expanded to number of species and tissues presented in this work, due to cost efficiency. We have identified legume specific small RNAs.
Publications
- No publications reported this period
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