Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) The objectives of this cooperative research project are to increase our understanding of genome organization of cereals and the QTLs associated with crop enhancement including response to the environment and nutritional improvement. Approach (from AD-416) 1) Development of genetic and physical markers for sorghum. Annotation of sorghum sequences will be used to identify SSR sequences to be used as genetic markers. In addition we will use the regions of synteny established between the rice genome sequence and sorghum genetic and physical map markers to identify rice gene models for genetic marker development. These resources will be used to enhance the emerging integrated sorghum genetic and physical map. 2) Molecular and quantitative trait loci (QTL) mapping of traits associated with improved crop performance and nutritional content in sorghum and other model species (i.e. Arabidopsis) will be carried out to isolate and characterize genes associated with these traits. We will leverage the near complete rice genome sequence and annotations and the known synteny among the grasses to identify candidate genes associated with QTLs and sequence these regions. This project is awaiting administrative close-out. There is no progress to report. Research was concluded on 9/14/2009.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) The objectives of this cooperative research project are to increase our understanding of genome organization of cereals and the QTLs associated with crop enhancement including response to the environment and nutritional improvement. Approach (from AD-416) 1) Development of genetic and physical markers for sorghum. Annotation of sorghum sequences will be used to identify SSR sequences to be used as genetic markers. In addition we will use the regions of synteny established between the rice genome sequence and sorghum genetic and physical map markers to identify rice gene models for genetic marker development. These resources will be used to enhance the emerging integrated sorghum genetic and physical map. 2) Molecular and quantitative trait loci (QTL) mapping of traits associated with improved crop performance and nutritional content in sorghum and other model species (i.e. Arabidopsis) will be carried out to isolate and characterize genes associated with these traits. We will leverage the near complete rice genome sequence and annotations and the known synteny among the grasses to identify candidate genes associated with QTLs and sequence these regions. Significant Activities that Support Special Target Populations The research during this past year has focused on the development and analysis of qPCR methods in sorghum to monitor expression of both the mature miRNA and the corresponding precursor miRNA from which it is derived. In addition, the group began to examine total RNA expression in sorghum using next generation sequencing technology (specifically Illumina� DGE-tagging protocol). Information about miRNA expression related to different stress treatments and the function of their gene targets will be invaluable for understanding the role of these non-coding RNAs in post-transcriptional gene regulation and for our ability to manipulate stress tolerance in plants. Using Sorghum as a model, 8-day old hydroponically grown sorghum seedlings are subjected to various stress conditions. Most of our work this past year has focused on abscisic acid (ABA) stress. The ABA treatment is used as a proxy for abiotic stress such as drought. In this treatment, 8-day old sorghum seedlings are subjected to 125�M ABA for 24hr and then both roots and shoots are harvested for RNA analysis. Mature miRNAs have traditionally been assayed using small RNA northerns, which require large amounts of input RNA. As an alternative, one of the collaborator groups has been developing protocols that quantified miRNA using hairpin primers in quantitative real time reverse transcription PCR (qPCR) reactions, allowing for rapid quantification of miRNAs using small amounts of RNA. In the past year, the group also has been working on optimizing this assay. In addition to developing the QPCR assays, the group has also optimized isolation protocols for mRNA and small RNAs and is now using a modified TriReagent protocol for the isolation of small RNA. As of this year, the group has developed 17 miRNA-specific primer sets and assayed in control shoot and root tissue. Once they have a validated set of 17 miRNA-specific primers, we will begin to analyze the expression of these mature miRNAs in response to various stress treatments including ABA, SA and JA. In collaboration, the groups have analyzed gene expression from RNA extracted from control and ABA-treated root tissue using Illumina�s DpnII digital gene expression tagging protocol. Preliminary analysis identified 14,420 different genes based on tags, were obtained in both the ABA and control treatments. Comparing the overall counts of these genes between the two treatment types suggested ~1100 genes were up-regulated more than 3-fold in response to treatment with ABA. Of interest are the 500 genes that were expressed in the ABA- treated root sample but were not expressed in the control root sample. In addition ~1200 genes were down-regulated more than 3-fold in ABA- treated roots. Research conducted under this agreement was monitored through in person meetings between the ADODR and the Cooperator.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) The objectives of this cooperative research project are to increase our understanding of genome organization of cereals and the QTLs associated with crop enhancement including response to the environment and nutritional improvement. Approach (from AD-416) 1) Development of genetic and physical markers for sorghum. Annotation of sorghum sequences will be used to identify SSR sequences to be used as genetic markers. In addition we will use the regions of synteny established between the rice genome sequence and sorghum genetic and physical map markers to identify rice gene models for genetic marker development. These resources will be used to enhance the emerging integrated sorghum genetic and physical map. 2) Molecular and quantitative trait loci (QTL) mapping of traits associated with improved crop performance and nutritional content in sorghum and other model species (i.e. Arabidopsis) will be carried out to isolate and characterize genes associated with these traits. We will leverage the near complete rice genome sequence and annotations and the known synteny among the grasses to identify candidate genes associated with QTLs and sequence these regions. Significant Activities that Support Special Target Populations This work focuses on increasing our understanding of the sorghum genome and QTL associated with crop enhancement. In 2007 the Klein laboratory developed and tested qPCR approaches to monitor gene expression of microRNA genes. In the last year the Klein lab has developed assays for more than 44 microRNA genes and monitored the expression of these in developing sorghum under two different treatments salicylic + jasmonic acid or ABA and two different time points, 3 and 27 hrs. In addition to the qPCR work, the Klein lab has also provided tissue for the development of small RNA libraries. Management and Communication: Discussion and review of the objectives are reviewed at least twice a year in conference calls or in person meetings. NP 301 Component 2 and Component 3.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) The objectives of this cooperative research project are to increase our understanding of genome organization of cereals and the QTLs associated with crop enhancement including response to the environment and nutritional improvement. Approach (from AD-416) 1) Development of genetic and physical markers for sorghum. Annotation of sorghum sequences will be used to identify SSR sequences to be used as genetic markers. In addition we will use the regions of synteny established between the rice genome sequence and sorghum genetic and physical map markers to identify rice gene models for genetic marker development. These resources will be used to enhance the emerging integrated sorghum genetic and physical map. 2) Molecular and quantitative trait loci (QTL) mapping of traits associated with improved crop performance and nutritional content in sorghum and other model species (i.e. Arabidopsis) will be carried out to isolate and characterize genes associated with these traits. We will leverage the near complete rice genome sequence and annotations and the known synteny among the grasses to identify candidate genes associated with QTLs and sequence these regions. Significant Activities that Support Special Target Populations This report serves to document research conducted under a Specific Cooperative Agreement between ARS and Texas A&M University. In the past year tissues were developed in replicate from drought stress conditions and developmental stage tissues and delivered to CSHL for small RNA libraries template. The tissues will also be used for other molecular techniques for downstream validation of microRNA predictions and targets associated with drought stress and developmental stages. Management and Communication: Discussion and review of the objectives are done at least twice a year in conference calls or in personal meetings as well as email exchanges and conference calls.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 4d Progress report. This report serves to document research conducted under a Specific Cooperative Agreement between ARS and Texas A&M University; ARS project 1907-21000-023-04S entitled "Improving Sorghum Genome Resources for Cereal Crop Improvement". Additional details of this research can be found in the report of the parent project 1907-21000-023-00D "Comparative Genomic Analyses, Bioinformatics and Resource Development for Cereal Genomes." This work focuses on increasing our understanding of the sorghum genome organization and QTL associated with crop enhancement. In 2005 the JGI announced that it would be producing an 8X whole genome shotgun and an assembly for Sorghum. After discussion with the collaborator, a decision was made to wait for the release of the assembly to make better use of the funds in the next year. No progress was reported for this period.
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Progress 10/01/04 to 09/30/05
Outputs
4d Progress report. This report serves to document research conducted under a Specific Cooperative Agreement between ARS and Texas A&M University. Additional details of this research can be found in the report of the parent project 1907-21000-014-00D Comparative Plant Genomics. This work focuses on increasing our understanding of the sorghum genome organization and QTL associated with crop enhancement. In the past year the major focus of research has been mapping gene sequences based on ESTs and GSS reads from sorghum to the sorghum genome, refining the tiling path of sorghum chromosome 3 and comparative analysis with rice. Seventy eight primers sets were developed to SSRs found in sorghum sequences and tested in the sorghum mapping populations. Of these 47 primers were polymorphic indicating these were suitable for genetic mapping. In conjunction with an NSF funded plant genome project, 612 EST-STS sequences were used to screen six-dimension BAC pools and HICF
fingerprinting was used to improve the TAMU physical map. Of the primer pairs 466 amplified, identifying 2000 sorghum BACs that were fingerprinted. Integration of the data sets has led to the construction of a high quality map of sorghum 3 that contains, 39 contigs covering a total size of 61 MB.
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