Source: N Y AGRICULTURAL EXPT STATION submitted to
GENOME-WIDE ANALYSIS OF DIVERSITY WITHIN PHYTOPHTHORA INFESTANS LINEAGES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1009070
Grant No.
2016-67011-25176
Project No.
NYG-625569
Proposal No.
2015-03627
Multistate No.
(N/A)
Program Code
A7101
Project Start Date
Apr 1, 2016
Project End Date
Mar 31, 2018
Grant Year
2016
Project Director
Hansen, Z. R.
Recipient Organization
N Y AGRICULTURAL EXPT STATION
(N/A)
GENEVA,NY 14456
Performing Department
Plant Path./Plant Microbe Biol
Non Technical Summary
Phytophthora infestans is a highly aggressive and destructive pathogen that causes late blight of potatoes and tomatoes. Although it has been studied for well over a century, it remains one of the most constraining factors in potato and tomato production. In the United States, P. infestans is primarily a clonal organism that reproduces by asexual spores which are non-persistent and aerially-dispersed. This underlies two important facts about P. infestans: (1) the pathogen can only overwinter on living host tissue, which in temperate regions is most likely infected potato tubers left in the ground after harvest or in cull piles; and (2) a few fit individuals, or clonal lineages, populate vast geographic regions and persist for a few to many years. There are currently two major gaps in our understanding of this important pathogen related to these facts: (i) what is the source of inoculum responsible for initiating annual late blight outbreaks; and (ii) how much diversity exists within P. infestans lineages. The goal of this project is to utilize advances in DNA sequencing technology to better understand the genetic diversity and population structure that exists within clonal lineages of P. infestans. This will be accomplished by (1) generating genotyping-by-sequencing (GBS) data from isolates within several clonal lineages; (2) identifying a large number of genetic differences, or single nucleotide polymorphisms (SNPs), from raw GBS data for downstream analyses; and (3) using those analyses to identify population structure within lineages and to make inferences about inoculum sources, dispersal patterns, and genetic divergence over time. This research project addresses both the AFRI Foundational Program Area of "Plant health and production and plant products" as well as the AFRI Challenge Area of "Food security" by generating information that will aid in the management of potato and tomato late blight. This project will also enhance my development as a plant pathology researcher, as well as provide me with opportunities to present my results at scientific and grower meetings, toward the goal of becoming a research and extension plant pathologist. Thus, the overall goal of the AFRI ELI Fellowships Grant Program, which is "to prepare the next generation of scientists through doctoral and post-doctoral fellowships", is fulfilled.
Animal Health Component
0%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21614691160100%
Knowledge Area
216 - Integrated Pest Management Systems;

Subject Of Investigation
1469 - Solanaceous and related crops, general/other;

Field Of Science
1160 - Pathology;
Goals / Objectives
The goals of this project are to determine the level of diversity that exists within clonal populations of Phytophthora infestans, and to use this information to better understand dispersal patterns, inoculum sources, and genetic divergence over time. This will be accomplished through two primary objectives: (1) use genotyping-by-sequencing (GBS) to identify single nucleotide polymorphisms (SNPs) among P. infestans isolates representing clonal lineages US-8, US-11, US-23, and US-24; and (2) identify structure within clonal populations of P. infestans using SNP data in order to make inferences about inoculum sources, dispersal patterns, and genetic divergence over time.
Project Methods
The first objective of this study is to use GBS to identify single nucleotide polymorphisms (SNPs) among P. infestans isolates representing clonal lineages US-8, US-11, US-23, and US-24. Many isolates are in hand, and others are being sent (with proper APHIS permits) by researchers with culture collections at Cornell, NC State and UC Riverside, with the goal of obtaining 80-100 isolates of diverse geographic origin and collection date, for each clonal lineage. Isolates will be grown on pea broth using standard practices. DNA will be extracted from mycelia using a Qiagen DNeasy Plant Mini Kit, and quantified. DNA will then be evaluated for quality and utility in GBS by digesting with ApeK1, followed by gel electrophoresis. All DNA samples that pass minimum quality standards will then be submitted for GBS analysis at the Cornell University Institute of Biotechnology Genomic Diversity Facility. Initial SNP calling will be performed in collaboration with bioinformaticist Katie Hyma at the Genomic Diversity Facility using the TASSEL pipeline. The second objective of this study is to identify structure within clonal populations of P. infestans using SNP data in order to make inferences about inoculum sources, dispersal patterns, and genetic divergence over time. Through these objectives, the following hypotheses will be addressed.Hypothesis 1: Isolates within lineages that are collected near one another, and from multiple years, are genetically more similar to each other than to isolates collected from distant geographic regions. Null hypothesis 1: There is not a genetic association between isolates within lineages and geographic origin.Support for hypothesis 1 would indicate that sub-populations within lineages exist, and remain geographically segregated, as the result of overwintering inoculum initiating regional outbreaks each year. Support for null hypothesis 1 would indicate that inoculum comes from diverse sources each year, and is likely spread via transport of infected seed potatoes and/or tomato seedlings.Hypothesis 2: There is not a genetic association between isolates within lineages and the year they were collected. Null hypothesis 2: Subpopulations of individuals within lineages are observed each year.Support for hypothesis 2 would indicate that many individuals survive the winter to repopulate the country each year, which would be most likely explained by P. infestans overwintering on potato tubers and initiating regional outbreaks each year. Support for null hypothesis 2 would indicate that few individuals survive the winter to repopulate the country each year, and there is a dominant subpopulation across the country each year, which would indicate spread via transport of infected seed potatoes and/or tomato seedlings.Hypothesis 3: Isolates within older clonal lineages (US-8 and US-11) will be more genetically diverse than those from younger clonal lineage (US-23 and US-24) due to the accumulation of mutations over time. Null hypothesis 3: Age of a given lineage is not associated with observed diversity.Knowing the level of genetic diversity within lineages of P. infestans may provide insight into why older genotypes decline. Hypotheses 1, 2, and 3 will be addressed through principle component analysis and the construction of NJ trees using the SNP data generated by GBS. Population genetic analyses will include calculation of genotypic diversity (including richness and evenness), population differentiation and structure (clustering and AMOVA). This work will be done by the PD in collaboration with members of Dr. Christine Smart's laboratory and members of the laboratory of Dr. Niklaus Grunwald (Department of Botany and Plant Pathology, Oregon State University) trained in statistical analysis of genomic data. SNPs will be identified using the TASSEL pipeline, which aligns sequencing reads to the P. infestans genome sequence and applies initial filtering steps to maximize SNP quality. SNP genotypes in variant call format (VCF) will be generated using the TASSEL pipeline, and filtered more stringently on minor allele frequency (.05), read depth (7), maximum missing data per site (0.1), and maximum missing data per individual (0.9) in VCFtools v0.1.12a. Principle component analysis (PCA) will be used to assess population structure both among and within lineages. Genomic regions associated with variation among and within lineages will be determined by identifying PCA-correlated SNPs. This procedure will also be used to determine the minimum number of SNPs capable of differentiating the four lineages. Efforts to disseminate the knowledge created during the course of the project will include presentations at scientific conferences, extension talks at grower meetings, and publication of results in a peer reviewed journal. The project will be evaluated by feedback from my annual PhD committee meeting (Smart and three other faculty, see recommendation letters), and by completion of appropriate milestones. Milestones include coursework (completed), research progress including obtaining P. infestans DNA and submitting for GBS in year 1 and analyzing genomic data and writing results in year 2 (assessed weekly with mentor meetings), student mentoring (summers of years 1 and 2), leadership/mentoring workshop (year 1), scientific, grower and general public presentations (throughout project), thesis preparation (throughout, and to be completed in year 2), and publications (year 2). Additionally, I submit an annual self-evaluation that provides an opportunity for assessment. Finally I will receive survey feedback from grower and public presentations so I can improve and ensure my talks are easily understood.

Progress 04/01/16 to 03/31/18

Outputs
Target Audience:The target audience for this project is primarily scientists in the fields of plant pathology, population biology, and agriculture. Our reseach efforts have resulted in a peer-reviewed publication that was accepted for publication on July 13 in the journal PLOS ONE. This publication will be available to the scientific community in the near future, and will contrbute to the existing body of work concerning Phytophthora infestans biology and late blight epidemiology and management. Additionally, the results obtained through this study have been shared by the PI through invited lectures at Ohio State University and Michigan State University. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This work gave the PI (Hansen) the opportunity to gain proficiency in working with and analyzing large genomic data sets. This is an invaluable skill for an early-career life scientist, especially as DNA sequencing technologies continue to advance. Additionally, this project allowed the PI to work closely with collaborators from across the country in the Phytophthora infestans and late blight research communities, expanding his professional network. This work was also presented by the PI the plant pathology departments of Ohio State University as well as Michigan State University. How have the results been disseminated to communities of interest?This project included a vast network of collaborators, many of whom are involved in extension and oureach efforts. These collaborators now have the opportunity to communicate the results of this study to potato and tomato growers and to reinforce the importance of starting each season with disease free planting material. The scientific results have been accepted for publication in the journal PLOS ONE. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Late blight is a devsastating disease of potato and tomato. This disease was the primary cause of the Irish potato famine of the 19th century and continues to devastate potato and tomato crops globally. Although much has been learned about the disease and its causal organism, Phytophthora infestans (p. infestans), much remains unknown. For example, we know that this pathogen has a clonal reroductive lifesyle in many parts of the world incuding the United States. Therefore, we know that members of a clonal lineage, or strain, are highly genetically similar. However, we also expect mutations to accumulate over time which would lead to some genetic diversity among individuals within clonal lineages. Advances in DNA sequencing technologies now make it possible to identify differences among very closely related individuals, including members of clonal lineages, in much greater detail than was previouslly possible. Our first goal was to use a relatively new approach to genotyping, called genotyping-by-sequencing (GBS), to identify diversity among individuals within four important P. infestans clonal lineages. Using this approach we identified a large amount of diversity within clonal lineages of this pathogen, represented by between 4,000 and 5,000 genetic markers (single nucleotide polymorphisms or SNPs) per lineage. We then analyzed these markers to increase our understanding of late blight epidemiology. The genetic data have been made publicly available to support future studies on P. infestans population biology. Additionlly, we know that this pathogen can spread through the air and on infected plant material (primarily seed potatoes), as well as overwinter in infected potato tubers left over after harvest. However, the relative contribution of each of these inoculum sources to annual late blight outbreaks is not well understood. The second objective of this study was to use the GBS data to analyze how the pathogen spreads throughout the United States on an annual basis. We discovered that the pathogen is regularly transported long distances, and that infected plant material likely plays an important role in initiating annual late blight outbreaks. This highlights the need for potato and tomato growers to begin each season with disease-free planting material. To obtain these results, we sequenced 257 P. infestans isolates originally collected from across the United States by collaborators, extension educators and specialists, as well as potato and tomato growers. These isolates belonged to four clonal lineages, called US-8, US-11, US-23, and US-24, all of which have caused significant late blight outbreaks in recent years. Of the 257 isolates included in the study, 166 belonged to lineage US-23, and so this lineage was a focus of the overall analysis. Isolates for the study were chosen to refelct the maximum breadth of geographic as well as temporal diversity that we had access to. Using GBS we identified between 3774 and 5070 SNPs per lineage. Using K-means hierarchical clustering as a means to identiify population structure within lineages, we discovered that US-23 individuals clustered together more by collection year than by geographic origin. This result indicates that P. infestans is regularly moving long distances throughout the United States rather than remaining geogrpahically isolated, which would have resulted in the detection of geographically-isolated sub-populations. Neighbor-joining trees were also constructed for each of the four lineages to assess population structure. All four neighbor-joining trees revealed evidence for regional pathogen dispersal and overwintering, as well as long distance pathogen transport. The US-23 neighbor-joining tree showed prominent groupings of individuals by year from distant geographic origins, which supported the K-means hierarchical clustering result and provided further evidence that late blight inoculum is spread over long distances through the transport of infected plant material. Finally, this work has demonstrated the feasibility of using genotyping-by-sequencing to conduct population studies of clonal individuals. This work will help to pave the way for future studies aimed at increasing our understanding of diversity within other important clonal organisms.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Hansen, Z.R., Everts, K.L., Fry, W.E., Gevens, A.J., Gr�nwald, N.J., Gugino, B.K., Knaus, B.J., Johnson, D.A., Johnson, S.B., Judelson, H.S., McGrath, M.T., Myers, K.L., Ristaino, J.B., Roberts, P.D., Secor, G.A., & Smart, C.D. 2016. Genetic variation within clonal lineages of Phytophthora infestans revealed through genotyping-by-sequencing, and implications for late blight epidemiology. Accepted by PLoSONE.