Source: UNIVERSITY OF ILLINOIS submitted to NRP
ADVANCING A GENETIC CONTROL STRATEGY FOR DIOECIOUS AMARANTHS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1027778
Grant No.
2022-67013-36142
Cumulative Award Amt.
$537,647.00
Proposal No.
2021-08438
Multistate No.
(N/A)
Project Start Date
Jan 1, 2022
Project End Date
Dec 31, 2025
Grant Year
2022
Program Code
[A1112]- Pests and Beneficial Species in Agricultural Production Systems
Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801
Performing Department
Crop Sciences
Non Technical Summary
Two Amaranthus species, waterhemp and Palmer amaranth, are among the worst agronomic weeds in the U.S. Their dioecious nature (plants are either male or female) ensures outcrossing, fostering great genetic diversity and an unprecedented ability to evolve resistances to multiple herbicides. In numerous cases, farmers have resorted to hand weeding as part of their waterhemp/Palmer amaranth control practices. My long-term goal is to exploit the dioecious nature of these two weeds to develop a novel genetic control strategy based on emerging gene-drive technology. My group recently identified the sex-determining regions of the genome for both species, but the causal genes remain unknown. I propose a comparative genomics approach, utilizing other dioecious Amaranthus species, to identify the specific genes that are most likely to mediate sex determination. Small-RNAs have been implicated as playing a role in sex determination in other dioecious species and, based on preliminary data, also in Amaranthus. Therefore, sRNAs are also the subject of the proposed research. Additionally, the stability of dioecy in waterhemp and Palmer amaranth will be evaluated using an artificial selection experiment. The proposed research builds on substantial previous research and preliminary data to further advance a truly novel weed control strategy. The proposed research also directly addresses Program Area Priority Code A1112, Pests and Beneficial Species in Agricultural Production Systems: it proposes "research on aspects of weed biology that impact reproductive biology" and it will "advance knowledge of invasive or established plant pests... leading to innovative and biologically based strategies to manage pests."
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21323001140100%
Knowledge Area
213 - Weeds Affecting Plants;

Subject Of Investigation
2300 - Weeds;

Field Of Science
1140 - Weed science;
Goals / Objectives
The long-term goal of this research is to develop a novel genetic control strategy for waterhemp (Amaranthus tuberculatus) and Palmer amaranth (Amaranthus palmeri). The strategy will take advantage of the dioecious nature of these two species, either using a maleness gene drive or a female-sterility gene drive, to decrease densities of local populations over time. Although theoretically feasible, there are several hurdles to making such a control strategy a reality. The first step was made with the identification of a male-specific genomic region (MSY) in both species and with the global gene expression analyses between males and females, which suggested potential involvement of small-RNAs (sRNAs). The next objectives are to identify the causal gene(s) within the male-specific regions and to determine the evolutionary stability of dioecy within the species. These objectives will be accomplished through the following specific aims:Identify conserved genes within the MSY region among dioecious Amaranthus species with a shared dioecy origin.Identify the role of sRNAs in the mechanism of sex determination in waterhemp and Palmer amaranth via sRNA sequencing.Determine the stability of dioecy in waterhemp and Palmer amaranth using an artificial selection experiment.In addition to moving us closer to the long-term goal of developing a novel genetic weed-control strategy, accomplishment of these specific aims will contribute fundamental knowledge to the reproductive biology of a key weed genus and to the evolution of dioecy, which is a largely unresolved question in plant biology.
Project Methods
To identify conserved genes within the MSY region among dioecious Amaranthus species, we will perform whole-genome sequencing of seven additional dioecious species, as well as with spiny amaranth (Amaranthus spinosus), a monoecious species closely related to Palmer amaranth. Resulting sequencing reads will be aligned to waterhemp and Palmer amaranth genomes and then subjected to variant calling followed by comparative genomics and phylogenetic analysis.Sequencing of sRNAs from early and late-stage flowers of both waterhemp and Palmer amaranth males and females will be performed. Differential expression analysis between flower gender and stages will be performed, and differentially expressed sRNAs will be confirmed using qRT-PCR. If differentially expressed sRNAs derived from the MSY regions are identified, they will be subjected to comparative sequence analysis using the homologous genes from the other dioecious species.An artificial selection experiment will be conducted in the greenhouse to evaluate the stability of dioecy in both waterhemp and Palmer amaranth. For this study, seed production by isolated females and males of both species will be monitored. When seed production is observed, selection for increased seed production will occur over a total of six generations.

Progress 01/01/24 to 12/31/24

Outputs
Target Audience:The target audience is the plant science research community. Changes/Problems:Our previously generated genome assembly of waterhemp still has several gaps in the sex-determiningregion, hindering our analysis of this region. Therefore, in collaboration with the International Weed Genomics Consortium, we generated a higher-quality, almost-gap-freeassembly. Because of this new assemble, we are now having to redo much of the bioinformatics analysis. For example, all the transcriptomic reads are being mapped back to this new assembly. What opportunities for training and professional development has the project provided?Two graduate students involved with this project have received training in bioinformatics with genomic and transcriptomic datasets, and in managing a research project and supervising undergraduate students. Three undergraduate students received training in general plant science research conducted in the greenhouse and laboratory. How have the results been disseminated to communities of interest?Preliminary findings of this research have been disseminated at scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?We will continue to analyze the RNA data sets, including differential gene expression analysis, identification of miRNA, and elucidation of miRNA-mRNA interaction networks. We will conduct a third generation of selection on select lines, and analyzethe genomic datasets generated from parents and their progeny. We will begin preparing manuscripts to publish our findings.

Impacts
What was accomplished under these goals? Progress was made towards specific aims 2 and 3; specific aim1 was completed previously. Towards aim 1, mRNA and sRNA datasets were generated and are being analzyed. Towards aim 3, two generations of selection have now been completed for both sexes of both species. Several interesting phenotypes (showing transitions from dioecy to monoecy) were recovered from the selection process and are being further evaluated. For example, whole-genome sequencing was performed on the parents and some of the progeny showing the aberrant phenotypes.

Publications


    Progress 01/01/23 to 12/31/23

    Outputs
    Target Audience:The target audience for this reporting period is the plant science research community. Changes/Problems:As described in the accomplishments section, a challengein RNA extraction from some tissue samplesdelayed progress towards specific aim 2. We have overcome the challenge, however, and expect progress to resume. What opportunities for training and professional development has the project provided?Two graduate students contributing to this project have gained experience in analyzing genome sequence data. One of the students also gained extensive experience in conducting greenhouse-based experiments. Both students also gained experience managing their own research projects and supervising undergraduate employees. Two undergraduate students received hands-on training in conducting greenhouse and plant science laboratory research. How have the results been disseminated to communities of interest?One peer-reviewed paper was published and other results have been disseminated at a scientific conference. What do you plan to do during the next reporting period to accomplish the goals?Analysis of the additional genome sequence we generated will be completed. RNA samples will be submitted for sequencing and resulting data will be analyzed. The third generation of the artificial selection cycle will be initiated. We anticipate writing three additional manuscripts to disseminate the results of this research project.

    Impacts
    What was accomplished under these goals? Regarding specific aim 1, in the previous reporting period we performed comparative genomics of nine dioecious amaranths to identify conserved genes within the MSY, as we originally proposed. Although this revealed candidate sex-determing genes, we have pursued this work further by generating chromosome-level assemblies of additional dioecious species and analyzingthese data. Regarding specific aim 2, tissue was harvest during the last reporting period for RNA extraction. However, challenges in extraction of RNA from some tissue samples (specifically, mature flowers) necessitated us growing the plants again. We have successfully modified our RNA extraction procedure and obtained RNA from the regrown plants; samples soon will be submitted for sequencing. Regaring specific aim 3, the second generation of artificial selection was completed during the current reporting period.

    Publications

    • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Lopez, AJ, LK Bobadilla, PJ Tranel. 2023. Evolutionary stability in the genus Amaranthus. Proc. North Central Weed Science Society. 78:49.
    • Type: Journal Articles Status: Published Year Published: 2023 Citation: Bobadilla, LK, Y Baek, PJ Tranel. 2023. Comparative transcriptomic analysis of male and females in the dioecious weeds Amaranthus palmeri and Amaranthus tuberculatus. BMC Plant Biology 23:339.


    Progress 01/01/22 to 12/31/22

    Outputs
    Target Audience:The target audience for this reporting period is the plant science research community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students contributingto this project have gained experience in bioinformatics analyses of genomic and transcriptomic data. They have also gained experience in disseminating research via conference presentations and writing papers. Two undergraduate students received hands-on training in conducting greenhouse and laboratory research, including isolation of DNA from plant samples. How have the results been disseminated to communities of interest?Three scientific papers associated with the project were written and two have already beensubmitted for peer review. Graduate students also presented results of their research at scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?RNA samples will be submitted for sequencing and resulting data will be analyzed to complete Specific Aim 2. Seed will be threshed from the first generation of the artificial selection experiment and used to initiate the second generation of the experiment to continue Specific Aim 3.

    Impacts
    What was accomplished under these goals? Regarding Specific Aim 1, weobtained whole-genome sequence data from seven dioecious Amaranthus species and compared these new genomes with those previously generated for waterhemp and Palmer amaranth. We wrote a paper describing this work and it has been accepted pending minor revisions. In addition, we utililzed our whole-genome sequence data to compare plastomes among the species to better understand their evolutionary relationships. We wrote a paper describing this work and it is currently under review.Regarding specific aim 2, harvesting of the appropriate tissues from flowering plants of both waterhemp and Palmer amaranth was completed, and RNA was extracted from the tissues. Regarding Specific Aim 3, the first generation of plant growth was completed, with all the plants harvested and awaiting seed threshing. In addition, we have completed a transcriptomics analysis of male and female floral tissues of waterhemp and Palmer amaranth. This work was an extension of our previous USDA grant associated with the broader project goals. A paper from this analysis was written and is nearly ready for submission.

    Publications

    • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Lopez, AJ, LK Bobadilla, and P Tranel. 2022. Evolutionary stability of dioecy in the genus Amaranthus. Proc. North Cent. Weed Sci. Soc. 77:100.
    • Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Raiyemo, DA, LK Bobadilla, and PJ Tranel. 2023. Genomic profiling of dioecious Amaranthus species provides novel insights into species relatedness and sex genes. Accepted pending minor revisions, BMC Biology.
    • Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Raiyemo, DA, and PJ Tranel. 2023. Comparative analysis of dioecious Amaranthus plastomes and phylogenomic implications within Amaranthaceae s.s. Submitted to BMC Ecology and Evolution.