University Research Proposal: Herbicide Resistance in Hairy Fleabane

This research proposal investigates herbicide resistance, focusing on glyphosate resistance in the weed Erigeron bonariensis (hairy fleabane). The study aims to understand the mechanisms of resistance, particularly target site and non-target site resistance, and their implications for agricultural practices. The methodology includes collecting samples from the San Joaquin Valley, germination, RNA extraction, and analysis using real-time quantitative PCR and RNA sequencing. The significance lies in providing critical information for effective and sustainable weed management strategies, especially in the context of increasing herbicide resistance. The researcher has successfully completed RNA extraction and initial analyses for several populations, with ongoing primer redesign to enhance the specificity of quantitative PCR. The findings are expected to contribute to the development of strategies to combat herbicide resistance and improve crop yields.

Running head: RESEARCH PROPOSAL ASSIGNMENT
Research proposal assignment
Name of the student:
Name of the university:
Author note:

1RESEARCH PROPOSAL ASSIGNMENT
Table of Contents
Introduction: 2
Material and methods: 3
Significance of the study: 3
My work done till now: 4
References: 5

2RESEARCH PROPOSAL ASSIGNMENT
Introduction:
Authors have recognized the herbicide resistance to be one of the heritable ability of the
weeds as one of the survival tactics. The very first record of herbicide resistance was discovered
in Senecio vulgari, and ever since the phenomenon of herbicide resistance become one of the
most crucial challenges to the agricultural farms, due to 187 weed species developing herbicide
resistance powers. Glyphosate however is one of the most abundantly used herbicides in the
world and with its power of targeting mostly the monocots and dicots, rapid microbial
degeneration, and cost effectiveness. However resistance to this particular multipurpose
herbicide has also been evolved in some of the weeds which has become a growing concern for
the agriculturists (Anders & Huber, 2010).
The mechanism of developing herbicide resistance in the weeds for Glyphosate involves two
key steps, target site resistance and non target site resistance. An exotic species of the family of
Erigeron, erigeron bonarensis is known to exhibit high resistance to glyphosate, however the
exact mechanism of developing the resistance is still obscure. The characterization of herbicide
resistance in E. Bonariensis or hairy fleabane will identify the presence of herbicide resistance
genes in the weed species (Anders, Pyl & Huber, 2015). This information is critical to the
cultivators to ensure that effective and sustainable weed management and control strategies are
put in place. Information about the genetic basis of herbicide resistance is key in effective weed
management and has the potential to bolster agricultural productivity. Elucidating the role of
both target site and non-target mutations in resistance to various chemicals is also important in
the genetic engineering of crop plant resistance, especially by plant transformation techniques.

3RESEARCH PROPOSAL ASSIGNMENT
Material and methods:
The sampling method will utilize ten different populations of Erigeron bonariensis for the
purpose of germinating, collected from the San Joaquin Valley in 2016 based on previous
fleabane collection sites. The germination process will follow a standard germination process of
imbibitions in 10 ml of distilled water and then sealing the plates with parafilm. Plants will be
randomized among treatment before spraying, and a complete randomized block design will be
set up after spraying, to minimize spatial effects in the greenhouse (Baldwin & Goldman, 2012).
The germination process will followed by the RNA extraction, by the means of harvesting
the leaves. The leaves will be harvested twice in the study, prior to the glyphosate spraying time
point, and twenty-four hours after glyphosate spraying occurs. The leaves that are middle4 aged
will be selected. Followed by RNA isolation by Qiagen Plant RNeasy Mini Kit, Potential
genomic DNA will be removed using DNase (FisherSci) and RNA will be measured using
Qubit® dsDNA HS Assay Kits. After that 1st Round of real-time quantitative PCRs will be
carried out followed by RNA-Sequencing and bioinformatic data analysis. After that the second
round of quantitative PCR and again Analysis of RNA sequence data will be carried out (Baylis,
2010).
Significance of the study:
It has to be understood that weeds are very competitive for resources; hence they contribute
largely to the crop yield reduction. And as herbicides is the most commonly utilized weed
controlling procedure, the growing resistance in the weed species is one of the most growing
concerns for the agriculturists to protect the crop yield. This particular study will attempt to
evaluate the presence of glyphosate resistance genes in the hairy fleabane in the central Valley to

4RESEARCH PROPOSAL ASSIGNMENT
evaluate the effectiveness of the resistance genes and the development of the herbicide resistance
in the effectiveness of the resistance genes and the development of the herbicide resistance in the
free species. It is common knowledge that is information will be a critical breakthrough for the
cultivators an agriculturist to ensure that effective and sustainable weight management and
controlling strategies can be facilitated keeping in mind the herbicide resistance capacity in the
weeds (Beckie & Tardif, 2012). The information about the genetic mechanism of herbicide
resistance involving the target site and non target site resistance mechanism will elucidate and
new wealth of knowledge regarding herbicide resistance mechanism in which and how to combat
them and the controlling strategies.
My work done till now:
Until now the RNA extraction of the population 1,2,5, and 9 has been completed successfully.
Followed by that checking the quantity of RNA with cubit and RNA gel electrophoresis have also been
carried out successfully. After the successful checking of the Anny quantity and viability the sea DNA
synthesis for the population one has also been completed, closely followed by checking the actin genes
with M10 genes. After the successful PC are running the electrophoresis gel short significant bands
performing the accuracy of our study so far. However in order to make the primers used more specific for
the quantitative PCR we are in the face of redesigning the primers with the help of direct sequencing to
make the primers much more target specific.

5RESEARCH PROPOSAL ASSIGNMENT
References:
Anders, S., & Huber, W. (2010). Differential expression analysis for sequence count
data. Genome biology, 11(10), R106.
Anders, S., Pyl, P. T., & Huber, W. (2015). HTSeq—a Python framework to work with high-
throughput sequencing data. Bioinformatics, 31(2), 166-169.
Baldwin, B. G., & Goldman, D. H. (2012). The Jepson manual: vascular plants of California.
University of California Press.
Beckie, H. J., & Tardif, F. J. (2012). Herbicide cross resistance in weeds. Crop Protection, 35,
15-28.
Haas, B. J., Papanicolaou, A., Yassour, M., Grabherr, M., Blood, P. D., Bowden, J. &
MacManes, M. D. (2013). De novo transcript sequence reconstruction from RNA-seq
using the Trinity platform for reference generation and analysis. Nature protocols, 8(8),
1494-1512.