Progress 12/15/08 to 12/14/11
Outputs
OUTPUTS: Objective 1: "sequencing the Eps-Am1 region." We sequenced and annotated five Triticum monococcum BAC clones covering the Eps-Am1 region. In addition, we sequenced and annotated three Aegilops tauschii BAC clones and the Brachypodium distachyon BAC clone encompassing the Eps-Am1 orthologous regions (GenBank, EU358773.2, EU358770.2, and EU358772.2). A major outcome of this objective was the identification of two candidate genes completely linked to the Eps-Am1 locus: FtsH4 & Mot1. Comparison of the early and late flowering genotypes showed the presence of two amino acid changes in Mot1 and no amino acid polymorphisms for FtsH4, suggesting that Mot1 is the strongest candidate for Eps-Am1. Mot1 is a global transcriptional regulator and changes in this gene are expected to have numerous pleiotropic effects, as observed in the Eps-Am1 isogenic lines. Objective 2: "characterization of the candidate genes." A semi-quantitative PCR experiment using SYBR Green systems showed that FtsH4 and Mot1 are transcribed in apices, crowns, leaves, and spikes, but not in roots. In addition, a quantitative RT-PCR study showed that only Mot1 transcript levels change during spike development. We completed the cloning of the wheat Mot1 gene to test its ability to complement mot1 mutants in yeast. Objective 3: "validate the candidate gene." To study the effect of Mot1 in commercial varieties of polyploid wheat we sequenced the hexaploid wheat Mot-A1, Mot-B1 and Mot-D1 copies. In tetraploid wheat, we identified truncation and amino acid mutants in Mot-A1 and Mot-B1. These mutations were backcrossed to the non-mutagenized parent to reduce the background mutations and now the A and B genome mutations are being combined to test their effect on flowering time and number of spikelets per spike. Objective 4: "introgress the Eps-Am1 allele into commercial wheat varieties." The outputs of this objective included hexaploid and tetraploid wheat lines with the Eps-Am1 allele introgressed by five backcrosses. We evaluated BC5F2 plants with and without the Eps-Am1 allele and found a small but significant increase in spikelet number and a delay in flowering time in the lines with the T. monococcum allele. We have now initiated the combination of the Eps-Am1 allele introgression with the Mot-B1 TILLING mutations to test if the effect of the T. monococcum allele increases in the absence of all the Eps1 alleles from cultivated wheat. In summary, the main objective of this project has been achieved by identifying an excellent candidate gene for Eps-Am1 and by transferring the useful allele from wild wheat to pasta and bread wheat. Results from these studies have been disseminated in two peer reviewed articles (J. Exp. Bot., 59: 3595-3607; Functional & Integrative Genomics. 10:293-306) and six presentations in national and international scientific meetings. PARTICIPANTS: Ph.D. student Maria E. Faricelli completed her doctorate thesis at UC Davis working on this project. The training received during this project was essential for her new position in Pioneer. A new graduate student, Alejandra Alvarez is now in charge of the project that will be the central theme of her PhD thesis. Two undergraduate students (Minh Phuc Nguyen and Thanhtoan Thai) were trained as part of this project. Collaborations in different aspects of the project have been established with Dr. Miroslav Valarik at the Laboratory of Molecular Cytogenetics and Cytometry, in the Czech Republic and Dr. Silvina Lewis from the Institute of Biological Resources, INTA in Argentina. BAC clone sequencing was done by Phillip San Miguel at the Genomics Center, Purdue University. TARGET AUDIENCES: One important target audience for this project are the wheat breeders. In most environments, there is a narrow window for optimum flowering time that will maximize wheat grain yield potential. The Eps genes have an indirect effect on yield potential by fine tuning wheat flowering time to this narrow window. In addition to this indirect effect, our results have shown that the Eps1 gene has a more direct impact on yield by affecting spike development and the number of grains in the spike. Therefore, the understanding of the regulation of this gene can contribute to a better understanding of wheat grain yield potential. An additional target audience of this project is the scientific community working in the understanding of the regulation of flowering in plants. The cloning of the first Eps gene in wheat and the new allelic variants generated by the TILLING mutant lines are expanding our understanding of the regulation of flowering regulation and its complex interactions with temperature. Finally, this project is of interest to scientists studying plant domestication, since the selection of the mutated Eps-Am1-l allele in the cultivated T. monococcum resulted in a 30% increase in the spikelet number in the cultivated forms relative to the wild type ancestors. This trait seems to be part of the domestication syndrome and may have contributed to the high productivity of the cultivated accessions of T. monococcum. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Variation in heading time among fully vernalized wheat plants grown under inductive photoperiod indicates that there are intrinsic differences in the rate of development between wheat varieties. We completed the genetic and physical maps of the Eps-Am1 region in T. monococcum, sequenced the complete region between flanking markers, and identified two candidate genes (FtsH4 & Mot1). A detailed comparative genomics study of the Eps-Am1 region in T. monococcum, Aegilops tauschii, Brachypodium, and rice was published. Among the two candidate genes only Mot1showed differences in the transcription profiles in the shoot apical meristems between the early and late spike development stages. Mot1 also showed differences in the coding region, which resulted in two amino acid changes in the cultivated parent. These amino acid differences together with the known function of this gene as a TATA-binding protein-associated factor that regulates genes involved in the organization of the shoot apical meristem in Arabidopsis, indicate that Mot1 is a strong candidate for the Eps-Am1 gene. We showed that in T. monococcum the Eps-Am1 gene affects not only flowering time but also the duration of spike development and the number of spikelets per spike, an important component of grain yield. Therefore, the characterization of the Eps-Am1 gene can contribute to our understanding of the regulation of an important grain yield component in wheat. In addition, we confirmed that the transfer of the Eps-Am1 allele to tetraploid wheat results in a small but significant increase in spikelet number under controlled growth conditions in field experiments. We hypothesize that the smaller effect of the Eps-Am1 allele in tetraploid than in diploid wheat is due to the presence of the Eps-B1 allele in the first one. To test this hypothesis we are combining the Eps-Am1 allele with TILLING mutants of Mot-B1 in a tetraploid genetic background. The allelic variation in wheat Mot1 genes was further increased by using our tetraploid and hexaploid wheat mutant populations. The Mot1 mutant lines are expected to generate new allelic variation in flowering time and spike development in polyploid wheat, broadening wheat breeder's options to modify these traits. Since genes involved in the regulation of flowering time in wheat are likely to be similar to those in barley and related Triticeae grasses, it is likely that the knowledge gained in wheat will be easy to transfer to other important agronomic species, as happened before with the vernalization genes. In addition to its practical applications, the understanding of the mechanisms, interactions, and allelic variation in the Eps-Am1 gene will also contribute to our knowledge of the basic mechanism of the regulation of flowering time by temperature in the temperate cereals.
Publications
- MA Alvarez, ME Faricelli, S Lewis, G Tranquilli, ML Appendino, and J. Dubcovsky. 2012. Eps-Am1, a locus regulating reproductive development in Triticum monococcum, has been delimited to a 50 kb region including two genes. Plant & Animal Genome XX Conference, January 14-18, 2012, San Diego, CA.
- Publications (total from the grant 2) Faricelli M.E., M. Valarik, and J. Dubcovsky. 2010. Control of flowering time and spike development in cereals: the earliness per se Eps-Am1 region in wheat, rice, and Brachypodium. Functional and Integrative Genomics. 10:293-306.
- Presentations (total from the grant 6) MA Alvarez, ME Faricelli, S Lewis, ML Appendino, M. Valarik and J. Dubcovsky. 2011. Physical map and candidate gene identification for the Triticum monococcum earliness per se locus EpsAm1. Plant & Animal Genome XIX Conference, January 15-19, 2011, San Diego, CA.
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Progress 12/15/09 to 12/14/10
Outputs
OUTPUTS: Objective 1: "sequencing the Eps-Am1 region." The output of this objective was the sequencing and complete annotation of the five Triticum monococcum BAC clones covering the Eps-Am1 region from the cultivated accession DV92. In addition, we sequenced and annotated three Aegilops tauschii BAC clones and the Brachypodium distachyon BAC clone encompassing the Eps-Am1 orthologous regions (sequences were deposited in GenBank, EU358773.2, EU358770.2, and EU358772.2). A major outcome of this objective was the identification of three candidate genes completely linked to the Eps-Am1 locus in the T. monococcum high density map. The three Eps-Am1 candidate genes, FtsH4, Mot1, and Fop1, were also sequenced in the other parental line of the mapping population (wild accession G3116) and were deposited in GenBank (accession numbers GQ422597.1, GQ422598, EU379332.2, and EU358774.2). Mot1 showed differences in the coding sequences resulting in two amino acid changes in the cultivated accession relative to the wild one. Based on these mutations and the known function of the gene, Mot1 is considered the strongest candidate for Eps-Am1. Objective 2: "characterization of the candidate genes." The output of this objective was the development of SYBR Green systems for the three candidate genes and the characterization of their transcription profiles. A semi-quantitative PCR experiment showed that FtsH4, Mot1, and Fop1, are transcribed in apices, crowns, leaves, and spikes, but not in roots. In addition, a quantitative RT-PCR study showed that only Mot1 transcript levels changed during spike development. We also completed the cloning of the wheat Mot1 gene to test its ability to complement mot1 mutants in yeast. Objective 3: "validate and characterize the candidate gene." An important output of this objective was the sequencing of the hexaploid wheat Mot-A1, Mot-B1 and Mot-D1 copies. In addition, we generated Mot-A1 and Mot-B1 specific primers that were used to screen our tetraploid wheat mutant populations by TILLING. Mutations in Mot-A1 and Mot-B1 with the highest probability of altered protein structure and function were selected for phenotypic evaluation and are being backcrossed to the non-mutagenized parent to reduce the background mutations. The two mutations will be combined to generate a null-Mot1 and test its effect on flowering time and number of spikelets per spike. Objective 4: "introgress the Eps-Am1 allele into bread wheat." The outputs of this objective included hexaploid and tetraploid wheat lines with the Eps-Am1 allele introgressed by five backcrosses. We evaluated BC5F2 plants with and without the Eps-Am1 allele and found a small but significant increase in spikelet number and a delay in flowering time in the lines with the T. monococcum allele. We have now initiated the combination of the Eps-Am1 allele introgression with the Mot-B1 TILLING mutations to test if the effect of the wild type alleles increases in the absence of all the Eps1 alleles from cultivated wheat. Results from these studies have been disseminated in one peer reviewed article in 2010 and two presentation in scientific meetings. PARTICIPANTS: Ph.D. student Maria E. Faricelli completed her doctorate thesis at UC Davis working on this project. The training received during this project was essential for her new position in Pioneer. A new graduate student, Alejandra Alvarez is now in charge of the project that will be the central theme of her PhD thesis. Two undergraduate students (Minh Phuc Nguyen and Thanhtoan Thai) were trained as part of this project. Collaborations in different aspects of the project have been established with Dr. Miroslav Valarik at the Laboratory of Molecular Cytogenetics and Cytometry, in the Czech Republic and Dr. Silvina Lewis from the Institute of Biological Resources, INTA in Argentina. BAC clone sequencing was done by Phillip San Miguel at the Genomics Center, Purdue University. TARGET AUDIENCES: One important target audience for this project are the wheat breeders. In most environments, there is a narrow window for optimum flowering time that will maximize wheat grain yield potential. The Eps genes have an indirect effect on yield potential by fine tuning wheat flowering time to this narrow window. In addition to this indirect effect, our results have shown that the Eps1 gene has a more direct impact on yield by affecting spike development and the number of grains in the spike. Therefore, the understanding of the regulation of this gene can contribute to a better understanding of wheat grain yield potential. An additional target audience of this project is the scientific community working in the understanding of the regulation of flowering in plants. The cloning of the first Eps gene in wheat and the new allelic variants generated by the TILLING mutant lines are expanding our understanding of the regulation of flowering regulation and its complex interactions with temperature. Finally, this project might be of interest to scientists studying plant domestication, since the selection of the mutated Eps-Am1-l allele in the cultivated T. monococcum resulted in a 30% increase in the spikelet number in the cultivated forms relative to the wild type ancestors. This trait seems to be part of the domestication syndrome and may have contributed to the high productivity of the cultivated accessions of T. monococcum. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Variation in heading time among fully vernalized wheat plants grown under inductive photoperiod indicates that there are intrinsic differences in the rate of development between wheat varieties. We completed the genetic and physical maps of the Eps-Am1 region in T. monococcum, sequenced the complete region between flanking markers, and identified three candidate genes (FtsH4, Mot1, and Fop1). No additional genes are present in this region in the related species Aegilops tauschii. A detailed comparative genomics study of the Eps-Am1 region in T. monococcum, Aegilops tauschii, Brachypodium, and rice was published in 2010. Among the three candidate genes only Mot1showed differences in the transcription profiles in the shoot apical meristems between the early and late spike development stages. Mot1 also showed differences in the coding region, which resulted in two amino acid changes in the cultivated parent. These amino acid differences together with the known function of this gene as a TATA-binding protein-associated factor that regulates genes involved in the organization of the shoot apical meristem in Arabidopsis, suggest that Mot1 is a strong candidate for the Eps-Am1 gene. We showed that in T. monococcum the Eps-Am1 gene affects not only flowering time but also the duration of spike development and the number of spikelets per spike, an important component of grain yield. Therefore, the characterization of the Eps-Am1 gene can contribute to our understanding of the regulation of an important grain yield component in wheat. In addition, we confirmed that the transfer of the Eps-Am1 allele to tetraploid wheat results in a small increase in spikelet number under controlled growth conditions. Field experiments were initiated this year. We hypothesize that the smaller effect of the Eps-Am1 allele in tetraploid than in diploid wheat is due to the presence of the Eps-B1 allele in the first one. To test this hypothesis we initiated the combination of the Eps-Am1 allele and the eps-B1 TILLING mutant in a tetraploid genetic background. The allelic variation in wheat Mot1 genes was further increased by using our tetraploid and hexaploid wheat mutant populations. The Mot1 mutant lines are expected to generate new allelic variation in flowering time and spike development in polyploid wheat, broadening wheat breeder's options to modify these traits. Since genes involved in the regulation of flowering time in wheat are likely to be similar to those in barley and related Triticeae grasses, it is likely that the knowledge gained in wheat will be easy to transfer to other important agronomic species, as happened before with the vernalization genes. In addition to its practical applications, the understanding of the mechanisms, interactions, and allelic variation in the Eps-Am1 gene will also contribute to our knowledge of the basic mechanism of the regulation of flowering time by temperature in the temperate cereals.
Publications
- Faricelli M.E., M. Valarik, and J. Dubcovsky. 2010. Control of flowering time and spike development in cereals: the earliness per se Eps-Am1 region in wheat, rice, and Brachypodium. Functional and Integrative Genomics. 10:293-306.
- Dubcovsky, J. 2011. Positional cloning of the Earliness Per Se 1 QTL in diploid wheat. Plant and Animal Genome XIX Conference, "QTL cloning" workshop, January 15-19, 2011, San Diego, CA.
- Faricelli M.E., S. Lewis, M.L. Appendino, M. Valarik, and J. Dubcovsky. 2009. The chromosome region including the earliness per se locus Eps-Am1 affects the duration of early developmental phases and spikelet number in diploid wheat. Plant and Animal Genome XVII Conference, January 2009, San Diego, CA.
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Progress 12/15/08 to 12/14/09
Outputs
OUTPUTS: Objective 1: "sequencing the Eps-Am1 region." The output of this objective was the sequencing and complete annotation of the five Triticum monococcum BAC clones covering the Eps-Am1 region from the cultivated accession DV92. In addition, we sequenced and annotated three Aegilops tauschii BAC clones and the Brachypodium distachyon BAC clone encompassing the Eps-Am1 orthologous regions (sequences were deposited in GenBank, EU358773.2, EU358770.2, and EU358772.2). A major outcome of this objective was the identification of three candidate genes completely linked to the Eps-Am1 locus in the T. monococcum high density map. The three Eps-Am1 candidate genes, FtsH4, Mot1, and Fop1, were also sequenced in the other parental line of the mapping population (wild accession G3116) and were deposited in GenBank (accession numbers GQ422597.1, GQ422598, EU379332.2, and EU358774.2). Only the Mot1 gene showed differences in the coding sequences resulting in two amino acid changes in the cultivated accession relative to the wild one. Based on these mutations and the known functions of these three genes, Mot1 is considered the strongest candidate for Eps-Am1. Objective 2: "characterization of the candidate genes." The output of this objective was the development of SYBR Green systems for the three candidate genes and the characterization of their transcription profiles. A semi-quantitative PCR experiment showed that FtsH4, Mot1, and Fop1, are transcribed in apices, crowns, leaves, and spikes, but not in roots. In addition, a quantitative RT-PCR study showed non-significant differences in the transcript levels of these three genes in the shoot apical regions between near isogenic lines carrying either the DV92 or the G3116 Eps-Am1 alleles. Objective 3: "validate and characterize the candidate gene." An important output of this objective was the sequencing of the hexaploid wheat complete Mot-A1 and Mot-D1 copies, and the discovery that the Mot-B1 copy is deleted in the polyploid wheat species. In addition, we generated Mot-A1 and Mot-D1 specific primers that were used to screen our wheat mutant populations by TILLING. We found 38 mutations in the Mot1 exons, 20 of which induced amino acid changes (13 for tetraploid Mot-A1, 5 for hexaploid Mot-A1, and 2 for Mot-D1). Mutations with the highest probability of altered protein structure and function were selected for phenotypic evaluation and are being backcrossed to the non-mutagenized parent to reduce the background mutations. Objective 4: "introgress the Eps-Am1 allele into bread wheat." The outputs of this objective included hexaploid and tetraploid wheat lines with the Eps-Am1 allele introgressed by five backcrosses. These plants are being grown in a greenhouse to obtain BC5F2 seeds, which will be evaluated for differences in heading time, duration of spike development, tiller number, spikelet number per spike, and yield potential. Results from these studies have been disseminated in two peer reviewed articles and one presentation in a scientific meeting. PARTICIPANTS: This project is currently led by the Ph.D. student Maria E. Faricelli, and is part of her doctorate thesis at UC Davis. Two undergraduate students (Minh Phuc Nguyen and Thanhtoan Thai) were trained as part of this project. Collaborations in different aspects of the project have been established with Dr. Miroslav Valarik at the Laboratory of Molecular Cytogenetics and Cytometry, in the Czech Republic and Dr. Silvina Lewis from the Institute of Biological Resources, INTA in Argentina. BAC clone sequencing was done by Phillip San Miguel at the Genomics Center, Purdue University. TARGET AUDIENCES: One important target audience for this project are the wheat breeders. In most environments, there is a narrow window for optimum flowering time that will maximize wheat grain yield potential. The Eps genes have an indirect effect on yield potential by fine tuning wheat flowering time to this narrow window. In addition to this indirect effect, our results have shown that the Eps1 gene has a more direct impact on yield by affecting spike development and the number of grains in the spike. Therefore, the understanding of the regulation of this gene can contribute to a better understanding of wheat grain yield potential. An additional target audience of this project is the scientific community working in the understanding of the regulation of flowering in plants. The cloning of the first Eps gene in wheat and the new allelic variants generated by the TILLING mutant lines are expanding our understanding of the regulation of flowering regulation and its complex interactions with temperature. Finally, this project might be of interest to scientists studying plant domestication, since the selection of the mutated Eps-Am1-l allele in the cultivated T. monococcum resulted in a 30% increase in the spikelet number in the cultivated forms relative to the wild type ancestors. This trait seems to be part of the domestication syndrome and may have contributed to the high productivity of the cultivated accessions of T. monococcum. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Variation in heading time among fully vernalized wheat plants grown under inductive photoperiod indicates that there are intrinsic differences in the rate of development between wheat varieties. During the first year of this project we have completed the genetic and physical maps of the Eps-Am1 region in T. monococcum, and sequenced the complete region between flanking markers. We identified three candidate genes (FtsH4, Mot1, and Fop1) and confirmed that no additional genes are present in this region in the related species Aegilops tauschii. A detail comparative genomics study of the Eps-Am1 region in T. monococcum, Aegilops tauschii, Brachypodium, and rice was published during this first year. The three candidate genes showed no differences in the transcription profiles in the shoot apical meristems between the early and late flowering parental lines. However, only Mot1 showed differences in the coding region, which resulted in two amino acid changes in the cultivated parent. These amino acid differences together with the known function of this gene as a TATA-binding protein-associated factor that regulates genes involved in the organization of the shoot apical meristem in Arabidopsis, suggest that Mot1 is a strong candidate for the Eps-Am1 gene. Based on the previous results we initiated the characterization of the Mot1 gene in polyploid wheat and discovered that the B genome copy of Mot1 is deleted both in the cultivated tetraploid and hexaploid germplasm as well as in the wild tetraploid wheats. This suggests that the B copy of Mot1 was eliminated early in the evolution of the tetraploid wheats. During this first year we showed that in T. monococcum the Eps-Am1 gene affects not only flowering time but also the duration of spike development and the number of spikelets per spike, an important component of grain yield (see publication list). Therefore, the characterization of the Eps-Am1 gene can contribute to our understanding of the regulation of an important grain yield component in wheat. In addition, the transfer of the Eps-Am1 allele for increased spikelet number from cultivated diploid wheat into common wheat will incorporate new allelic diversity in this important gene into cultivated wheat. The allelic variation in wheat Mot1 genes was further increased by using our tetraploid and hexaploid wheat mutant populations. The Mot1 mutant lines are expected to generate new allelic variation in flowering time and spike development in polyploid wheat, broadening wheat breeder's options to modify these traits. Since genes involved in the regulation of flowering time in wheat are likely to be similar to those in barley and related Triticeae grasses, it is likely that the knowledge gained in wheat will be easy to transfer to other important agronomic species, as happened before with the vernalization genes. In addition to its practical applications, the understanding of the mechanisms, interactions, and allelic variation in the Eps-Am1 gene will also contribute to our knowledge of the basic mechanism of the regulation of flowering time by temperature in the temperate cereals.
Publications
- Lewis, S., Faricelli, M.E., Appendino, M.L., Valarik, M., Dubcovsky, J. (2008). The chromosome region including the earliness per se locus Eps-Am1 affects the duration of early developmental phases and spikelet number in diploid wheat. Journal of Experimental Botany, 59: 3595-3607.
- Faricelli, M.E., Valarik, M., Dubcovsky, J. (2009). Control of flowering time and spike development in cereals: the earliness per se Eps-1 region in wheat, rice, and Brachypodium. Functional and Integrative Genomics, in press DOI 10.1007/s10142-009-0146-7.
- Presentations Faricelli, M.E., M.L., Valarik, Lewis, S., Appendino, M., Dubcovsky, J. (2008). Physical map of the Eps-Am1 gene region in Triticum monococcum L. The 11th International Wheat Genetics Symposium proceedings, edited by R. Appels, R. Eastwood, E. Lagudah, P. Langridge, M. Mackay Lynne, http://hdl.handle.net/2123/3333
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