Progress 07/01/16 to 06/30/21
Outputs
Target Audience:Researchers and students in the fields of molecular genetics and agronomy, located at academic and industry institutions Changes/Problems: Normal lab progress was limited due to Covid pandemic Field program suffered considerable losses due to plant lodging caused by high winds during Derecho windstorm, August 2020. What opportunities for training and professional development has the project provided?This project provided training for two graduate students in classical and molecular genetic approaches, as well as computational methods. The project also provided research experience and on-the-job training for two undergraduate students. These educational activities provide training and experience that is otherwise unavailable to young students and future scientists, and will thereby help to enhance the potential for scientific and technological research in the USA. How have the results been disseminated to communities of interest?The results have been disseminated by publications in peer-reviewed journals, by oral and poster presentations at international scientific conferences, and by creation of a website containing animated models showing how transposable elements induce genome rearrangements. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
IMPACT: This project investigatedthe role of transposable elements, or jumping genes, in generating duplications and other genome rearrangements. This project has identified Alternative Transposition as a new mechanism by which transposons duplicate and rearrange functional segments, ranging from individual genic elements (enhancers and exons) and whole genes, up to large duplications containing hundreds of genes. Once the mechanism by which transposable elements induce genome rearrangements is known, it may be possible to use this mechanism to generate diversity; for example, by making duplications of beneficial genes, or by deleting harmful genes. Objective 1... Isolate and analyze alleles derived from alternative transposition and DNA re-replication events in maize, to identify the sizes and breakpoint junctions of chromosome rearrangements and transposon Composite Insertions. As described in previous project reports, we have isolated Composite Insertions from two unstable maize alleles: p1-ovov454 and p1-wwB54. These CI alleles were isolated based on their ability to induce ectopic expression in kernel pericarp of the maize p2 gene, a paralog of p1, which is normally not expressed in kernel pericarp. Objective 2... Clone and sequence Composite Insertions (CIs) to determine the extents of re-replication and the mechanism of DSB repair in each case. As described in previous project reports, we isolated 16 CI alleles from p1-ovov454, and 30 from p1-wwB54 alleles. All of the CIs contain Ac, fAc, and variable lengths of DNA derived from the original p1 donor site in the p1-wwB54 progenitor; these CIs range in size from 12.8 kb to 23.6 kb. The internal junctions have sequences characteristic of DSB repair by NHEJ (Non Homologous End Joining) without additional insertions (5 cases) or with insertions of up to 50 bp of Filler DNA. Filler DNA sequences were mostly copied from nearby p1 sequence, similar to other reports of Filler DNA in maize. The results were published in the respected academic journal Genetics. Objective 3... Isolate and compare meiotic and mitotic products of RET and DNA re-replication to learn how the different nuclear cycles affect the processes of alternative transposition, DNA replication fork processivity, and DSB repair. We are especially keen to identify any cases that may contain tandem duplications of Ac element sequences, as this could provide insight into the mechanism of gene amplification. Further analysis of this material identified one allele, P*-353, that has a tandem duplication of sequences flanking the p1 gene. We are characterizing this allele to try to understand the duplication mechanism. Long-range sequencing analysis confirmed the presence of a tandem duplication involving Ac element and p1 gene sequences. However, the precise mechanism of duplication could not be discerned. Due to constraints caused by the ongoing pandemic, work on this goal was suspended. Objective 4... Test the hypothesis that transposition-induced duplications and DNA re-replication events contributed to maize genetic diversification and genome evolution. We developed a new program termed TIRLearner to annotate TIR TEs in sequenced genomes. Applying this pipeline, we have shown that maize genomes have a considerably larger proportion of TIR TEs than previously reported. Also, TIR TEs exhibit large diversity in presence and location in the maize inbreeds B73, Mo17 and W22, indicating their potential role in generating genetic diversity. In further work, the TIR-Learner program was incorporated into a comprehensive TE annotation pipeline termed EDTA (Extensive De Novo TE Annotator). This work is in collaboration with Drs. Shujun Ou and Matthew Hufford, Iowa State University. This work has been completed and was published in 2019 in Genome Biology. In addition, we showed that alternative transposition reactions can induce chromosomal inversions that can activate ectopic gene expression. These results show that TEs can play important roles in generating adaptive diversity.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Sharma, S.P. and T. Peterson. 2020. Transposon-induced inversions activate gene expression in maize. Virtual Maize Genetics Conference, March 8 - 12, 2021
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Sharma, S.P., Zuo, T. and T. Peterson. 2021. Transposon-induced inversions activate gene expression in the maize pericarp. Genetics, 218(2), iyab062. https://doi.org/10.1093/genetics/iyab062
|
Progress 10/01/19 to 09/30/20
Outputs
Target Audience:Researchers and students in the fields of molecular genetics and agronomy, located at academic and industry institutions Changes/Problems: Normal lab progress was limited due to Covid pandemic Field program suffered considerable losses due to plant lodging caused by high winds during Derecho windstorm, August 2020. What opportunities for training and professional development has the project provided?This project provided training for two graduate students in classical and molecular genetic approaches, as well as computational methods. The project also provided research experience and on-the-job training for two undergraduate students. These educational activities provide training and experience that is otherwise unavailable to young students and future scientists, and will thereby help to enhance the potential for scientific and technological research in the USA. How have the results been disseminated to communities of interest?The results have been disseminated by publications in peer-reviewed journals, by oral and poster presentations at international scientific conferences, and by creation of a website containing animated models showing how transposable elements induce genome rearrangements. What do you plan to do during the next reporting period to accomplish the goals?Work on this project will continue using the established protocols in our lab. Transposon-induced rearrangements in maize will be analyzed using both molecular biological approaches, and via computational techniques. These approaches include 1) identification of candidate rearrangement alleles via an efficient visible phenotype screen; 2) PCR-based approaches to molecularly test for rearrangement events; 3) study selected cases in detail by molecular isolation of the rearrangement breakpoints, followed by sequence analysis to infer the mechanism(s) of rearrangement; and by genomic DNA gel blot hybridizations to confirm rearrangement structures predicted by analysis of PCR junctions; 4) computational approaches include the development of software to search sequenced genomes for sequences bearing the structural hallmarks of transposon-induced rearrangements.
Impacts
What was accomplished under these goals?
IMPACT:This project investigates the role of transposable elements, or jumping genes, in generating duplications and other genome rearrangements. This project has identified Alternative Transposition as a new mechanism by which transposons duplicate and rearrange functional segments, ranging from individual genic elements (enhancers and exons) and whole genes, up to large duplications containing hundreds of genes. Once the mechanism by which transposable elements induce genome rearrangements is known, it may be possible to use this mechanism to generate diversity; for example, by making duplications of beneficial genes, or by deleting harmful genes.The results will provide new insight into the forces that continue to shape plant genomes and may provide insight into how plant species may respond and adapt to climate change. Objective 1... Isolate and analyze alleles derived from alternative transposition and DNA re-replication events in maize, to identify the sizes and breakpoint junctions of chromosome rearrangements and transposon Composite Insertions. As described in previous project reports, we have isolated Composite Insertions from two unstable maize alleles: p1-ovov454 and p1-wwB54. These CI alleles were isolated based on their ability to induce ectopic expression in kernel pericarp of the maize p2 gene, a paralog of p1, which is normally not expressed in kernel pericarp. Objective 2... Clone and sequence Composite Insertions (CIs) to determine the extents of re-replication and the mechanism of DSB repair in each case. As described in previous project reports, we isolated 16 CI alleles from p1-ovov454, and 30 from p1-wwB54 alleles. All of the CIs contain Ac, fAc, and variable lengths of DNA derived from the original p1 donor site in the p1-wwB54 progenitor; these CIs range in size from 12.8 kb to 23.6 kb. The internal junctions have sequences characteristic of DSB repair by NHEJ (Non Homologous End Joining) without additional insertions (5 cases) or with insertions of up to 50 bp of Filler DNA. Filler DNA sequences were mostly copied from nearby p1 sequence, similar to other reports of Filler DNA in maize. The results were published in the December issue of Genetics. Objective 3... Isolate and compare meiotic and mitotic products of RET and DNA re-replication to learn how the different nuclear cycles affect the processes of alternative transposition, DNA replication fork processivity, and DSB repair. We are especially keen to identify any cases that may contain tandem duplications of Ac element sequences, as this could provide insight into the mechanism of gene amplification. Further analysis of this material identified one allele, P*-353, that has a tandem duplication of sequences flanking the p1 gene. We are characterizing this allele to try to understand the duplication mechanism. Objective 4... Test the hypothesis that transposition-induced duplications and DNA re-replication events contributed to maize genetic diversification and genome evolution. We developed a new program termed TIRLearner to annotate TIR TEs in sequenced genomes. Applying this pipeline, we have shown that maize genomes have a considerably larger proportion of TIR TEs than previously reported. Also, TIR TEs exhibit large diversity in presence and location in the maize inbreeds B73, Mo17 and W22, indicating their potential role in generating genetic diversity. In further work, the TIR-Learner program was incorporated into a comprehensive TE annotation pipeline termed EDTA (Extensive De Novo TE Annotator). This work is in collaboration with Drs. Shujun Ou and Matthew Hufford, Iowa State University. This work has been completed and was published in 2019 in Genome Biology
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Wang, D., Zhang, J., Zuo, T., Lisch, D. and T. Peterson. 2020. siRNA-mediated de novo silencing of Ac/Ds transposons is initiated by alternative transposition in maize. Genetics 215: 393406. doi:10.1534/genetics.120.303264
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Su W., and Peterson T. 2020. The impact of Terminal Inverted Repeat (TIR) Transposable Elements in maize genomes. 62nd Annual Maize Genetics Conference, June 25-26, Online.
- Type:
Theses/Dissertations
Status:
Awaiting Publication
Year Published:
2020
Citation:
Su, Weijia. "The impact of Terminal Inverted Repeat (TIR) transposable elements on plant genomes."
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Sharma, S.P. and T. Peterson. 2020. Transposon-induced inversions activate tissue-specific gene expression in maize. Virtual Maize Genetics Conference, June 25-26
|
Progress 10/01/18 to 09/30/19
Outputs
Target Audience:Researchers and students in the fields of molecular genetics and agronomy, located at academic and industry institutions. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided training for two graduate students in classical and molecular genetic approaches, as well as computational methods. The project also provided research experience and on-the-job training for four undergraduate students. These educational activities provide training and experience that is otherwise unavailable to young students and future scientists, and will thereby help to enhance the potential for scientific and technological research in the USA. How have the results been disseminated to communities of interest?The results have been disseminated by publications in peer-reviewed journals, by oral and poster presentations at international scientific conferences, and by creation of a website containing animated models showing how transposable elements induce genome rearrangements. What do you plan to do during the next reporting period to accomplish the goals?Work on this project will continue using the established protocols in our lab. Transposon-induced rearrangements in maize will be analyzed using both molecular biological approaches, and via computational techniques. These approaches include 1) identification of candidate rearrangement alleles via an efficient visible phenotype screen; 2) PCR-based approaches to molecularly test for rearrangement events; 3) study selected cases in detail by molecular isolation of the rearrangement breakpoints, followed by sequence analysis to infer the mechanism(s) of rearrangement; and by genomic DNA gel blot hybridizations to confirm rearrangement structures predicted by analysis of PCR junctions; 4) computational approaches include the development of software to search sequenced genomes for sequences bearing the structural hallmarks of transposon-induced rearrangements.
Impacts
What was accomplished under these goals?
IMPACT: In corn and other crop plants, traits are determined by genes carried in the plant genome and encoded in the DNA (genotype). Plant breeding involves making crosses among diverse genotypes and selecting for desirable combinations of genes. For this reason, understanding sources of genetic variation is important for modern plant breeding. A major source of genetic variation in crop plants is the presence of genome rearrangements. These rearrangements include large changes in genome structure that affect multiple genes, such as duplications (gains of genetic material). Duplications are very important because the duplicated genes may be over-expressed, leading to important changes in plant phenotypes. Additionally, having multiple gene copies allows one copy to change its expression or function, while one copy maintains the original function. This provides genetic flexibility and is thought to be an important mechanism by which plants can adapt to changing environments over time. Duplications are very common in crop plant genomes, and there are several known mechanisms for generating new duplications. However, in many cases the molecular origin of the duplications is unknown. This project investigates the role of transposable elements, or jumping genes, in generating duplications and other genome rearrangements. Transposons are well known to have expanded genomes over time, and to modulate gene expression in various ways. This project has identified Alternative Transposition as a new mechanism by which transposons duplicate and rearrange functional segments, ranging from individual genic elements (enhancers and exons) and whole genes, up to large duplications containing hundreds of genes. One goal of this current project is to determine the number of duplications in the corn genome generated by transposable elements. Another goal is to investigate an unusual duplication mechanism associated with a novel structure termed the Composite Insertion (CI). The CI is a transposable element that duplicates pieces of genes and then inserts them back into the corn genome. Different CIs contain different lengths of genetic sequence, and they are inserted at diverse sites in the genome. CIs can influence the expression of genes near their insertion site. This project will determine the origin and effects of CIs and their role in generating duplications over time. This hypothesis-driven research takes advantage of an active transposable element system coupled with visual markers to enable extremely efficient screening and selection of genome rearrangements. The generality of results from experimental materials will be tested by bioinformatic searches of sequenced genomes for structures with the characteristic features of these events. The results could have a major impact on methods of plant breeding. Once the mechanism by which transposable elements induce genome rearrangements is known, it may be possible to use this mechanism to generate diversity; for example, by making duplications of beneficial genes, or by deleting harmful genes. Objective 1... Isolate and analyze alleles derived from alternative transposition and DNA re-replication events in maize, to identify the sizes and breakpoint junctions of chromosome rearrangements and transposon Composite Insertions. As described in previous project reports, we have isolated Composite Insertions from two unstable maize alleles: p1-ovov454 and p1-wwB54. These CI alleles were isolated based on their ability to induce ectopic expression in kernel pericarp of the maize p2 gene, a paralog of p1, which is normally not expressed in kernel pericarp. Objective 2... Clone and sequence Composite Insertions (CIs) to determine the extents of re-replication and the mechanism of DSB repair in each case. As described in previous project reports, we isolated 16 CI alleles from p1-ovov454, and 30 from p1-wwB54 alleles. All of the CIs contain Ac, fAc, and variable lengths of DNA derived from the original p1 donor site in the p1-wwB54 progenitor; these CIs range in size from 12.8 kb to 23.6 kb. The internal junctions have sequences characteristic of DSB repair by NHEJ (Non Homologous End Joining) without additional insertions (5 cases) or with insertions of up to 50 bp of Filler DNA. Filler DNA sequences were mostly copied from nearby p1 sequence, similar to other reports of Filler DNA in maize. To gain further insight into the regulation of the p2 gene, we isolated colorless derivatives of two CI alleles (termed S7 and E3). These derivatives were termed S7-M and E3-M, respectively. Structural analysis of S7-M indicates that the Composite Insertion has excised; this is consistent with the model that the p1 enhancer contained within the S7 CI is required to activate p2 expression. In contrast, E3-M shows no apparent structural change from its progenitor E3. We hypothesize that the change in E3-M expression is a result of epigenetic effects, and further tests are ongoing. Objective 3... Isolate and compare meiotic and mitotic products of RET and DNA re-replication to learn how the different nuclear cycles affect the processes of alternative transposition, DNA replication fork processivity, and DSB repair. Maize plants expected to generate meiotic and mitotic CIs were grown in an isolation field in Ames in summer 2016. Candidate CI-containing kernels were selected and grown in the summer of 2017. Ears have been harvested and a sample of putative CI's of meiotic origin have been selected and are currently undergoing molecular testing. Plants containing these derivative alleles are being screened by genomic Southern blot to detect structural rearrangements. We are especially keen to identify any cases that may contain tandem duplications of Ac element sequences, as this could provide insight into the mechanism of gene amplification. Objective 4... Test the hypothesis that transposition-induced duplications and DNA re-replication events contributed to maize genetic diversification and genome evolution. As described in previous reports, we have used computational programs termed STRAND (Search for Transposon-Induced Duplications) and DDS2 to identify Transposon-Induced Duplications. These programs did identify duplications, but it became clear that the published Transposon annotations were incomplete. Therefore we developed a new program termed TIR-Learner to annotate TIR TEs in sequenced genomes. Applying this pipeline, we have shown that maize genomes have a considerably larger proportion of TIR TEs than previously reported. Also, TIR TEs exhibit large diversity in presence and location in the maize inbreeds B73, Mo17 and W22, indicating their potential role in generating genetic diversity. In further work, the TIR-Learner program was incorporated into a comprehensive TE annotation pipeline termed EDTA (Extensive De Novo TE Annotator). This work is in collaboration with Drs. Shujun Ou and Matthew Hufford, Iowa State University.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Su W., Gu X., and Peterson T. 2019. TIR-Learner, a New Ensemble Method for TIR Transposable Element Annotation, Provides Evidence for Abundant New Transposable Elements in the Maize Genome. Mol. Plant. 12: 447460.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2019
Citation:
Ou, S., Su, W., Liao, Y., Chougule, K., Ware, D., Peterson, T., Jiang, N., Hirsch, C.N., and Hufford, M.B. Benchmarking Transposable Element Annotation Methods for Creation of a Streamlined, Comprehensive Pipeline. bioRxiv:657890.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Sharma, S.P. and T. Peterson. 2019. Transposon-Induced Inversions activate Tissue-specific gene expression in maize. 61st Annual Maize Genetics Conference, March 14 17, St. Louis, Missouri, USA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Su W., and Peterson T. 2019. TIR-Learner, a New Ensemble Method for TIR Transposable Element Annotation. 61st Annual Maize Genetics Conference, March 14 17, St. Louis, Missouri, USA.
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Researchers and students in the fields of molecular genetics and agronomy, located at academic and industry institutions. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided training for two graduate students in classical and molecular genetic approaches, as well as computational methods. The project also provided research experience and on-the-job training for five undergraduate students. These educational activities provide training and experience that is otherwise unavailable to young students and future scientists, and will thereby help to enhance the potential for scientific and technological research in the USA. How have the results been disseminated to communities of interest?The results have been disseminated by poster presentations at international scientific conferences, by publication of a book chapter, and by creation of a website containing animated models showing how transposable elements induce genome rearrangements. What do you plan to do during the next reporting period to accomplish the goals?Analysis of transposon-induced rearrangements in maize will be continued using both molecular biological approaches, and via computational techniques. These approaches include 1) identification of candidate rearrangement alleles via an efficient visible phenotype screen; 2) PCR-based approaches to molecularly test for rearrangement events; 3) study selected cases in detail by molecular isolation of the rearrangement breakpoints, followed by sequence analysis to infer the mechanism(s) of rearrangement; and by genomic DNA gel blot hybridizations to confirm rearrangement structures predicted by analysis of PCR junctions; 4) computational approaches include the development of software to search sequenced genomes for sequences bearing the structural hallmarks of transposon-induced rearrangements.
Impacts
What was accomplished under these goals?
Overall impact statement: In corn and other crop plants, traits are determined by genes carried in the plant genome, and encoded in the DNA (genotype). Plant breeding involves making crosses among diverse genotypes and selecting for desirable combinations of genes. For this reason, understanding sources of genetic variation is important for modern plant breeding. A major source of genetic variation in crop plants is the presence of genome rearrangements. These rearrangements include large changes in genome structure that affect multiple genes, such as duplications (gains of genetic material). Duplications are very important because the duplicated genes may be over-expressed, leading to important changes in plant phenotypes. Additionally, having multiple gene copies allows one copy to change its expression or function, while one copy maintains the original function. This provides genetic flexibility and is thought to be an important mechanism by which plants can adapt to changing environments over time. Duplications are very common in crop plant genomes, and there are several known mechanisms for generating new duplications. However, in many cases the molecular origin of the duplications is unknown. This project investigates the role of transposable elements, or jumping genes, in generating duplications and other genome rearrangements. Transposons are well known to have expanded genomes over time, and to modulate gene expression in various ways. This project has identified Alternative Transposition as a new mechanism by which transposons duplicate and rearrange functional segments, ranging from individual genic elements (enhancers and exons) and whole genes, up to large duplications containing hundreds of genes. One goal of this current project is to determine the number of duplications in the corn genome generated by transposable elements. Another goal is to investigate an unusual duplication mechanism associated with a novel structure termed the Composite Insertion (CI). The CI is a transposable element that duplicates pieces of genes and then inserts them back into the corn genome. Different CIs contain different lengths of genetic sequence, and they are inserted at diverse sites in the genome. CIs can influence the expression of genes near their insertion site. This project will determine the origin and effects of CIs and their role in generating duplications over time. This hypothesis-driven research takes advantage of an active transposable element system coupled with visual markers to enable extremely efficient screening and selection of genome rearrangements. The generality of results from experimental materials will be tested by bioinformatic searches of sequenced genomes for structures with the characteristic features of these events. The results could have a major impact on methods of plant breeding. Once the mechanism by which transposable elements induce genome rearrangements is known, it may be possible to use this mechanism to generate diversity; for example, by making duplications of beneficial genes, or by deleting harmful genes. Objective 1... Isolate and analyze alleles derived from alternative transposition and DNA re-replication events in maize, to identify the sizes and breakpoint junctions of chromosome rearrangements and transposon Composite Insertions. Genetic methods to isolate alleles derived from alternative transposition and DNA re-replication were described in the 2017 report. We are continuing with that strategy and have a number of alleles under investigation. Objective 2... Clone and sequence Composite Insertions (CIs) to determine the extents of re-replication and the mechanism of DSB repair in each case. Characterization of CIs was described in the 2017 Progress Report. Objective 3... Isolate and compare meiotic and mitotic products of RET and DNA re-replication to learn how the different nuclear cycles affect the processes of alternative transposition, DNA replication fork processivity, and DSB repair. Maize plants expected to generate meiotic and mitotic CIs were grown in an isolation field in Ames in summer 2016. Candidate CI-containing kernels were selected and grown in the summer of 2017. Ears have been harvested and a sample of putative CI's of meiotic origin have been selected and are currently undergoing molecular testing. Objective 4... Test the hypothesis that transposition-induced duplications and DNA re-replication events contributed to maize genetic diversification and genome evolution. Based on the predictions of our models, we have developed computational methods to identify Transposon-Induced Duplications (TIDs) in sequenced genomes. The program termed STRAND (Search for Transposon-Induced Duplications) takes known transposon and genome sequences as input, and outputs a list of transposon-associated duplications and their graphical structures. For maize B73 the program returned 28 genomic duplications, ranging in size from 158 bp to 47 kb, initiated by DNA transposable elements. The frequency of Transposon-Induced Duplications returned by the program may appear low, considering the large number of duplications present in higher plants. However, it is important to consider that we are currently using very stringent search parameters, requiring highly-homologous duplications, perfect TSDs, etc.; i.e. the program detects only recent duplications that have maintained a pristine structure. Older duplications would likely erode quickly by deletion as observed in plant genomes. In further work we used the program DDS2 in a computational pipeline to detect additional TIDs. In preliminary tests, 163 TIDs were detected in maize B73 genome. Work is ongoing to characterize these cases, and to compare with TIDs in other maize inbred lines including W22 and Mo17. In order to test the hypothesis that Transposable Elements are involved in generating duplications and other genome rearrangements, it is essential to have an accurate annotation of TEs. Our investigations indicated that the currently published TIR TE (Terminal Inverted Repeat Transposable Element) annotations are largely inaccurate. Therefore we developed a new software pipeline (TIR-Learner) to annotate TIR TEs in sequenced genomes. Applying this pipeline, we have shown that maize genomes have a considerably larger proportion of TIR TEs than previously reported. Also, TIR TEs exhibit large diversity in presence and location in the maize inbreds B73, Mo17 and W22, indicating their potential role in generating genetic diversity.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2018
Citation:
Su, W., Sharma, S. P., & Peterson, T. (2018). Evolutionary Impacts of Alternative Transposition. In Origin and Evolution of Biodiversity (pp. 113-130). Springer, Cham.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Thomas Peterson, Weijia Su, Dafang Wang, Sharu Paul Sharma and David F. Weber (2018) Chromosome Rearrangements Induced by hAT Transposable Elements: Mechanisms and Genetic Impacts. International Chromosome Conference, Prague, Czech Republic, 2 ⿿ 5 September 2018.
|
Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Researchers and students in the fields of molecular genetics and agronomy, located at academic and industry institutions. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided training for two graduate students in classical and molecular genetic approaches, as well as computational methods. The project also provided research experience and on-the-job training for five undergraduate students. These educational activities provide training and experience that is otherwise unavailable to young students and future scientists, and will thereby help to enhance the potential for scientific and technological research in the USA. How have the results been disseminated to communities of interest?The results have been disseminated by oral and poster presentations at international scientific conferences, and by creation of a website containing animated models showing how transposable elements induce genome rearrangements. What do you plan to do during the next reporting period to accomplish the goals?Analysis of transposon-induced rearrangements in maize will be continued using both molecular biological approaches, and via computational techniques. These approaches include 1) identification of candidate rearrangement alleles via an efficient visible phenotype screen; 2) PCR-based approaches to molecularly test for rearrangement events; 3) study selected cases in detail by molecular isolation of the rearrangement breakpoints, followed by sequence analysis to infer the mechanism(s) of rearrangement; 4) computational approaches include the development of software to search sequenced genomes for sequences bearing the structural hallmarks of transposon-induced rearrangements.
Impacts
What was accomplished under these goals?
Overall impact statement: In corn and other crop plants, traits are determined by genes carried in the plant genome, and encoded in the DNA (genotype).Plant breeding involves making crosses among diverse genotypes and selecting for desirable combinations of genes.For this reason, understanding sources of genetic variation is important for modern plant breeding.A major source of genetic variation in crop plants is the presence of genome rearrangements.These rearrangements include large changes in genome structure that affect multiple genes, such as duplications (gains of genetic material).Duplications are very important because the duplicated genes may be over-expressed, leading to important changes in plant phenotypes.Additionally, having multiple gene copies allows one copy to change itsexpression or function, while one copy maintains the original function.This provides genetic flexibility and is thought to be an important mechanism by which plants can adapt to changing environments over time. Duplications are very common in crop plant genomes, and there are several known mechanisms for generating new duplications. However, in many cases the molecular origin of the duplications is unknown. This project investigates the role of transposable elements, or jumping genes, in generating duplications and other genome rearrangements. Transposons are well known to have expanded genomes over time, and to modulate gene expression in various ways. This project has identified Alternative Transposition as a new mechanism by which transposons duplicate and rearrange functional segments, ranging from individual genic elements (enhancers and exons) and whole genes, up to large duplications containing hundreds of genes.One goal of this current project is to determine the number of duplications in the corn genome generated by transposable elements.Another goal is to investigate an unusual duplication mechanism associated with a novel structure termed the Composite Insertion (CI).The CI is a transposable element that duplicates pieces of genes and then inserts them back into the corn genome.Different CIs contain different lengths of genetic sequence, and they are inserted at diverse sites in the genome. CIs can influence the expression of genes near their insertion site.This project will determine the origin and effects of CIs and their role in generating duplications over time. Once the mechanism by which transposable elements induce genome rearrangements is known, it may be possible to use this mechanism to generate diversity; for example, by making duplications of beneficial genes, or by deleting harmful genes. Objective 1... Isolate and analyze alleles derived from alternative transposition and DNA re-replication events in maize, to identify the sizes and breakpoint junctions of chromosome rearrangements and transposon Composite Insertions. In previous work used a well-characterized allele termed P1-ovov454 to study Alternative Transposition in maize. For screening, plants of genotype P1-ovov454/p1-ww[4Co63] are grown in large isolated fields, detasseled, and allowed to pollinate with adjacent plants of genotype p1-ww[4Co63] C r1-m3::Ds.The pollen parent lacks pericarp color, but has genes (C and r1-m3::Ds) that produce purple aleurone sectors in the presence of the Ac element in P1-ovov454. The dosage of Ac can be inferred from the sizes of sectors, due to the Ac negative dosage effect described by McClintock. However, the p1-ovov454 allele is not optimal for detecting CIs because they are not easily distinguished from the background pericarp color. Fortunately, we have identified a new allele that is ideal for detecting chromosome rearrangements induced by RET. The p1-wwB54 allele has a deletion of the first two exons of p1, hence the pericarp is colorless. It has reverse-orientated Ac/fAc insertions in the maize p1 gene, similar to that of p1-ovov454, except that the two ends are separated by only 331 bp; apparently this close proximity facilitates AT reactions, as it exhibits a high frequency of chromosome breakage and produces frequent red pericarp sectors. Nearby the p1 gene is a paralog termed p2; the p2 gene encodes a functionally equivalent protein, but p2 is not expressed in kernel pericarp. Because p1-wwB54 has a deletion of p1 exons 1 and 2, red sectors cannot originate from simple Ac transpositions; only RET events can join the broken p1 gene with the unexpressed (in pericarp) p2 gene to produce a gene expressed in pericarp.By selecting sectors of red kernels on p1-wwB54 ears, we have identified over 20 p2-expressing alleles that contain CI insertions into the p2 promoter or intron 2.We hypothesize that these CI insertions bring the p1 3' enhancer sufficiently close to the p2 promoter to induce p2 expression in kernel pericarp.This class usually has two Ac copies: one at the original site in p1-wwB54, and one in the CI. Objective 2... Clone and sequence Composite Insertions (CIs) to determine the extents of re-replication and the mechanism of DSB repair in each case. From the p1-ovov454 allele we identified 16 CI alleles; these CIs are bounded by Ac and fAc, range in size from ~ 4 kb to over 23 kb, and contain various lengths of DNA segments derived from the sequences flanking the original Ac/fAc donor elements.The CI internal sequences were isolated by PCR of genomic DNA using primers positioned within each CI structure.Most of the internal junctions coincide with small microhomology regions consistent with formation by Non-Homologous End Joining (NHEJ).In some cases, junctions occur within directly-oriented homologous repeat sequences, and appear to be generated by Homologous Recombination (HR). From the p1-wwB54 allele, we identified more than 30 CI alleles and mapped 24 of them to the p2 gene. Among these 24 CIs, 10 of them inserted into p2 promoter region, and 14 inserted into p2 intron 2. All of the CIs contain Ac, fAc, and variable lengths of DNA derived from the original p1 donor site in the p1-wwB54 progenitor; these CIs range in size from 12.8 kb to 23.6 kb. The internal junctions have sequences characteristic of DSB repair by NHEJ (Non Homologous End Joining) without additional insertions (5 cases) or with insertions of up to 50 bp of Filler DNA. Filler DNA sequences were mostly copied from nearby p1 sequence, similar to other reports of Filler DNA in maize. Objective 3... Isolate and compare meiotic and mitotic products of RET and DNA re-replication to learn how the different nuclear cycles affect the processes of alternative transposition, DNA replication fork processivity, and DSB repair. Maize plants expected to generate meiotic and mitotic CIs were grown in an isolation field in Ames in summer 2016.Candidate CI-containing kernels were selected and grown in the summer of 2017.Ears have been harvested and are awaiting further screening and analysis. Objective 4... Test the hypothesis that transposition-induced duplications and DNA re-replication events contributed to maize genetic diversification and genome evolution. Based on the predictions of our models, we have developed computational methods to identify Transposon-Induced Duplications (TIDs) in sequenced genomes. The program termed STRAND (Search for Transposon-Induced Duplications) takes known transposon and genome sequences as input, and outputs a list of transposon-associated duplications and their graphical structures. For maize B73 the program returned 28 genomic duplications, ranging in size from 158 bp to 47 kb, initiated by DNA transposable elements. In further work we used the program DDS2 in a computational pipeline to detect additional TIDs. In preliminary tests, 163 TIDs were detected in maize B73 genome. Work is ongoing to characterize these cases, and to compare with TIDs in other maize inbred lines including W22 and PH207.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Composite Insertions (CIs) and the Evolutionary Impact of Reversed-Ends Transposition (RET) in Maize. Weijia Su and Thomas Peterson. Annual Maize Genetics Meeting, St. Louis Missouri, 2017
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Genome Rearrangements Induced by hAT Transposable Elements: Mechanisms and Genetic Impacts. Thomas Peterson, Tao Zuo, Jianbo Zhang, Weijia Su, Dafang Wang, Sharu Paul Sharma, and David F. Weber. 21st Annual Evolutionary Biology Meeting, Marseilles, France 2017
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Transposon-Induced Genome Rearrangements in Maize. Weijia Su, Sharu Paul Sharma, Dafang Wang, Tao Zuo, Jianbo Zhang, and Thomas Peterson. 2017 Keystone Symposia Conference Z1: Genomic Instability and DNA Repair, Santa Fe, New Mexico, 2017
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Peterson, T. 2017 Maize Transposon Storm Kicks up a White Cap. Genetics 206: 87-89; https://doi.org/10.1534/genetics.116.199638
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Progress 07/01/16 to 09/30/16
Outputs
Target Audience:Researchers and students in the fields of molecular genetics and agronomy, located at academic and industry institutions. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided training for two graduate students in classical, cytogenetic, and molecular genetic approaches. The project also provided laboratory work experience and on-the-job training for three undergraduate students. These educational activities provide training and experience that is otherwise unavailable to young students and future scientists, and will thereby help to enhance the potential for scientific and technological research in the USA. How have the results been disseminated to communities of interest?The results have been disseminated by oral presentation at an international scientific conference, and by creation of a website containing animated models showing how transposable elements induce genome rearrangements. What do you plan to do during the next reporting period to accomplish the goals?Analysis of transposon-induced rearrangements in maize will be continued using both molecular biological approaches, and via computational techniques. These approaches include 1) identification of candidate rearrangement alleles via an efficient visible phenotype screen; 2) PCR-based approaches to molecularly test for rearrangement events; 3) study selected cases in detail by molecular isolation of the rearrangement breakpoints, followed by sequence analysis to infer the mechanism(s) of rearrangement; 4) computational approaches include the development of software to search sequenced genomes for sequences bearing the structural hallmarks of transposon-induced rearrangements.
Impacts
What was accomplished under these goals?
In corn and other crop plants, traits are determined by genes carried in the plant genome, and encoded in the DNA (genotype). Plant breeding involves making crosses among diverse genotypes and selecting for desirable combinations of genes. For this reason, understanding sources of genetic variation is important for modern plant breeding. A major source of genetic variation in crop plants is the presence of genome rearrangements. These rearrangements include large changes in genome structure that affect multiple genes, such as duplications (gains of genetic material). Duplications are very important because the duplicated genes may be over-expressed, leading to important changes in plant phenotypes. Additionally, having multiple gene copies allows one copy to change its expression or function, while one copy maintains the original function. This provides genetic flexibility and is thought to be an important mechanism by which plants can adapt to changing environments over time. Duplications are very common in crop plant genomes, and there are several known mechanisms for generating new duplications. However, in many cases the molecular origin of the duplications is unknown. This project investigates the role of transposable elements, or jumping genes, in generating duplications. Transposons are well known to have expanded genomes over time, and to modulate gene expression in various ways. This project has identified Alternative Transposition as a new mechanism by which transposons duplicate and rearrange functional segments, ranging from individual genic elements (enhancers and exons) and whole genes, up to large duplications containing hundreds of genes. One goal of this current project is to determine the number of duplications in the corn genome generated by transposable elements. Another goal is to investigate an unusual duplication mechanism associated with a novel structure termed the Composite Insertion (CI). The CI is a transposable element that duplicates pieces of genes and then inserts them back into the corn genome. Different CIs contain different lengths of genetic sequence, and they are inserted at diverse sites in the genome. CIs can influence the expression of genes near their insertion site. This project will determine the origin and effects of CIs and their role in generating duplications over time. This hypothesis-driven research takes advantage of an active transposable element system coupled with visual markers to enable extremely efficient screening and selection of genome rearrangements. The generality of results from experimental materials will be tested by bioinformatic searches of sequenced genomes for structures with the characteristic features of these events. The results could have a major impact on methods of plant breeding. Once the mechanism by which transposable elements induce genome rearrangements is known, it may be possible to use this mechanism to generate diversity; for example, by making duplications of beneficial genes, or by deleting harmful genes. Objective 1... Isolate and analyze alleles derived from alternative transposition and DNA re-replication events in maize, to identify the sizes and breakpoint junctions of chromosome rearrangements and transposon Composite Insertions. We have used a well-characterized allele termed P1-ovov454 to study Alternative Transposition in maize. This allele contains Ac and fAc inserted into intron 2 of the p1 gene; the p1 gene is expressed at a moderate level to give dark orange kernel pericarp. The Ac and fAc elements can undergo various transposition events to generate gains or losses of p1 function, leading to red/white variegation on the orange background. The alternative transposition frequency in P1-ovov454 is very high (>0.5%), presumably due to the short distance (823 bp) between Ac and fAc in this allele. For screening, plants of genotype P1-ovov454/p1-ww[4Co63] are grown in large isolated fields, detasseled, and allowed to pollinate with adjacent plants of genotype p1-ww[4Co63] C r1-m3::Ds. The pollen parent lacks pericarp color, but has genes (C and r1-m3::Ds) that produce purple aleurone sectors in the presence of the Ac element in P1-ovov454. The dosage of Ac can be inferred from the sizes of sectors, due to the Ac negative dosage effect described by McClintock. Objective 2... Clone and sequence Composite Insertions (CIs) to determine the extents of re-replication and the mechanism of DSB repair in each case. We identified 16 CIs by screening p1-ovov454 for twinned sectors and whole ear mutants with altered Ac dosage. The CIs are bounded by Ac and fAc, range in size from ~ 4 kb to over 23 kb, and contain various lengths of DNA segments derived from the sequences flanking the original Ac/fAc donor elements. The CI internal sequences were isolated by PCR of genomic DNA using primers positioned within each CI structure. Most of the internal junctions coincide with small microhomology regions consistent with formation by Non-Homologous End Joining (NHEJ). In some cases, junctions occur within directly-oriented homologous repeat sequences, and appear to be generated by Homologous Recombination (HR). Objective 3... Isolate and compare meiotic and mitotic products of RET and DNA re-replication to learn how the different nuclear cycles affect the processes of alternative transposition, DNA replication fork processivity, and DSB repair. Maize plants expected to generate meiotic and mitotic CIs were grown in an isolation field in Ames in summer 2016. Ears of the expected genotypes and phenotypes were produced and are currently in storage. The ears will be screened to identify and pick the individual kernels containing new CIs. These will be planted and grown in the summer of 2017. Plants that appear to contain novel CIs will be identified by the phenotypes of the kernels they produce. These will be then screened using molecular methods to determine the structures of the individual CIs. Objective 4... Test the hypothesis that transposition-induced duplications and DNA re-replication events contributed to maize genetic diversification and genome evolution. Based on the predictions of our models, we developed a protocol to identify Transposon-Induced Duplications (TIDs) in sequenced genomes. First, DNA TE sequences were used as queries to BLAST with maize B73 reference genome to identify TE insertion sites via local BLASTN with default parameters. Second, the TE flanking sequences were extracted to identify loci containing two or more TE copies in the same orientation within a 100 kbp interval, as well as homologous segments immediately flanking one side of the TEs. These cases were manually inspected to determine whether they possess the other features of TIDs, such as the presence of flanking TSDs (Target Site Duplications generated by TE insertion). This protocol was successfully applied to identify four TIDs in the maize B73 reference genome sequence. We automated this approach to conduct more extensive and systematic searches of genomic sequence data. The program termed STRAND (Search for Transposon-Induced Duplications) takes known transposon and genome sequences as input, and outputs a list of transposon-associated duplications and their graphical structures. For maize B73 the program returned 28 genomic duplications, ranging in size from 158 bp to 47 kb, initiated by DNA transposable elements. The frequency of Transposon-Induced Duplications returned by the program may appear low, considering the large number of duplications present in higher plants. However, it is important to consider that we are currently using very stringent search parameters, requiring highly-homologous duplications, perfect TSDs, etc.; i.e. the program detects only recent duplications that have maintained a pristine structure. Older duplications would likely erode quickly by deletion as observed in plant genomes.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Thomas Peterson, Tao Zuo, Jianbo Zhang, Weijia Su, Dafang Wang, Sharu Paul Sharma. Transposon-induced genome rearrangements in maize: mechanisms and genetic impacts. The Allied Genetics Conference, July 13-17, 2016. Orlando, Florida USA.
- Type:
Websites
Status:
Other
Year Published:
2016
Citation:
http://thomasp.public.iastate.edu/transposition.html
Animations of Alternative Ac/Ds Transposition and Chromosomal Rearrangements in Maize
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