Progress 01/01/15 to 12/31/16
Outputs
Target Audience:Target audiences of graduate and undergraduate students in plant breeding and genetics were served by instructional activities in formal educational programs. Two lectures and one laboratory instruction were created and delivered in graduate Biometrical Genetics. A lecture was delivered to both resident and online undergraduate and graduate students in Germplasm and Genes. Online instructional activities were developed for Breeding Self-Pollinated Crops and Germplasm and Genes utilizing USDA GRIN and ERS databases and a NIFA-supported database. A laboratory module on multiple-trait selection was developed for Biometrical Genetics using grain mineral concentration and protein data from the PI's research. Practicum experience was provided to a group of three graduate students in analysis of next-generation sequencing data using high performance computing resources. Graduate and undergraduate student target audiences also were served by mentoring activities. An undergraduate student developed a funded grant proposal for an undergraduate research project. Mentoring activities included support on experimental design and analysis, greenhouse propagation of wheat, and molecular biology approaches for functional genomics. High school students were served by an experiential learning opportunity at the University of Nebraska Youth World Food Prize. Activities were centered around the improvement of wheat nutritional and manufacturing quality through plant breeding. A high school student was served through mentoring in the Young Nebraska Scientist project (EPSCoR), providing laboratory experience in analysis of mineral composition of wheat grain. Peer scientists were reached by seminars and poster presentations during the reporting period. Changes/Problems:PI transfered positions (Research Geneticist, Plants, USDA-ARS HWWGRU, Manhattan, KS)Dec 13, 2015. What opportunities for training and professional development has the project provided?Software Carpentry Workshop: Python, Bash, and Git. University of Nebraska Holland Computing Center, Omaha, NE. April 22-23, 2015 Workshop: Innovation in Pedagogy and Technology Symposium. University of Nebraska Online Worldwide. May 14, 2015. Training: Adobe Connect - Creating and Editing Recordings. University of Nebraska, Lincoln. June 29, 2015. Participation in American Society of Agronomy/Crop Science Society of America/Soil Science Society of America annual meeting. Minneapolis, MN. Nov. 15-18, 2015. How have the results been disseminated to communities of interest?Seminars (3) "Breeding Wheat for Improved Mineral Nutrition and Food Safety." University of Minnesota Department of Agronomy and Plant Genetics. May 4, 2015. "Zinc and Cadmium in Great Plains Hard Winter Wheat." USDA-ARS and Kansas State University Department of Agronomy. August 18, 2015. "Toward a more healthful wheat crop: Zinc and cadmium in Great Plains hard winter wheat." University of Nebraska Lincoln, Department of Agronomy and Horticulture. November 13, 2015. Poster Presentations (2) "Cadmium and Zinc in Bread Wheat" NIFA Fellows Program. Project Director's Meeting. Washington, DC. August 4, 2015. "Distribution of Cadmium, Iron, and Zinc in Millstreams of Hard Winter Wheat (Triticum aestivum L.)" 2015 ASA, CSSA, and SSSA Annual Meeting. Minneapolis, MN. November 17, 2015. Outreach Activities Youth World Food Prize, University of Nebraska Lincoln. 31 high school students plus parents and teachers. Sept. 11, 2015. Immersion experience in cereal science/wheat breeding with a focus on mineral nutrition quality. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Winter wheat varieties grown in the Great Plains of the United States can accumulate cadmium, a toxic heavy metal, in wheat grain in concentrations that exceed international standards for food safety. This research aims to explain why some wheat varieties accumulate less cadmium than others so that safe, low-cadmium wheat varieties can be developed. We learned that winter wheat varieties that accumulate high concentrations of cadmium in grain also keep less of the cadmium that they absorb stored in their roots and move more of the cadmium that they absorb into their shoots, compared to a winter wheat variety that does not accumulate high concentrations of cadmium in grain. We also found minor variation among winter wheats in a gene that has been shown to differentiate low and high-cadmium durum (pasta) wheat varieties. We can use this variation to track this gene in wheat breeding programs. Objective 1 (Research Goal): In Year 1 of the project, the specific objective was to use RNA sequencing (RNA-seq) and selected diverse genotypes to identify root genes involved in differential accumulation of cadmium (Cd) in winter wheat grain. The major activities completed / experiments conducted were hydroponic experiments with three winter wheat genotypes (Panhandle, TAM111, OK1068026) that vary for grain Cd accumulation (low, high, very high), characterization of transcriptional response to Cd, and sequencing of a candidate gene, Heavy Metal Associated3_A (HMA3_A). Data collected included root and shoot dry weights and tissue cadmium concentrations after treatment at varying media pH, Cd concentration, and time. RNA was collected from roots of plants of three winter wheat genotypes (OK1068026, TAM 111, and Panhandle) grown with and without 1 uM Cd. Differentially expressed genes were identified by sequencing this RNA using next-generation sequencing technology, then counting the sequencing reads that aligned to predicted genes in the International Wheat Genome Consortium reference sequence. Expression of specific genes also was measured by qRTPCR. Summary statistics/discussion of results. Low and high grain-Cd varieties differed in how Cd was partitioned between shoots and roots in hydroponically grown seedings. The low grain-Cd variety consistently kept a greater proportion of Cd in roots. For example, after 28 days treatment with 1 uM Cd, 62% of the Cd in a high-Cd variety was found in shoots, but only 35% of the Cd in the low-Cd variety was found in shoots. The sequencing project produced over 335 million raw reads. An average of 76% of reads aligned to the IWGSC reference genome. Cadmium treatment decreased abundance of 551 transcripts and increased abundance of 326 transcripts in one or more of the genotypes. The low-Cd genotype, Panhandle, had the fewest Cd-responsive transcripts, and the high-Cd genotype, OK1068026, had the most Cd-responsive transcripts. Of the 326 upregulated transcripts, 170 had gene ontology (GO) annotations, and 22 of these terms were significantly enriched. Among these were a set of 38 transcripts associated with metal ion binding. Of the 551 downregulated transcripts, 328 had GO annotations, and 94 of the GO terms were significantly enriched. The most significant enrichment of GO terms was related to nicotianamine biosynthetic processes. We identified 134 transcripts that responded differently to Cd treatment in the low-Cd variety, Panhandle, than in the highest- Cd genotype, OK1068026. Of these 134 transcripts that were differentially expressed, 105 had GO annotations and 28 of the GO terms were significantly enriched. Enriched transcripts were particularly associated with the biological process of transmembrane transport and with heme binding and nutrient reservoir activity. The transcripts associated with nutrient reservoir activity often were associated with manganese binding. Transcript levels of specific candidate genes for differential Cd accumulation in winter wheat, based on work in durum wheat and barley, were analyzed by RT-PCR. Expression of transcripts for Heavy Metal Associated3 (HMA3) and Phytochelatin Synthase (PCS), which were expected to be associated with differential Cd response because they are associated with sequestration, was not significantly different among the wheat genotypes. Nor was expression of these transcripts affected by Cd treatment. Genomic sequence of HMA3_A from the three wheat genotypes identified a 3 bp deletion in the third intron in the sequences of the high-Cd wheats OK1068026 and TAM111, relative to Panhandle. HMA3_A is a candidate gene based both on its location on chromosome 5A, the site of a quantitative trait locus for grain Cd in winter wheat, and based on the effect of HMA3 mutants on Cd accumulation in other crops including durum wheat and rice. PCR primers were designed to detect the deletion, and their utility was demonstrated in progeny of a cross of a high-Cd wheat, 'Wesley,' with Panhandle. Key outcomes. A low-volume hydroponic protocol was developed to measure uptake and translocation of Cd by wheat seedlings. A key change in knowledge was that a low grain-Cd wheat genotype consistently accumulated less Cd in shoots and more Cd in roots than high grain-Cd wheat genotypes, although total Cd accumulation was not significantly different. A simple PCR marker to detect polymorphism in HMA3_A alleles of winter wheat genotypes was developed to track HMA3_A alleles in segregating populations. Skills in analyzing genome-wide RNA sequence information were developed, a skills in utilizing databases of wheat genome information were substantially improved. Objective 2 (grant writing skills) was to construct a follow-up grant proposal that will be submitted to a funding agency. A proposal, "Decreasing Cadmium and Increasing Zinc in Winter Wheat," was submitted to AFRI Plant Growth and Development, Composition and Stress Tolerance Program (Program Code A1101, proposal 2015-06559). The proposal received high priority for funding, but was not funded. Synthesis Comments of Reviews: This is a well-written proposal that addresses an important nutrient uptake issue in wheat, and will likely have successful outcomes in identifying molecular markers associated with reduced cadmium accumulation. The proposal could be improved by evaluating resulting lowcadmium lines for pleiotropic effects in important agronomic traits. Key learning experiences: complex and time-consuming process of assembly of a full-scale proposal, helpful reviews for future submission. Objective 3 (teaching competencies) is to develop a teaching portfolio, develop mentoring relationships with undergraduate and graduate students, and prepare and provide guest lectures in undergraduate and graduate classes. Major activities completed: New mentoring relationships were developed with two graduate, one undergraduate student (leading to development of a funded undergraduate research grant and a graduate thesis project) and a high school student. Lectures were delivered to graduate and undergraduate students in two classes (Biometrical Genetics, Germplasm and Genes) and learning activities were developed for both resident and online instruction, including an instructional video. Data collected: student evaluations were collected following the two lectures and the laboratory in Biometrical Genetics. Lecture in Germplasm and Genes was recorded using Adobe Connect for subsequent evaluation. Course evaluations were collected following Breeding Self Pollinated Crops and Germplasm and Genes. Teaching reviews were highly encouraging in Biometrical Genetics. Key outcomes: improved skills in instructional design and implementation, particularly for courses that integrate resident and distance education; improved skills with learning management software systems; improved skills for video recording short instructions and full lectures.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Guttieri, Mary; Seabourn, Bradford; Liu, Caixia; Baenziger, P.; Waters, Brian M. Distribution of Cadmium, Iron, and Zinc in
Millstreams of Hard Winter Wheat (Triticum aestivum L.). Journal of Agricultural and Food Chem. doi:
10.1021/acs.jafc.5b04337. Nov. 21, 2015.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Cadmium and Zinc in Bread Wheat NIFA Fellows Program. Project Directors Meeting. Washington, DC. August 4,
2015.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Distribution of Cadmium, Iron, and Zinc in Millstreams of Hard Winter Wheat (Triticum aestivum L.) 2015 ASA, CSSA,
and SSSA Annual Meeting. Minneapolis, MN. November 17, 2015.
|
Progress 01/01/15 to 12/31/15
Outputs
Target Audience:Target audiences of graduate and undergraduate students in plant breeding and genetics were served by instructional activities in formal educational programs. Two lectures and one laboratory instruction were created and delivered in graduate Biometrical Genetics. A lecture was delivered to both resident and online undergraduate and graduate students in Germplasm and Genes. Online instructional activities were developed for Breeding Self-Pollinated Crops and Germplasm and Genes utilizing USDA GRIN and ERS databases and a NIFA-supported database. A laboratory module on multiple-trait selection was developed for Biometrical Genetics using grain mineral concentration and protein data from the PI's research. Practicum experience was provided to a group of three graduate students in analysis of next-generation sequencing data using high performance computing resources. Graduate and undergraduate student target audiences also were served by mentoring activities. An undergraduate student developed a funded grant proposal for an undergraduate research project. Mentoring activities included support on experimental design and analysis, greenhouse propagation of wheat, and molecular biology approaches for functional genomics. High school students were served by an experiential learning opportunity at the University of Nebraska Youth World Food Prize. Activities were centered around the improvement of wheat nutritional and manufacturing quality through plant breeding. A high school student was served through mentoring in the Young Nebraska Scientist project (EPSCoR), providing laboratory experience in analysis of mineral composition of wheat grain. Peer scientists were reached by seminars and poster presentations during the reporting period. Changes/Problems:Unexpected outcomes: Transcripts that have been associated with cadmium response in other crops did not respond differently to cadmium in accumulating and non-accumulating wheat genotypes. Transcripts that responded differently to cadmium in accumulating and non-accumulating wheat genotypes often had unexpected functions (Hydrolase activity, hydrolyzing O-glycosyl compounds GO:0004553; nutrient reservoir activity - Mn binding - germin-like proteins GO:0045735; nicotianamine biosynthetic process GO:0030418). We will further explore the potential relationship of these molecular functions to the key phenotype of movement of cadmium into above-ground plant tissues. What opportunities for training and professional development has the project provided?Software Carpentry Workshop: Python, Bash, and Git. University of Nebraska Holland Computing Center, Omaha, NE. April 22-23, 2015 Workshop: Innovation in Pedagogy and Technology Symposium. University of Nebraska Online Worldwide. May 14, 2015. Training: Adobe Connect - Creating and Editing Recordings. University of Nebraska, Lincoln. June 29, 2015. Participation in American Society of Agronomy/Crop Science Society of America/Soil Science Society of America annual meeting. Minneapolis, MN. Nov. 15-18, 2015. How have the results been disseminated to communities of interest?Seminars (3) "Breeding Wheat for Improved Mineral Nutrition and Food Safety." University of Minnesota Department of Agronomy and Plant Genetics. May 4, 2015. "Zinc and Cadmium in Great Plains Hard Winter Wheat." USDA-ARS and Kansas State University Department of Agronomy. August 18, 2015. "Toward a more healthful wheat crop: Zinc and cadmium in Great Plains hard winter wheat." University of Nebraska Lincoln, Department of Agronomy and Horticulture. November 13, 2015. Poster Presentations (2) "Cadmium and Zinc in Bread Wheat" NIFA Fellows Program. Project Director's Meeting. Washington, DC. August 4, 2015. "Distribution of Cadmium, Iron, and Zinc in Millstreams of Hard Winter Wheat (Triticum aestivum L.)" 2015 ASA, CSSA, and SSSA Annual Meeting. Minneapolis, MN. November 17, 2015. Outreach Activities Youth World Food Prize, University of Nebraska Lincoln. 31 high school students plus parents and teachers. Sept. 11, 2015. Immersion experience in cereal science/wheat breeding with a focus on mineral nutrition quality. What do you plan to do during the next reporting period to accomplish the goals?Goal 1(Research): Conduct the experiments proposed for Year 2 of the project to characterize the wheat root's transcriptomic response to N limitation by RNAseq. Bring to publication the results from the Year 1 transcriptome experiments on differential cadmium accumulation. Deposit sequence information in NCBI Short Read Archive. Goal 3 (Teaching Competencies): Package the educational materials developed in Year 1 into a Teaching Portfolio.
Impacts
What was accomplished under these goals?
Winter wheat varieties grown in the Great Plains of the United States can accumulate cadmium, a toxic heavy metal, in wheat grain in concentrations that exceed international standards for food safety. This research aims to explain why some wheat varieties accumulate less cadmium than others so that safe, low-cadmium wheat varieties can be developed. We learned that winter wheat varieties that accumulate high concentrations of cadmium in grain also keep less of the cadmium that they absorb stored in their roots and move more of the cadmium that they absorb into their shoots, compared to a winter wheat variety that does not accumulate high concentrations of cadmium in grain. We also found minor variation among winter wheats in a gene that has been shown to differentiate low and high-cadmium durum (pasta) wheat varieties. We can use this variation to track this gene in wheat breeding programs. Objective 1 (Research Goal): In Year 1 of the project, the specific objective was to use RNA sequencing (RNA-seq) and selected diverse genotypes to identify root genes involved in differential accumulation of cadmium (Cd) in winter wheat grain. The major activities completed / experiments conducted were hydroponic experiments with three winter wheat genotypes (Panhandle, TAM111, OK1068026) that vary for grain Cd accumulation (low, high, very high), characterization of transcriptional response to Cd, and sequencing of a candidate gene, Heavy Metal Associated3_A (HMA3_A). Data collected included root and shoot dry weights and tissue cadmium concentrations after treatment at varying media pH, Cd concentration, and time. RNA was collected from roots of plants of three winter wheat genotypes (OK1068026, TAM 111, and Panhandle) grown with and without 1 uM Cd. Differentially expressed genes were identified by sequencing this RNA using next-generation sequencing technology, then counting the sequencing reads that aligned to predicted genes in the International Wheat Genome Consortium reference sequence. Expression of specific genes also was measured by qRT-PCR. Genomic sequence of HMA3_A genes was determined. Summary statistics/discussion of results. Low and high grain-Cd varieties differed in how Cd was partitioned between shoots and roots in hydroponically grown seedings. The low grain-Cd variety consistently kept a greater proportion of Cd in roots. For example, after 28 days treatment with 1 uM Cd, 62% of the Cd in a high-Cd variety was found in shoots, but only 35% of the Cd in the low-Cd variety was found in shoots. The sequencing project produced over 335 million raw reads. An average of 76% of reads aligned to the IWGSC reference genome. Cadmium treatment decreased abundance of 551 transcripts and increased abundance of 326 transcripts in one or more of the genotypes. The low-Cd genotype, Panhandle, had the fewest Cd-responsive transcripts, and the high-Cd genotype, OK1068026, had the most Cd-responsive transcripts. Of the 326 upregulated transcripts, 170 had gene ontology (GO) annotations, and 22 of these terms were significantly enriched. Among these were a set of 38 transcripts associated with metal ion binding. Of the 551 downregulated transcripts, 328 had GO annotations, and 94 of the GO terms were significantly enriched. The most significant enrichment of GO terms was related to nicotianamine biosynthetic processes. We identified 134 transcripts that responded differently to Cd treatment in the low-Cd variety, Panhandle, than in the highest-Cd genotype, OK1068026. Of these 134 transcripts that were differentially expressed, 105 had GO annotations and 28 of the GO terms were significantly enriched. Enriched transcripts were particularly associated with the biological process of transmembrane transport and with heme binding and nutrient reservoir activity. The transcripts associated with nutrient reservoir activity often were associated with manganese binding. Transcript levels of specific candidate genes for differential Cd accumulation in winter wheat, based on work in durum wheat and barley, were analyzed by RT-PCR. Expression of transcripts for Heavy Metal Associated3 (HMA3) and Phytochelatin Synthase (PCS), which were expected to be associated with differential Cd response because they are associated with sequestration, was not significantly different among the wheat genotypes. Nor was expression of these transcripts affected by Cd treatment. Genomic sequence of HMA3_A from the three wheat genotypes identified a 3 bp deletion in the third intron in the sequences of the high-Cd wheats OK1068026 and TAM111, relative to Panhandle. HMA3_A is a candidate gene based both on its location on chromosome 5A, the site of a quantitative trait locus for grain Cd in winter wheat, and based on the effect of HMA3 mutants on Cd accumulation in other crops including durum wheat and rice. PCR primers were designed to detect the deletion, and their utility was demonstrated in progeny of a cross of a high-Cd wheat, 'Wesley,' with Panhandle. Key outcomes. A low-volume hydroponic protocol was developed to measure uptake and translocation of Cd by wheat seedlings. A key change in knowledge was that a low grain-Cd wheat genotype consistently accumulated less Cd in shoots and more Cd in roots than high grain-Cd wheat genotypes, although total Cd accumulation was not significantly different. A simple PCR marker to detect polymorphism in HMA3_A alleles of winter wheat genotypes was developed to track HMA3_A alleles in segregating populations. Skills in analyzing genome-wide RNA sequence information were developed, a skills in utilizing databases of wheat genome information were substantially improved. Objective 2 (grant writing skills) was to construct a follow-up grant proposal that will be submitted to a funding agency. A proposal, "Decreasing Cadmium and Increasing Zinc in Winter Wheat," was submitted to AFRI Plant Growth and Development, Composition and Stress Tolerance Program (Program Code A1101, proposal 2015-06559). The proposal received high priority for funding, but was not funded. Synthesis Comments of Reviews: This is a well-written proposal that addresses an important nutrient uptake issue in wheat, and will likely have successful outcomes in identifying molecular markers associated with reduced cadmium accumulation.The proposal could be improved by evaluating resulting low-cadmium lines for pleiotropic effects in important agronomic traits.Key learning experiences: complex and time-consuming process of assembly of a full-scale proposal, helpful reviews for future submission. Objective 3 (teaching competencies) is to develop a teaching portfolio, develop mentoring relationships with undergraduate and graduate students, and prepare and provide guest lectures in undergraduate and graduate classes. Major activities completed: New mentoring relationships were developed with two graduate, one undergraduate student (leading to development of a funded undergraduate research grant and a graduate thesis project) and a high school student. Lectures were delivered to graduate and undergraduate students in two classes (Biometrical Genetics, Germplasm and Genes) and learning activities were developed for both resident and online instruction, including an instructional video. Data collected: student evaluations were collected following the two lectures and the laboratory in Biometrical Genetics. Lecture in Germplasm and Genes was recorded using Adobe Connect for subsequent evaluation. Course evaluations were collected following Breeding Self Pollinated Crops and Germplasm and Genes. Teaching reviews were highly encouraging in Biometrical Genetics. Key outcomes: improved skills in instructional design and implementation, particularly for courses that integrate resident and distance education; improved skills with learning management software systems; improved skills for video recording short instructions and full lectures.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2015
Citation:
Guttieri, Mary; Seabourn, Bradford; Liu, Caixia; Baenziger, P.; Waters, Brian M. Distribution of Cadmium, Iron, and Zinc in Millstreams of Hard Winter Wheat (Triticum aestivum L.). Journal of Agricultural and Food Chem. doi: 10.1021/acs.jafc.5b04337. Nov. 21, 2015.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Cadmium and Zinc in Bread Wheat NIFA Fellows Program. Project Directors Meeting. Washington, DC. August 4, 2015.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Distribution of Cadmium, Iron, and Zinc in Millstreams of Hard Winter Wheat (Triticum aestivum L.) 2015 ASA, CSSA, and SSSA Annual Meeting. Minneapolis, MN. November 17, 2015.
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