A TRANSLATIONAL GENOMICS APPROACH FOR CROP TRAIT IMPROVEMENT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1016297
• Project Start Date: Jul 1, 2018
• Project End Date: Sep 30, 2022
• Program Code: [(N/A)]- (N/A)

Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695

Performing Department
Horticultural Science

Non Technical Summary
We intend to translate novel discoveries in functional genomics into applications for crop trait improvement. To meet this goal, we will conduct translational genomics research to develop novel means, such as genes or methods that will facilitate the translation of basic discoveries into crop improvement. The proposed research includes, but is not limited to, genetic engineering and genome editing of camelina for non-opening petals, genetic engineering of Arabidopsis, soybean, and camelina for improved seed size, and identification of the key genes in the whole carotenoid biosynthesis pathway responsible for high lycopene content in tomato fruit. Other projects relating to translational genomics of horticultural crops will be developed as opportunities arise. Research based knowledge and information will be disseminated via publications, conference presentations and meetings to the stakeholders and the general public.

Animal Health Component

100%

Research Effort Categories

Basic

(N/A)

Applied

100%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	2499	1080	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
2499 - Plant research, general;

Field Of Science
1080 - Genetics;

Keywords

tomato

camelina

soybean

arabidopsis

genetic engineering

genome editing

crop trait improvement

Goals / Objectives
We seek to develop novel means, such as genes, promoter sequences or methods that can be used directly or indirectly for crop trait improvement. Specific examples of the projects include: 1) Genetic engineering and genome editing of camelina to prevent flowers from opening (i.e., cleistogamy; funded by a USDA NIFA grant) as a means to inhibit gene flow to other plants; 2) genetic engineering of Arabidopsis, soybean, and camelina for improved seed size; and 3) Identification of the key genes in the carotenoid biosynthesis pathway responsible for high lycopene content in tomato fruit.

Project Methods
1) Genetic engineering and genome editing of camelina for cleistogamy:Genetic engineering will be used to overexpress a peach cleistogamy gene. This gene had been overexpressed in tobacco and caused non-opening petals (Sherif et al., 2015) The CRISPR-Cas9-assisted genome editing will be used to create a point mutation in the camelina cleistogamy gene, resulting in cleistogamy. Phenotypic and molecular analysis will be conducted to analyze the mutant phenotype and mutation efficiency of the GE cleistogamous events. Next generation sequencing will be used to study the off-target effects of the genome editing method. The best events will be used in greenhouse study to test phenotypic stability and pollen-mediated gene flow (PMGF) to non-GE camelina. Field trials using the Nelder wheel design (Hanson et al., 2005) will be performed to study the phenotypic stability and PMGF in a real world setting. PCR will be used to test the presence of the transgenes in the resulting seeds which will be collected from the non-GE recipients in both the greenhouse study and the field trials. By doing these, we will engineer camelina plants to have self-pollinating and non-opening flowers for the prevention of PMGF from transgenic plants to non-transgenic plants or closely related species, and then bioconfinement in genetically engineered camelina. Cleistogamy could be used for bioconfinement of transgenes responsible for improvements in any traits (such as yield, seed oil content and composition, drought tolerance, pest resistance, etc.) that need to be improved in horticultural crops, especially bioenergy crops, for sustainability.2) Genetic engineering of Arabidopsis, soybean, and camelina for improved seed size:Using genome wide association study (GWAS) and functional genomics, we have identified a novel function of an Arabidopsis MYB transcription factor gene in controlling seed size in both transgenic Arabidopsis and soybean (Liu et al., under revision). Now we intend to identify the direct target genes of the MYB gene in the Arabidopsis genome. We plan to use the MYB transcription factor for Yeast Reverse One Hybrid (Zeng et al., 2008) and Electrophoretic Mobility Shift Assay (EMSA) in order to identify which genes' promoters could bind to the transcription factor. Once the binding promoter sequences and then the target genes are identified, genetic engineering and T-DNA insertion mutants will be used to further confirm the functions of the target genes in seed size controlling.We also obtained a few candidate genes in Arabidopsis which are linked to enhanced seed size by using GWAS. Now we are using genetic engineering to further investigate the functions of seven candidate genes in controlling seed size in transgenic Arabidopsis and camelina. T-DNA insertion mutants will be obtained to study the knockout effects of the candidate genes on controlling seed size.The novel functions of the MYB gene and its direct targets as well as the candidate genes could be directly used in different horticultural crops for seed size improvement whereas seed size is desirable.3) Identification of the key genes in the whole carotenoid biosynthesis pathway responsible for high lycopene content in tomato fruit:Lycopene is a dietary antioxidant carotenoid which contributes to tomato red color and reduces the risks of cancer, cardiovascular disease and diabetes (Burton-Freeman et al., 2014; Rao and Rao, 2007), and tomato contributes 85% of dietary lycopene in the U.S. (Rao and Agarwal, 1999). As an intermediate in the carotenoid biosynthesis pathway, lycopene content in tomatoes is largely under genetic control and varies greatly among tomato genotypes (Dorais et al., 2008; George et al., 2004). We have collected about 25 high-lycopene lines all over the world. All of these lines contain different mutated genes in different genetic backgrounds. Plants will be grown at the same time under the same conditions. Fruits will be harvested at six developmental stages, i.e., immature green, mature green, breaker, orange, pink, and ripe. Real-time RT-PCR will be used to investigate the expression patterns of the individual genes in the complete pathway at different fruit developmental stages. Identification of the key genes controlling lycopene content will enable the genetic engineering and genome editing of tomato fruits for lycopene content improvement.

Progress 10/01/20 to 09/30/21

Outputs
Target Audience:Plant scientists and biotechnologiests, plant breeders, professionals in horticutural science and biotechnology, farmers,horticultural companies and others in the private sector interested in horticultural crops. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Postdocs Drs. Debao Huang,Paul Kosenta and Nathan Maren, graduate students James Duduit, Sihui Ni, Davis Harmon, Meghan Roche,Emily Brooks, and Anna Nelson, visiting PhD graduate students Fangzhou Zhao and Hongyan Lu, and undergradue workers Jeremy McAdams, Paulina Bollina, Rhet Pasour, and Anna Nelsonwere actively involved in many of the projects. How have the results been disseminated to communities of interest?So far, part of the results hve been disseminated to communities of interest through seminars, presentations and publications. What do you plan to do during the next reporting period to accomplish the goals?Continue and expand these efforts.

Impacts
What was accomplished under these goals? Project 1): A second field trial has been conducted in2020-2021 for the best line overexpressing the peach cleistogamous gene without interfering seed yield has been used for a field trial, which was completed, in order to investigate whether the engineered cleistogous trait dramatically restricted pollenmediated gene flow from transgenic plants to non-transgenic plants. A manuscript has been drafted for submission for publication. Project 2):A novel qPCR method has been published for analysis of relative expression levels of genes. Project 3): Confirmation of the function of the first bacterial wilt resistance gene in tomato using overexpression of the gene in tomato. Project 4): Successfully conducted de novo motif discovery to look for constitutive expression promoter motifs. Project 5): Publication of a transcriptomics paper for tripidum. Project 6): Publication of a invited review paper for plant synthetic biology.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Yang, Y., Lee, J., Poindexter, M., Shao, Y., Liu, W., Lenaghan, S., Ahkami, A., Blumwald, E., Stewart, C.N. Jr. (2021) Rational design and testing of abiotic stress inducible synthetic promoters from poplar cis-regulatory elements. Plant Biotechnology Journal 19:1354-1369.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Zhao, F., Maren, N.A., Kosentka, P.Z., Liao, Y.-Y., Lu, H., Duduit, J.R., Huang, D., Ashrafi, H., Zhao, T., Huerta, A.I., Ranney, T.G., Liu, W.* (2021) An optimized protocol for stepwise optimization of real-time RT-PCR analysis. Horticultural Research 8:179.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Huang, D., Kosentka, P.Z., Liu, W.* (2021) Synthetic biology approaches in regulation of targeted gene expression. Current Opinion in Plant Biology 63:102036.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Maren, N.A, Zhao, F., Aryal, R., Touchell, D.H., Liu, W., Ranney, T.G., Ashrafi, H. (2021) Reproductive developmental transcriptome analysis of Tripidium ravennae (Poaceae). BMC Genomics 22:483

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Plant scientists and biotechnologiests, plant breeders, professionals in horticutural science and biotechnology, farmers,horticultural companies and others in the private sector interested in horticultural crops. Changes/Problems:Nothing to report. What opportunities for training and professional development has the project provided?Postdocs Drs. Debao Huang and Paul Kosenta, graduate students James Duduit, Sihui Ni, Davis Harmon, Meghan Roche,visiting PhD graduate students Fangzhou Zhao and Hongyan Lu, and undergradue workers Morgan Miller, Via Snow Abiera, and Jeremy McAdams were actively involved in many of the projects. How have the results been disseminated to communities of interest?So far, part of the results hve been disseminated to communities of interest through seminars, presentations and publications. What do you plan to do during the next reporting period to accomplish the goals?Continue and expand these efforts.

Impacts
What was accomplished under these goals? Project 1): The best line overexpressing the peach cleistogamous gene without interfering seed yield has been used for a field trial, which was completed, in order to investigate whether the engineered cleistogous trait dramatically restricted pollen-mediated gene flow from transgenic plants to non-transgenic plants. A second field trial will be planned for 2020-2021. In addition, some homologous transgenic lines containing the CRISPR/Cas9 for genome editing were obtained. Project 2): Three genes from Arabidopsis and one gene from soybean were identifed to significantly affect seed weight when overexpressed or knocked out in transgenic Arabidopsis, camelina and/or soybean. Project 3): Eight upsteam genes and seven downstream genes in the carotenoid biosynthesis pathway had been identified to be responsible for high fruit lycopene content in 42 tomato high lycopene breeding lines collected from all over the world. A manuscript is prepared for submission for publication. DIC microscopy is still underway to investigate the size and numbers ofchromoplasts per cell for each line. Project 4): A novel lypofection method has been published to effectively deliver the Cas9/gRNA ribonucleoprotein (RNP) into plant protoplasts for gene editing.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Yu, J., Gao, L., Liu, W., Song, L., Xiao, D., Liu, T., Hou, X., Zhang, C. (2019) Transcription coactivator ANGUSTIFOLIA3 (AN3) regulates leafy head formation in Chinese cabbage. Frontiers in Plant Science 10:520.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Ondzighi-Assoume, C.A., Willis, J.D., Ouma, W.K., Allen, S.M., King, Z., Parrott, W.A., Liu, W., Burris, J.N., Lenaghan, S.C., Stewart, C.N., Jr. (2019) Embryogenic cell suspensions for high capacity genetic transformation and regeneration of switchgrass (Panicum virgatum L.). Biotechnology for Biofuels 12:1-14.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Wang, H., Xie, Y., Liu, W., Tao, G., Sun, C., Sun, X., Zhang, S. (2020) Transcription factor LkWOX4 is involved in adventitious root development in Larix kaempferi. Gene 758:144942.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Liu, W*, Rudis, M.R., Cheplick, M.H., Millwood, R.J., Yang, J.-P., Ondzighi-Assoume, C.A., Montgomery, G.A., Burris, K.P., Mazarei, M., Chesnut, J.D., Stewart, C.N., Jr.* (2020) Lipofection mediated genome editing using DNA-free delivery of the Cas9/gRNA ribonucleoprotein into plant cells. Plant Cell Reports 39:245-257.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Plant scientists and biotechnologiests, plant breeders, professionals in horticutural science and biotechnology, farmers, horticultural companies and others in the private sector interested in horticultural crops. Changes/Problems:Nothing to report. What opportunities for training and professional development has the project provided?Postdocs Drs. Debao Huang and Paul Kosenta, graduate students David Denton, James Duduit, Sihui Ni and Meghan Roche, visiting PhD graduate students Liwei Gao and Xianlian Chen, and undergradue workers Morgan Miller and Via Snow Abiera were actively involved in many of the projects. How have the results been disseminated to communities of interest?So far, part of the results hve been disseminated to communities of interest through seminars and presentations. What do you plan to do during the next reporting period to accomplish the goals?Continue and expand these efforts.

Impacts
What was accomplished under these goals? Project 1): Some homologous transgenic lines overexpressing a peach cleistogamous gene were obtained. These lines exhibited half-closed to closed petals. PCR and real-time RT-PCR were used to further confirm the existance of the transgene and the relative expression levels of the transgene in those lines. Pollen germination analysis was conducted to compare the pollen germination rate between the transgenic lines and the non-transgenic plants in order to explore whether the overexpression of the transgene would affect pollen viability. In addition, generation of transgenic lines for genome editing is still underway to obtain T2 or T3 homozygous transgenic lines. A field trial of the transgenic lines is underway in Sandhills with a USDA APHIS release permit. Project 2): About 10 genes from Arabidopsis and soybean are used to make stable transgenic Arabidopsis and camelina lines in order to investigate whether overexpression of each of the genes will impact seed size and seed number. Some of the genes showed significantly enlarged seed size when overexpressed in Arabidopsis and camelina when compared to non-transgenic plants. The T-DNA insertional knockout mutants were also purchased from the SALK Institutie Collection and propogated to obtain homozygous lines. PCR and real-time RT-PCR were used to further confirm the existance of the transgene and the relative expression levels of the transgene in those lines. Yeast two hybrid was used for two genes in order to identify the interating promoters or genes. A manuscript preparation is underway. Project 3): About 46 tomato high lycopene breeding lines were collected and planted together in greenhouse. Fruits adifferent developmental stages were harvested. Real-time RT-PCR and HPLC were used to investigate which carotenoid biosynthesis pathway genes most impact the fruit lycopene content. DIC microscopy is underway to investigate the size and numbers of chromoplasts per cell for each line. A manuscript preparatio is underway.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Liu, W., Mazarei, M., Ye, R., Peng, Y., Shao, Y., Baxter, H.L., Sykes, R.W., Turner, G.B., Davis, M.F., Wang, Z.-Y., Dixon, R.A, Stewart, C.N., Jr. (2018) Switchgrass (Panicum virgatum L.) promoters for green tissue-specific expression of the MYB4 transcription factor for reduced-recalcitrance transgenic switchgrass. Biotechnology for Biofuels 11:122
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Ondzighi-Assoume, C.A., Willis, J.D., Ouma, W.K., Allen, S.M., King, Z., Parrott, W.A., Liu, W., Burris, J.N., Lenaghan, S.C., Stewart, C.N., Jr. (2019) Embryogenic cell suspensions for high capacity genetic transformation and regeneration of switchgrass (Panicum virgatum L.). Biotechnology for Biofuels 12:1-14
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Yu, J., Gao, L., Liu, W., Song, L., Xiao, D., Liu, T., Hou, X., Zhang, C. (2019) Transcription coactivator ANGUSTIFOLIA3 (AN3) regulates leafy head formation in Chinese cabbage. Frontiers in Plant Science 10:520.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Liu, W.� , Rudis, M.R., Cheplick, M.H., Millwood, R.J., Yang, J.-P., Ondzighi-Assoume, C.A., Montgomery, G.A., Burris, K.P., Mazarei, M., Chesnut, J.D., Stewart, C.N., Jr.� (2019) Lipofection-mediated genome editing using DNA-free delivery of the Cas9/gRNA ribonucleoprotein into plant cells. Plant Cell Reports 39:245-257

Progress 07/01/18 to 09/30/18

Outputs
Target Audience:Commercial growers, breeders, extension professionals, technical representatives, scientists, graduate students, undergraduate students, and high school students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Postdocs Drs. Debao Huang and Paul Kosenta, graduate students David Denton and James Duduit, visiting PhD graduate students Liwei Gao and Xianlian Chen, and undergradue worker Morgan Miller were actively involved in many of the projects. How have the results been disseminated to communities of interest?So far, part of the results hve been disseminated to communities of interest through seminars and presentations. What do you plan to do during the next reporting period to accomplish the goals?Continue and expand these efforts.

Impacts
What was accomplished under these goals? Project 1): Some homologous transgenic lines overexpressing a peach cleistogamous gene were obtained. These lines exhibited half-closed to closed petals. PCR and real-time RT-PCR are underway to further confirm the existance of the transgene and the relative expression levels of the transgene. Pollen germination analysis is underway to compare the pollen germination rate between the transgenic lines and the non-transgenic plants in order to explore whether the overexpression of the transgene would affect pollen viability. In addition, generation of transgenic lines for genome editing is still underway to obtain T0 transgenic lines. Project 2): About 10 genes from Arabidopsis and soybean are used to make stable transgenic Arabidopsis and camelina lines in order to investigate whether overexpression of each of the genes will impact seed size and seed number. Some of the genes showed significantly enlarged seed size when overexpressed in Arabidopsis and camelina when compared to non-transgenic plants. The T-DNA insertional knockout mutants were also purchased from the SALK Institutie Collection and propogated to obtain homozygous lines. PCR and real-time RT-PCR are underway to further confirm the existance of the transgene and the relative expression levels of the transgene. Yeast one or two hybrid analysis is underway for two genes in order to identify the interating promoters or genes. Project 3): About 46 tomato high lycopene breeding lines were collected and planted together in greenhouse. Fruits at different developmental stages were harvested. Real-time RT-PCR and HPLC are underway to investigate which carotenoid biosynthesis pathway genes most impact the fruit lycopene content. Primers for each of the pathway genes had been designed and optimized for real-time RT-PCR analysis.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Liu, W., Mazarei, M., Ye, R., Peng, Y., Shao, Y., Baxter, H.L., Sykes, R.W., Turner, G.B., Davis, M.F., Wang, Z.-Y., Dixon, R.A, Stewart, C.N., Jr. (2018) Switchgrass (Panicum virgatum L.) promoters for green tissue-specific expression of the MYB4 transcription factor for reduced-recalcitrance transgenic switchgrass. Biotechnology for Biofuels 11:122.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Xu, W., Liu, W., Ye, R., Mazarei, M., Zhang, X., Stewart, C.N., Jr. (2018) A profilin gene promoter from switchgrass (Panicum virgatum L.) directs strong and specific transgene expression to vascular bundles in rice. Plant Cell Reports 37:587-597.