DEVELOPMENT OF GENOMIC AND MOLECULAR TOOLS TO ACCELERATE THE IDENTIFICATION AND DELIVERY OF DURABLE GENETIC RESISTANCE TO FOV4 IN UPLAND COTTON

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1019776
• Grant No.: 2019-67029-29915
• Cumulative Award Amt.: $294,000.00
• Proposal No.: 2018-09116
• Project Start Date: Aug 15, 2019
• Project End Date: Aug 14, 2022
• Grant Year: 2019
• Program Code: [A1701]- Critical Agricultural Research and Extension: CARE

Recipient Organization
CLEMSON UNIVERSITY
(N/A)
CLEMSON,SC 29634

Performing Department
Plant and Environmental Scienc

Non Technical Summary
Cotton production across the United States is under serious threat from a soil-inhabiting, density dependent fungus, called Fusarium oxysporum f. sp. Vasinfectum race 4 (FOV4), which causes Fusarium wilt. Previously, it was found only in California, but during the past decade the devastating effect of FOV4 has moved into the wider cotton belt and is a systemic problem. Left unchecked, FOV4 could decimate the entire cotton growing industry in the United States. Stable and durable genetic resistance to FOV4 in Upland cotton is not available, despite focused efforts. Fortunately, in Gossypium barbadense (Pima cotton), stable and durable resistance to the same FOV4 pathogen was discovered by our team and is currently deployed in commercial varieties. This approach includes robust protocols for building inoculum density and including proprietary checks to map the severity of disease across the field over the past two decades, which are reliable and repeatable across years. Field screening of 1,000 Upland cotton lines began in 2018, and we have identified promising FOV4 resistant Upland germplasm. In this project, we will continue screening Upland germplasm and to improve FOV4 resistance by stacking favorable alleles from multiple resistance sources, and to elucidate the genetics of resistance. This project will deliver new sources of FOV4 resistant germplasm, highly resistant lines, Quantitative Trait Loci (QTL)s and biomarkers to track and predict resistance.

Animal Health Component

80%

Research Effort Categories

Basic

20%

Applied

80%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	1710	1080	50%
201	1710	1081	50%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1710 - Upland cotton;

Field Of Science
1081 - Breeding; 1080 - Genetics;

Keywords

dna biomarkers

fusarium wilt

breeding

molecular genetics

upland cotton

Goals / Objectives
The major project goals are to Identify multiple sources of durable genetic resistance to FOV4 in Upland cotton through reliable and proven screening methodology in an FOV4 hotspot and to release these new resistant germplasms to the public. Additional goals include the development of a rapid intercross mating design to quickly stack favorable resistance alleles to release highly resistant germplasm to the public and to deveop an understanding of resistance genetics to develop discreet biomarkers that can be used to track and predict resistance to FOV4 to rapidly introgress this trait into elite breeding lines. The objectives of this project are to:1. Identify sources of resistance to FOV4 in upland cotton by continuing a wide Upland cotton germplasm screen in proven high FOV4 pathogen load fields.2. Improve resistance to FOV4 in Upland cotton through breeding and genetics.3. Elucidate resistance genetics and develop prognostic biomarkers

Project Methods
In this project, we will employ a rigorously vetted disease screening system to quickly screen diverse Upland germplasm accessions for sources of resistance which will be quickly publicly released. In our experience in screening for the same FOV4 resistance in Gossypium barbadense, we anticipate to discover multiple resistance sources which will be utilized in an advanced breeding design to transgressively stack favorable alleles to improve FOV4 resistance in Upland cotton (develop elite or high levels of resistance); which will also be publicly released in the medium time-frame (end of the grant period). We will use molecular genotyping and genome wide association analysis to elucidate the genetics of FOV 4 resistance to release QTLs and prognostic biomarkers to track and predict resistance alleles. We have found that high inoculum load and verification of uniformity in the field is absolutely crucial in determining and tracking resistance. The use of multiple field screening measurements such as early season productivity, vascular staining, and boll load in both the rigorous checks and the germplasm being evaluated accurately quantifies pathogen pressure and disease progression. Combining this large-scale screening/phenotyping approach with molecular genetics to identify and deliver resistant germplasm sources early in the project, and to stack favorable resistance alleles to develop highly resistant lines in combination with prognostic biomarkers, is a novel and aggressive approach to mitigate FOV4 in Upland cotton. The identified biomarkers can be used to screen Upland germplasm collections for new sources of resistance, in addition to tracking and monitoring resistant alleles in breeding programs. Thus, the project will complement breeding programs for developing FOV4 resistant varieties to ensure continued and sustainable cotton production with durable sources of resistance to the devastating FOV4 pathogen. The efforts that we will use to communicate project data will be made at extension meetings, and distributed through the Cotton Incorporated network to reach growers across all 17 states where cotton is grown in the U.S. Furthermore, project outcome reports will be delivered to the Cotton Incorporated Board of Directors, made up of 70+ growers, on strategy, successes, and failures on projects that affect their farming operations. Results will be assessed by the scientific advisory panel (composed of cotton breeding, genetics and extension experts) during the course of each year as critical data is collected, and in annual online meetings. The advisory panel will give feedback to the PD's and recommendations on moving the project forward to maintain alignment with the overall goals.

Progress 08/15/19 to 08/14/22

Outputs
Target Audience:The target audiences reached by our efforts for this project include the following: All major cotton seed company representatives. These representatives were engaged during each year of the project at various meetings/conferences/field days (described in other sections). Cotton Incorporated Board of Directors Agricultural Research Committee. This committee consists of 70 U.S. cotton growers and represents all Upland cotton growing regions in the U.S. The audience at the annual Beltwide Cotton Conference. This conference is attended by allied research scientists, cotton breeders, plant pathologists, industry representatives and is considered the primary annual cotton meeting. Attendees of field days. These include all major cotton seed companies and cotton growers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project supported Stephen Parris (PhD student) who participated in one-on-one training activities with Co-Project Directors (Drs. Jim Olvey and Don Jones) and mentor Mike Olvey. These activities include field work in El Paso, TX. This project also mentored Dr. Whitney Huang (an Assistant Professor of Mathematics and Statistics at Clemson University) who was trained by all project PI's in plant breeding approaches, disease screening in a field and the associated challenges. This training led to a newly formed collaboration to integrate statistical methods to improve plant phenotyping. This idea also translated to a special topics class offered by Dr. Huang to Stephen Parris to develop this concept further as part of his Minor in Statistics accreditation to his Ph.D. degree. Our project team also educated the newly hired Cotton Incorporated pathology director Dr. Kaitlyn Bissonnette on the proper protocol for FOV4 evaluation. Likewise, the Chairmen of Cotton Incorporated (James Johnson) and the Cotton Board (Jimmy Webb) attended both classroom and field based sessions to learn about our protocols. The below professional develoment opportunites were supported by this project: i.Jim and Mike Olvey hosted FOV4 session at 2022 Beltwide Cotton Conference ii.Dr. Jones attended and presented at the Beltwide Cotton Conference in 2022. iii.Stephen Parris attended and presented at the Beltwide Cotton Conference in 2022 iv.Dr. Saski attended the American Society for Plant Biology Meetign in July 2022 v.Drs. Saski and Jones attended the National Association for Plant Breeders meeting in August 2022 vi.Dr. Jones presented the teams findings in invited oral presentations at the 2022 International Cotton Conference in Bremen, Germany vii.and at the 7th World Cotton Research Conference in Cairo, Egypt How have the results been disseminated to communities of interest?All breeders who submitted lines for screening in the FOV4 screening trial were provided data of early season death and late season vascular staining. The data has been distributed to the larger cotton breeding community as well.Dr. Olvey has conveyed screening results to specific breeders who submitted lines for screening. Afterwards Dr. Jones requested of each breeder if those results could be shared with the larger community. If the breeder(s) agreed, those results were shared with the entire public sector cotton breeding community. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In the final year of this project, we discovered discreet knowledge of the genes underlying FOV4 resistance in several Upland cotton lines. Our results show that FOV4 resistance is not controlled by nearly as many genes as has been published over the last 15 years. We advanced 46 lines to expanded field trials that includes adozen more F8 RIL lines that are resistant, but we follow a conservative calling regimen and require three (3) years of screening data before publicly releasing them as 'FOV4 resistant'. We designed and validated diagnostic biomarkers for FOV4 resistance. We also finalized development of a statistical interpolation method to improve field phenotyping that functions to reduce experimental error.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Stephen M. Parris, Steven N. Jeffers, James M. Olvey, James M. Olvey , Jeffrey W. Adelberg, Li Wen, Joshua A. Udall, Jeffrey J. Coleman, Don C. Jones, and Christopher A. Saski. 2022. An in vitro co-culture system for rapid differential responses to Fusarium oxysporum f. sp. vasinfectum Race 4 in domesticated cotton (Plant Disease).
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Stephen�Parris, Whitney Huang, William Bridges, Mike Olvey, Jim Olvey, Don Jones, Christopher Saski. 2022. Statistical modeling of FOV4 field distribution to develop spore-adjusted phenotype scores for genomic analyses. Cotton Beltwide Meeting.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Stephen Parris, Jim Olvey, Don Jones, Christopher Saski. 2022. Transcriptomic analysis of a resistance response to FOV4 in Pima cotton
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Stephen�Parris, Don Jones, Jeffrey Adelberg, Steven Jeffers, Mike Olvey, Jim Olvey, Christopher Saski. 2022. Building better cotton through genomics, engineering, and advanced molecular breeding tools

Progress 08/15/20 to 08/14/21

Outputs
Target Audience:Target audiences reached by our efforts during this reporting period include the following: All major cotton seed company representatives Cotton Incporporated Board of Directors Agricultural Research Committee (consists of 70 U.S. cotton growers) - Results were presented at 3 separate occasions. The audience at the annual Beltwide Cotton Conference (allied scientists, growers, breeders, industry representatives), which is the primary annual national cotton meeting - The PI team were authors in seven presentations at this conference. We presented at five field days directly to all the major cotton seed companies and cotton growers. Our team also gave a report to the California Cotton Growers Changes/Problems:No changes or problems anticipated What opportunities for training and professional development has the project provided?Training activities Stephen Parris (PhD student involved in the project) participated in one-on-one training activities with Co-Project Director (Dr. Jim Olvey) and mentor Mike Olvey. These activities include field work in Clint, TX. Dr. Whitney Huang (an Assistant Professor of Mathematics and Statistics at Clemson University) was trained by all project PI's in plant breeding approaches, disease screening in a field and the associated challenges. This trainng led to a newly formed collaboration to integrated statistical methods to improve plant phenotyping. This idea also translated to a special topics class offered by Dr. Huang to Stephen Parris to develop this concept further as part of his Minor in Statistics accredationto his Ph.D. degree. Undergraduate students were trained in genomic DNA extraction in the Saski lab at Clemson University Dr. Josh Udall and team (USDA-ARS) trained in leaf field leaf sampling and operations at FOV4 nursery Professional Development Jim and Mike Olvey hosted FOV4 session at 2021 Beltwide Cotton Conference Drs. Saski and Jones attended and presented at the Beltwide Cotton Conference in 2021. Stephen Parris attended and presented at the Beltwide Cotton Converence in 2021 Dr. Saski attended the American Society for Plant Biology Meetign in July 2021 Drs. Saski and Jones attended the National Association for Plant Breeders meeting in August 2021 Dr. Saski attended and presented at the Bayer Crop Sciences Academy of Plant Biotechnology in June 2021 Dr. Saski attended and presented at the Univeristy of Illinois Urbana-Champagne Plant Sciences Symposium Dr. Saski attended and presented at the Southeast regional Fruit & Vegetable Conference in December 2020 How have the results been disseminated to communities of interest?All breeders who submitted lines for screening in the FOV4 screening trial were provided data of early season death and late season vascular staining. The data has been distributed to the larger cotton breeding community as well. In addition, development and data on U1 has been released to the larger cotton breeding community. Cotton growers, 70 of whom make up the Cotton Incorpoarted Board of Directors, have attended presentations on the development and success of U1. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will assemble and publish biparental QTL maps of each of the 3 families and make public release of diagnostic molecular markers for FOV4. We will continue line advancement of the 45 lines selected in 2021 with expanded evaluations at the FOV4 nursery We will also continue to advance our populations at the CWN to develop recombinant inbred lines (RILs).

Impacts
What was accomplished under these goals? Leaf samples collected in duplicate from 288 individuals from each of the 3 families. Total samples collected 864 x 2 = 1,728 DNA extracted and genotyped using whole genome skim sequencing from 864 individuals at the HudsonAlpha Instituite for Biotechnology Detailed phenotyping of 864 individulas for vascular stain height and severity, boll load, early and late season productivity 45 lines with FOV4 resistance advanced to expanded field trials Public release of U1 - an Upland genotype with the highest known resistance profile to FOV4 Developed a unique phenotyping approach that integrates surface interpolation statistics with rigorous field check multi-year performance data that mathematically predicts spore density and distribution in the screening field. The result is a spore load adjusted phenotype score that reduces environmental error in the Phenotype = Genotype(G) + Environment(E) + GxE equation. Advanced the 3 populations to the F8 generation

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Stephen M. Parris, Steven N. Jeffers, James M. Olvey, James M. Olvey , Jeffrey W. Adelberg, Li Wen, Joshua A. Udall, Jeffrey J. Coleman, Don C. Jones, and Christopher A. Saski. 2021. An in vitro co-culture system for rapid differential responses to Fusarium oxysporum f. sp. vasinfectum Race 4 in domesticated cotton (Plant Disease)
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Chris Saski, Stephen Parris, Jim Olvey, Mike Olvey, Joshua Udall, and Don Jones. 2021. Deciphering Genetic Resistance Mechanisms to Fusarium Oxysporum Race 4 in Domesticated Cotton (Beltwide Cotton Conference).
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Stephen Parris, Jim Olvey, Mike Olvey, Don Jones, Chris Saski. 2021. An in vitro co-culture system for and rapid differential responses to Fusarium oxysporum f. sp. vasinfectum Race 4 in domesticated cotton (Beltwide Cotton Conference).
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Don Jones, Jim Olvey, and Mike Olvey. 2021. Private / Public Sector Partnership to Address FOV4 (Beltwide Cotton Conference).
• Type: Other Status: Published Year Published: 2021 Citation: Don Jones. 2021. FOV4 - Significant Progress to Date in NIFA/Cotton Board Project (Cotton Incorporated Board of Directors).
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: David W. Albers and Mike Olvey. 2021. Yield of select upland cotton varieties grown in moderate to high disease pressure of Fusarium Race 4 (FOV4): 2018 to 2020 (Beltwide Cotton Conference).
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Jim Olvey. 2021. Origin and Worldwide Distribution of Fusarium Race-4 (FOV-4) (Beltwide Cotton Conference).
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Jim Olvey, Mike Olvey, Don Jones, and Geoff Toledo. 2021. Public Release of OA-FOV4-U1 (Beltwide Cotton Conference).
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Mike Olvey, Jim Olvey, and Don Jones. 2021. Fusarium Race-4 Upland Screening and Evaluation (Beltwide Cotton Conference)

Progress 08/15/19 to 08/14/20

Outputs
Target Audience:Target audiences reached by our efforts during this reporting period include the following: All major cotton seed company representatives Cotton incorporated Board of Directors Agricultural Research Committee (consists of 25 U.S. cotton growers) Audience at the annual Beltwide Cotton Conference (allied scientists, growers, breeders, industry representatives) Changes/Problems:We do not anticipate changes/problems in our approach. What opportunities for training and professional development has the project provided? Training activities. Stephen Parris (PhD student involved in the project) participated in one-on-one training activities with Co-Project Director (Dr. Jim Olvey) and mentor Mike Olvey. These activities include field work in El Paso, TX. In addition, Dr. Joshua Udall (USDA-ARS Crop Germplasm Research Leader, College Station, TX) was trained in plant leaf harvesting for DNA genotyping at the FOV4 hot site in El Paso TX. Professional Development. Drs. Saski and Jones attended the International Plant and Animal Genome meeting, San Diego, CA January of 2020. Drs. Jones and Olvey attended the Beltwide Cotton Conference in Austin, TX 2020. How have the results been disseminated to communities of interest?The FOV4 hot site in El Paso, TX has been made available for access to the broader cotton industry that includes public/private breeders, allied scientists, and non-profits. All breeders who submitted lines for screening in the FOV4 screening trial were provided data of early season death and lateseason vascular staining. The data has been distributed to the larger cotton breeding community as well. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will genotype approximately 300 (or more) F3 individuals segregating for resistance using whole genome skim sequencing for each population. We will collect phenotypes (final stand readings) that will be combined for use in detailed genetic mapping to identify resistance quantitative trait loci (QTLs) and genes of interest. We will also continue to advance our populations at the CWN to develop recombinant inbred lines (RILs).

Impacts
What was accomplished under these goals? i. Massive germplasm screen on FOV4 hotspot identified 2 candidate sources of resistance to FOV4 (a resistant line SA3208 TWICE Reselectioned and moderately tolerant UGA2016016 line). Development of biparental crosses (3 populations developed). Biparental crosses were generated between each resistant/tolerant parent and a susceptible genotype, in addition to a RxR cross at the Cotton Winter Nursery (CWN) that led to the generation of F3 seed. ii. Screening and selection of individuals for genotyping and phenotyping segregating progeny. F3 seed from CWN activities were planted in the FOV4 hot site in El Paso, TX in the Spring of 2020. A total of 288 rows were planted for each population (864 total rows). Each plant was tagged for tracking individual progeny for genotyping and phenotyping. iii. The 3 populations alsoremained at the CWN for the development of inbreds (longer term, higher resolution mapping) were advanced to F5 and 1 boll from a single F5 plant will be soon advanced to make F6. iv. Conducted deep whole genome sequencing of the resistant, tolerant, and susceptible parents. v. We have developed a sterile, in vitro culture system that can support growth of both domesticated cottons (g. barbadense and g. hirsutum) and FOV4. We have demonstrated that a susceptible genotype will succumb to FOV4 in as few as 20 days, and are optimizing this system as a tool for further understanding the interaction between the host and the pathogen.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: "FOV4 NIFA Delivery of Durable Genetic Resistance to FOV4 in Upland Cotton" presented to the Cotton Inc. Board of Directors Agricultural Research Committee
• Type: Journal Articles Status: Other Year Published: 2020 Citation: Stephen Parris, Steven Jeffers, Jim Olvey, Mike Olvey, Jeffrey Adelberg, Li Wen, Joshua A. Udall, Jeffrey Coleman, Don Jones, and Christopher A. Saski. An in vitro co-culture system and rapid differential responses to Fusarium oxysporum f.sp. vasinfectum Race 4 in domesticated cotton. In Submission to Plant Diseases.