Progress 09/15/02 to 09/14/06
Outputs
Progress Report 4d Progress report. This report serves to document research conducted under a trust agreement between ARS and USDA-CSREES NRI Competitive Grants program. Additional details of research can be found in the report for the parent project #3625-21000-035-00D, Cereal Genomics and Pathology. Transposable elements are a type of mobile DNA found throughout eukaryotic organisms. Although abundance is extremely high in some organisms, little is known about the processes governing the distribution of transposable elements across the genome. In 2006, bioinformatic software was developed to perform automated annotation in the complex repeated regions of the maize genome. This is especially important as the $30 million maize sequencing project started this year. Eighty percent of the maize genome (equivalent in size to the human genome) consists of transposable elements (TEs) and their associated long terminal repeats (LTRs). A majority of TEs occur in clusters
of nested repeats, where a transposable element inserts into the genome within the boundaries of an existing element. In maize, more than 80% of TEs are found nested within one another, necessitating an accurate nested TE identification tool for complete annotation of the maize genome. Current software does not address nested TEs, making it especially laborious to resolve and determine the evolution of even moderately clustered repeat regions. "TE Nest" software was developed to facilitate the annotation of our 1.5 Megabase rf1-spanning sequence in the centromeric region of chromosome 3, constructed from 19 contiguous BAC (Bacterial Artificial Chromosome) clones. TE Nest contains an up-to-date database of hundreds of maize TEs and their LTRs. With use of the TE database, TE Nest identifies and maps repeat-incorporations into the original genome sequence while also providing chronology of insertion events in millions of years (Mya) based on the base pair substitution rate of LTRs.
A triangle insertion graph is produced to give an accurate visual representation of the TE integration history by showing timeline, location, and classes of each TE identified, thus creating a framework from which evolutionary comparisons can be made among various regions of the maize genome.
Impacts
(N/A)
Publications
- Kronmiller, B., Gobelman Werner, K.S., Wise, R.P. 2005. Sequencing a 1.3 Mb contig spanning the rf1 fertility restorer locus as a prototype to assess complex-genome coverage strategies. 47th Annual Maize Genetics Conference Abstracts. P184.
- Williams, A., Caldo, R., Hanna, D., McLean, L., Tanimoto, G., Wise, R.P. 2005. Affymetrix maize pilot expression array. Plant and Animal Genome XIII Conference Proceedings. P897.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Cytoplasmic male sterility (CMS) is invaluable in the breeding of hybrid seed. CMS systems are found in 150 plant species and are often attributed to chimeric open-reading frames in the mitochondrial genome. These open- reading frames encode unique proteins that can interfere with mitochondrial function and pollen development and also can confer disease sensitivity. Nuclear restorer (Rf) genes suppress CMS-associated male sterility. In many instances, this suppression is concurrent with Rf- gene-dependent mitochondrial RNA processing. To characterize these mechanisms, we are investigating the T-cytoplasm maize restorers that mediate the processing of T-urf13 mitochondrial transcripts and the concurrent reduction of the mitochondrial URF13 protein. Rf/T-urf13 interactions in maize serve as a model
for restorer-mediated processing of mitochondrial transcripts because this process occurs in many CMS systems such as sorghum, rice, wheat, sunflower, oilseed rape, petunia, and common bean. Investigation of these interactions will therefore contribute to the basic understanding of mitochondrial RNA processing and fertility restoration in crop plants. In many of these economically important plants, CMS is the only efficient way to produce hybrid seed. How does it relate to the National Program(s) and National Program Component(s)? Up until 1970, approximately 85% of the U.S. hybrid maize was produced with the Texas or T-cytoplasmic male-sterile (cms-T) system. However, in 1970, maize produced via this system succumbed to a severe epidemic of southern corn leaf blight. One of the long-term consequences of the epidemic was the development of cms-T as a model system to study mechanisms underlying disease susceptibility, male sterility, and fertility restoration. This research
addresses NP302, Plant Biological and Molecular Processes and is supported by USDA-CSREES-NRI competitive grant 2002-35301-12064. 2. List the milestones (indicators of progress) from your Project Plan. Objective 2 of parent CRIS 3625-21000-035-00D. Characterize the rf1, rf8, and rf* genomic regions for pollen-fertility restoration in cytoplasmic male sterile maize via recombination and sequence analyses. Analyze the expression of candidate Rf1 sequences via complementation in our T-cytoplasm transformation system. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Objective 2 of parent CRIS 3625-21000-035-00D. Characterize the rf1, rf8, and rf* genomic regions for pollen-fertility restoration in cytoplasmic male sterile maize via recombination and sequence analyses. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3
years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? 2006 Objective 2 Characterize the rf1, rf8, and rf* genomic regions for pollen-fertility restoration in cytoplasmic male sterile maize via recombination and sequence analyses. 4d Progress report. This report serves to document research conducted under a trust agreement between ARS and USDA-NRI. Additional details of research can be found in the report for the parent project #3625-21000-035-00D, Cereal Genomics and Pathology. Cytoplasmic male sterility (CMS) is invaluable in the breeding of hybrid seed. CMS systems are found in 150 plant species and are often attributed to chimeric open-reading frames in the mitochondrial genome. These open-reading frames encode unique proteins that can interfere with mitochondrial function and pollen development and also can confer disease sensitivity. Nuclear restorer (Rf) genes suppress CMS-associated male sterility. In
many instances, this suppression is concurrent with Rf- gene-dependent mitochondrial RNA processing. To characterize these mechanisms, we are investigating the T-cytoplasm maize restorers that mediate the processing of T-urf13 mitochondrial transcripts and the concurrent reduction of the mitochondrial URF13 protein. In T-cytoplasm maize, CMS is attributed to the presence of the unique mitochondrial gene, T-urf13. Full suppression of T-urf13-mediated CMS is directed by the combined action of dominant alleles of the nuclear (fertility restorer) genes, rf1 and rf2a. To facilitate a candidate approach towards identification of the rf1 gene, three B73 BAC libraries were used to create a physical map of 794 clones from the centromeric region of chromosome 3 anchored to the rf1 locus. A minimum-tiling path of 14 contiguous BACs covering 1.3 megabases were shotgun sequenced, assembled and finished to completion for annotation and display in the GBrowse viewer. Eighty-seven percent of the
sequence has been identified as repetitive, with most transposable elements found in large nested clusters spanning up to 300 kb with insertion chronologies of 0.19 to 8.50 million years. GeneSeqer, Fgenesh, and GeneMark.hmm were used to predict consensus locations and structures for 53 genes. Thirty-seven of these are positioned in gene clusters with as many as 8 members. Two hundred fifty- four GSS assemblies (including MAGIs, TIGRs AZM and PlantGDBs GSS) aligned to the 1.3 Mb contig, 36 of which aligned to predicted genes. Two hundred eighteen GSS assemblies aligned to regions not predicted as genes, revealing that only 15% of GSS contigs align to genes in this region. Seventeen predicted genes did not correspond to any GSS assembly indicating that at least in the centromeric region of chromosome 3, finished sequence can provide a significant number of previously undescribed gene predictions. 5. Describe the major accomplishments over the life of the project, including their
predicted or actual impact. Over the course of this USDA-NRI competitively funded project, we have performed a systematic genetic analysis of three independent nuclear restorers that direct mitochondrial RNA processing in maize. Through these analyses we have 1) isolated transposon-tagged, mutant alleles of the Rf1 nuclear restorer as a tool to clone this nuclear-restorer gene, 2) generated extensive mapping populations segregating for four independent rf1-m alleles, Rf8 and Rf*, 3) recovered over 50 recombinants (from 10, 564 backcross progeny) between the rf1-m alleles and a dominant visible gene, Lg3, to facilitate cosegregation analysis and map-based, gene- discovery, and 4) used the population segregating for Rf8 to identify closely linked DNA markers via AFLP bulk-segregant analysis. Completion of this project will provide a physical foundation for the investigation of nuclear-mitochondrial interaction and RNA processing in plant systems. Because CMS remains a widely used tool
for the production of hybrid seed in a number of economically important crops, we anticipate that a common mechanism among Rf1-, Rf8-, and Rf*-like restorers will be applicable to these crops as well. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? This information has been transferred to other scientists and breeders through publication in peer-reviewed journals and by presentation at invited seminars at universities, national, and international meetings.
Impacts
(N/A)
Publications
- Kronmiller, B., Gobelman Werner, K.S., Wise, R.P. 2005. Sequencing a 1.3 Mb contig spanning the rf1 fertility restorer locus as a prototype to assess complex-genome coverage strategies. 47th Annual Maize Genetics Conference Abstracts. P184.
- Williams, A., Caldo, R., Hanna, D., McLean, L., Tanimoto, G., Wise, R.P. 2005. Affymetrix maize pilot expression array. Plant and Animal Genome XIII Conference Proceedings. P897.
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Cytoplasmic male sterility (CMS) is invaluable in the breeding of hybrid seed. CMS systems are found in 150 plant species and are often attributed to chimeric open-reading frames in the mitochondrial genome. These open- reading frames encode unique proteins that can interfere with mitochondrial function and pollen development and also can confer disease sensitivity. Nuclear restorer (Rf) genes suppress CMS-associated male sterility. In many instances, this suppression is concurrent with Rf- gene-dependent mitochondrial RNA processing. To characterize these mechanisms, we are investigating the T-cytoplasm maize restorers that mediate the processing of T-urf13 mitochondrial transcripts and the concurrent reduction of the mitochondrial URF13 protein. Rf/T-urf13 interactions in maize serve as a model
for restorer-mediated processing of mitochondrial transcripts because this process occurs in many CMS systems such as sorghum, rice, wheat, sunflower, oilseed rape, petunia, and common bean. Investigation of these interactions will therefore contribute to the basic understanding of mitochondrial RNA processing and fertility restoration in crop plants. In many of these economically important plants, CMS is the only efficient way to produce hybrid seed. How does it relate to the National Program(s) and National Program Component(s)? Up until 1970, approximately 85% of the U.S. hybrid maize was produced with the Texas or T-cytoplasmic male-sterile (cms-T) system. However, in 1970, maize produced via this system succumbed to a severe epidemic of southern corn leaf blight. One of the long-term consequences of the epidemic was the development of cms-T as a model system to study mechanisms underlying disease susceptibility, male sterility, and fertility restoration. This research
addresses NP302, Plant Biological and Molecular Processes and is supported by USDA-CSREES-NRI competitive grant 2002-35301-12064. 2. List the milestones (indicators of progress) from your Project Plan. Objective 2 of parent CRIS 3625-21000-035-00D. Characterize the rf1, rf8, and rf* genomic regions for pollen-fertility restoration in cytoplasmic male sterile maize via recombination and sequence analyses. Analyze the expression of candidate Rf1 sequences via complementation in our T-cytoplasm transformation system. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004, and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. Objective 2 of parent CRIS 3625-21000-035-00D. Characterize the rf1, rf8, and rf* genomic regions for pollen-fertility restoration in cytoplasmic male sterile maize via recombination and sequence analyses.
Completed draft sequence of 1.2 megabase region of the rf1 locus. This establishes a structural foundation from which the Rf1 gene will be isolated and characterized. B. List the milestones that you expect to address over the next 3 years (FY 2005, 2006, and 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY 2005 Objective 2 Characterize the rf1, rf8, and rf* genomic regions for pollen-fertility restoration in cytoplasmic male sterile maize via recombination and sequence analyses. FY 2006 Objective 2 Analyze the expression of candidate Rf1 sequences via complementation in our T-cytoplasm transformation system. FY 2007 Objective 2 Analyze the expression of candidate Rf1 sequences via complementation in our T-cytoplasm transformation system. 4. What were the most significant accomplishments this past year? D. Progress Report. This report serves to document research conducted under a trust agreement between ARS and NRI-CGP.
Additional details of research can be found in the report for the parent project #3625-21000-035-00D, Cereal Genomics and Pathology. Cytoplasmic male sterility (CMS) is invaluable in the breeding of hybrid seed. CMS systems are found in 150 plant species and are often attributed to chimeric open-reading frames in the mitochondrial genome. These open-reading frames encode unique proteins that can interfere with mitochondrial function and pollen development and also can confer disease sensitivity. Nuclear restorer (Rf) genes suppress CMS-associated male sterility. In many instances, this suppression is concurrent with Rf- gene-dependent mitochondrial RNA processing. To characterize these mechanisms, we are investigating the T-cytoplasm maize restorers that mediate the processing of T-urf13 mitochondrial transcripts and the concurrent reduction of the mitochondrial URF13 protein. In T-cytoplasm maize, cytoplasmic male sterility (CMS) is attributed to the presence of the unique
mitochondrial gene, T-urf13. Full suppression of T-urf13-mediated CMS is directed by the combined action of dominant alleles of the nuclear (fertility restorer) genes, rf1 and rf2. As a first step in the positional cloning of the rf1 gene, 1.2 megabases of maize chromosome 3 spanning the rf1 locus was sequenced and annotated. Three B73 BAC libraries were used to create a physical map of 439 clones anchored to the rf1 locus. A minimum-tiling path of 12 sequenced BACs comprise 79% transposable elements as well as other repetitive sequences, including centromeric-specific repeats. One-hundred fourteen unique gene families from the megabase maize sequence were compared to the TIGR rice pseudomolecules. One-hundred sixty one maize sequences that correspond to rice genes were found dispersed throughout the genome, suggesting that large-scale synteny has dissipated since species divergence. In contrast, mico-colinearity is still intact, shown by groups of multiple genes that have
retained relative distribution and orientation in both species. Additionally, several single copy maize genes from the rf1 region are found in multiple positions on the rice genome, restricted to either single chromosomes or particular areas within chromosomes. This sequence contig facilitates a candidate gene approach towards identification of the rf1 family while also providing a basis for comparison of functionally related regions in cereals. Because CMS remains a widely used tool for the production of hybrid seed in a number of economically important crops, we anticipate that a common mechanism among Rf1-like restorers will be applicable to these systems as well. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Over the course of this USDA-NRI competitively funded project, we have performed a systematic genetic analysis of three independent nuclear restorers that direct mitochondrial RNA processing in maize. Through
these analyses we have 1) isolated transposon-tagged, mutant alleles of the Rf1 nuclear restorer as a tool to clone this nuclear-restorer gene, 2) generated extensive mapping populations segregating for four independent rf1-m alleles, Rf8 and Rf*, 3) recovered over 50 recombinants (from 10, 564 backcross progeny) between the rf1-m alleles and a dominant visible gene, Lg3, to facilitate cosegregation analysis and map-based, gene- discovery, and 4) used the population segregating for Rf8 to identify closely linked DNA markers via AFLP bulk-segregant analysis. Completion of this project will provide a physical foundation for the investigation of nuclear-mitochondrial interaction and RNA processing in plant systems. Because CMS remains a widely used tool for the production of hybrid seed in a number of economically important crops, we anticipate that a common mechanism among Rf1-, Rf8-, and Rf*-like restorers will be applicable to these crops as well. 6. What science and/or technologies
have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? This information has been transferred to other scientists and breeders through publication in peer-reviewed journals and by presentation at invited seminars at universities, national, and international meetings.
Impacts
(N/A)
Publications
- Kronmiller, B., Gobelman Werner, K.S., Wise, R.P. 2004. Comparative analysis of a one-megabase sequence spanning the maize Rf1 fertility restorer with the rice genome. 46th Maize Genetics Conference. p. 112.
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