Progress 09/01/15 to 08/31/19
Outputs
Target Audience:The target audience is primarily researchers and students involved in the development of woody bioenergy crops in the US and internationally. This work also benefits professionals in agriculture, horticulture, and associated businesses that have interest in on-farm production of renewable feedstocks for heat, power, and biofuels. This work is engaging researchers and students in the field of plant pathology, especially those with interest in rust fungi and host resistance mechanisms. It is also engaging students and researchers in plant breeding, especially those interested in woody perennials. Changes/Problems:Several of the project objectives were based on marker analysis using genotyping-by-sequencing originally planned and budgeted to be done at the Cornell Institute of Biotechnology in Ithaca, NY. However, Cornell University lost a law suit brought by KeyGene for patent violation in conducting GBS and has ceased to conduct GBS on a service basis. Instead, we contracted for plant DNA isolation and GBS services through the University of Wisconsin-Madison at higher costs. The DNA isolation method they used was not effective for certain species hybrids of willow, and the GBS failed for a high proportion of samples. The Wisconsin lab ran GBS analysis at no additional cost using DNA isolated in our lab - at additional cost, but these failures severely delayed the completion of the F1 species hybrid maps. What opportunities for training and professional development has the project provided?A Ph.D. student in Plant Pathology, Chase Crowell, is working with co-PI Chris Smart on the pathogen side of the project, and he has gained experience in identifying and isolating Melampsora, propagating single-pustule isolates, collecting spores, extracting DNA, and analysis of GBS data. He has participated in regular project meetings and has gained a strong understanding of project aims and objectives. An undergraduate research intern, Patrick McMullen, worked on the project for nine weeks in summer 2018 in Chris Smart's lab to characterize the genetic diversity of Melampsora isolates. A Ph.D student in Plant Breeding, Dustin Wilkerson, started working in PI Larry Smart's lab in January 2017 to study the plant breeding and genomics side of the project. He is gaining experience in statistical analysis of rust survey data, bioinformatic processing of GBS data, genetic mapping and QTL analysis, and differential expression and mapping of eQTL using RNAseq data. He has participated in regular Smart lab meetings, regular project meetings, presented his research within the department and at scientific conferences and is gaining an understanding of willow genetics and genomics and its application to other crops. How have the results been disseminated to communities of interest?Results on Melampsora genetic diversity and pathogenicity were presented in poster presentations at the DOE Genomic Science PI Meeting in Crystal City, VA in Feb 2017 and at the Annual JGI Users Meeting in Walnut Creek, CA in March 2017. Carlson, et al. (2019) reports detected QTL for rust resistance from the S. purpurea F2 population. Preliminary results of rust severity and population structure of the F1 species hybrid families were presented via poster at the Plant and Animal Genome Conference in San Diego, CA. in January 2018, while more expanded results and preliminary QTL were presented in both poster and presentation form at PAG in 2019. Preliminary results of differential expression and eQTL for infection response will be presented at PAG 2020. Project activities have also been disseminated via Twitter through the handles @CornellWillow and @ChristineSmart6. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
To address Objective 1, we collected Melampsora leaf rust from Salix purpurea and commercial hybrids from sites in NY, WV, PA, VT, and MI in the summer/fall of 2015 and purified 184 single-spore isolates through in planta propagation on detached Salix purpurea leaves. Very little rust disease was observed in the 2016 growing season, so only 10 additional collections were made. From July to November 2017, infected leaves from 40 shrubs resulting in 120 isolates were collected representing four locations, with the hopes to investigate within-field diversity. DNA was extracted following spore collection for 268 of the total isolate collection for genotyping by sequencing. Raw reads were received September 12, 2017, May 21, 2018, and August 9, 2019. A Community Science Program project submitted by co-PI Chris Smart was approved by the DOE Joint Genome Institute to sequence the genome of our M. americana isolate R15-033-03. We prepared 40 µg of high quality gDNA, which was used by JGI to construct two 10 kb libraries that were sequenced using PacBio. Additionally, 10 µg of total RNA from leaves of the reference willow genotype 94006 infected with M. americana R15-033-03 and sampled at 10 different time intervals then pooled was submitted to generate RNA-Seq data to aid in gene annotation of the M. americana genome assembly. This reference genome was made available July 31, 2018, made public on the JGI portal July 20, 2019, and was used as a reference in the Tassel 5 pipeline for variant calling. Resulting variants were used for a preliminary assessment of the population with a principal component analysis. This analysis identified some separation by location across large geographic distances ranging from Vermont to Pennsylvania as well as scattered individuals with no population structure. However, significant clonality does exist within a single field, suggesting relatively high homogeneity among inoculum within a single infected field. To further address within-field pathogen epidemiology, early season rust isolates were collected in 2018, and surrounding shrubs from original collection sites were also samples later in the season to investigate within-field rust spread. A manuscript from this work is in preparation for peer review. To address Objective 2, in late summer 2017 we surveyed the progeny, parents, and grandparents in a S. purpurea F2 mapping population planted in a replicated trial in Geneva, NY. This population has been genotyped by Genotyping-by-Sequencing (GBS), filtered for the highest quality markers and individuals, and used to construct a linkage map for the population. Using the rust severity survey data, quantitative trait loci (QTL) were discovered in 2015 on chromosome 1, 5, and 10 and a single overlapping QTL in 2017 on chromosome 1. These results and QTL for many other traits produced an article (Carlson, et al., 2019). Further analysis into candidate genes and associated markers within these QTL intervals is underway to determine the potential for marker assisted selection for rust resistance. This process is aided exponentially by the availability of the S. purpurea v5.1 reference genome of '94006' on Phytozome. The approach for objective 3 utilized field rust severity surveys collected from the S. purpurea F2 mapping population in 2015 and 2017 to identify 28 resistant and 28 susceptible Salix spp. genotypes. These genotypes along with the parents and grandparents were included in a randomized complete block design with two replications of two treatment groups, inoculated and uninoculated (control), and three time points of leaf collection for 3'RNAseq. Starting six weeks after planting in the greenhouse, T0 is the uninoculated control for both treatments, collected just prior to the inoculation of the inoculated treatment group with Melampsora reference isolate R15-033-03. Both treatments were placed in separate mist chambers overnight and removed 12 hours later. Additional leaf collections occurred at 42 and 66 hours post inoculation, time points determined by a small pilot study to be the most informative. Rust severity ratings were collected seven days post inoculation and correlated to field measurements of the same plants. The correlations for 2015 and 2017 are 0.48 and 0.53 for Rep 1, and 0.66 and 0.64 for Rep 2, suggesting strong relationship between greenhouse and field ratings. Extracted RNA was sent to the Cornell Institute of Biotechnology for 3'RNA sequencing. Analysis of differential expression and eQTL detection are currently underway but preliminary results suggest that our experimental design is producing strong differentially expressed genes between treatment groups that relate to their divergence through time as infection progresses. Objective 4 involves mapping rust resistance genes in eight F1 species hybrid families that share a common Salix purpurea parent, '94006' or '94001'. Each population is an F1 species hybrid with S. viminalis, S. suchowensis, S. koriyanagi, S. integra, or S. alberti all demonstrating a varied susceptibility to M. americana, with S. viminalis displaying the greatest resistance and S. suchowensis the least. Three of the eight families were established in previous field trials in 2013 and 2014 with 88, 100, and 100 individuals. In 2016, we planted 150 individuals from each of the remaining five F1 mapping populations in field trials in Geneva, NY. Leaf rust severity ratings were collected in 2017, the first-year of post-coppice regrowth, and 2019. Rust presence in 2018 was negligible and therefore no survey was conducted. These mapping populations have been genotyped using GBS at Univ. of Wisconsin Biotech Center although significant difficulty performing quality extractions greatly delayed this project. Called variants for each of the eight families were used to develop linkage maps for each parent using backcross markers resulting in 16 separate maps for use in QTL mapping for rust resistance. These maps are near completion.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Carlson, C. H., Gouker, F. E., Crowell, C. R., Evans, L., DiFazio, S. P., Smart, C. D., Smart, L. B. 2019. Joint linkage and association mapping of complex traits in shrub willow (Salix purpurea L.). Annals of Botany. doi:10.1093/aob/mcz047.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Wilkerson, D. G, Crowell, C. R., Carlson, C. H., Gouker, F. E., Smart, C. D., Smart, L. B. Multiple Approaches to Mapping Resistance to Melampsora Leaf Rust in Shrub Willow (Salix spp.) . January 2019. Poster. 2019 Plant and Animal Genome Conference, San Diego, CA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Wilkerson, D. G, Crowell, C. R., Carlson, C. H., Gouker, F. E., Smart, C. D., Smart, L. B. Multiple Approaches to Mapping Resistance to Melampsora Leaf Rust in Shrub Willow (Salix spp.) . January 2019. Presentation. 2019 Plant and Animal Genome Conference: Forest Tree Workshop, San Diego, CA.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
Wilkerson, D. G., Crowell, C. R. Smart, C. D., Smart, L. B. Salix purpurea eQTL for Response to Infection by Willow Leaf Rust, Melampsora americana . January 2020. Presentation. 2020 Plant and Animal Genome Conference: Plant Disease Resistance Workshop, San Diego, CA.
|
Progress 09/01/17 to 08/31/18
Outputs
Target Audience:The target audience is primarily researchers and students involved in the development of woody bioenergy crops in the US and internationally. This work also benefits professionals in agriculture and horticulture and associated businesses that have interest in on-farm production of renewable feedstocks for heat, power, and biofuels. This work is engaging researchers and students in the field of plant pathology, especially those with interest in rust fungi and host resistance mechanisms. It is also engaging students and researchers in plant breeding, especially those interested in woody perennials. Changes/Problems:Several of the project objectives were based on marker analysis using genotyping-by-sequencing originally planned and budgeted to be done at the Cornell Institute of Biotechnology in Ithaca, NY. However, Cornell University lost a law suit brought by KeyGene for patent violation in conducting GBS and has ceased to conduct GBS on a service basis. Instead, we contracted for plant DNA isolation and GBS services through the University of Wisconsin-Madison at higher costs. The DNA isolation method they used was not effective for certain species hybrids of willow, and the GBS failed for a high proportion of samples. The Wisconsin lab ran GBS analysis at no additional cost using DNA isolated in our lab - at additional cost, but these failures severely delayed the completion of the F1 species hybrid maps. What opportunities for training and professional development has the project provided?A Ph.D. student in Plant Pathology, Chase Crowell, is working with co-PI Chris Smart on the pathogen side of the project, and he has gained experience in identifying and isolating Melampsora, propagating single-pustule isolates, collecting spores, extracting DNA, and analysis of GBS data. He has participated in regular project meetings and has gained a strong understanding of project aims and objectives. An undergraduate research intern, Patrick McMullen, worked on the project for nine weeks in summer 2018 in Chris Smart's lab to characterize the genetic diversity of Melampsora isolates. A Ph.D student in Plant Breeding, Dustin Wilkerson, started working in PI Larry Smart's lab in January 2017 to study the plant breeding and genomics side of the project and is gaining experience in statistical analysis of rust survey data, bioinformatic processing of GBS data, and genetic mapping and QTL analysis. He has participated in regular Smart lab meetings, regular project meetings, and is gaining an understanding of willow genetics and genomics. How have the results been disseminated to communities of interest?Results on Melampsora genetic diversity and pathogenicity were presented in poster presentations at the DOE Genomic Science PI Meeting in Crystal City, VA in Feb 2017 and at the Annual JGI Users Meeting in Walnut Creek, CA in March 2017. Preliminary results of rust severity and genomic population structure of the F1 species hybrid families were presented via poster at the Plant and Animal Genome Conference in San Diego, CA. in January 2018. Project activities have also been disseminated via Twitter through the handles @CornellWillow and @ChristineSmart6. What do you plan to do during the next reporting period to accomplish the goals?In the next year, we plan to complete the following tasks to meet our objectives. For objective 1, we plan to complete the genotyping-by-sequencing marker analysis of at least 95 additional isolates of Melampsora americana and use the reference genome developed by JGI for additional pathogenomic investigation.. For objective 2, we will map QTL from 2017 rust severity data in association and F2 mapping populations and compare with mapping results from 2015 data. With improved PacBio assemblies of S. purpurea we will focus on identifying candidate genes at QTL loci and developing marker-assisted selection strategies. For objective 3, we will complete the RNA extraction of leaf tissue and submit these samples for sequencing. Once completed, we will identify eQTL to characterize the response to infection. For objective 4, we will complete the development of pseudo testcross maps for each of the eight F1 families. Then, we will use to maps to identify QTL for rust resistance.
Impacts
What was accomplished under these goals?
To address Objective 1, we collected Melampsora leaf rust from Salix purpurea and commercial hybrids from sites in NY, WV, PA, VT, and MI in the summer/fall of 2015 and purified 184 single-spore isolates through in planta propagation on detached Salix purpurea leaves. Very little rust disease was observed in the 2016 growing season, so only 10 additional collections were made. From July to November 2017, infected leaves from 40 shrubs were collected representing four locations, with the hopes to investigate within-field diversity. For 166 of the total isolate collection, spores were collected, DNA extracted, and genotyping by sequencing was performed. Raw reads were received September 12, 2017, and May 21, 2018. A Community Science Program project submitted by co-PI Chris Smart was approved by the DOE Joint Genome Institute to sequence the genome of our M. americana isolate R15-033-03. We prepared 40 µg of high quality gDNA, which was used by JGI to construct two 10 kb libraries that were sequenced using PacBio. Additionally, 10 µg of total RNA from leaves of the reference willow genotype 94006 infected with M. americana R15-033-03 and sampled at 10 different time intervals then pooled was submitted to generate RNA-Seq data to aid in gene annotation of the M. americana genome assembly. This reference genome was made available July 31st, 2018 and was used as a reference in the Tassel 5 pipeline for variant calling. Resulting variants were used for a preliminary assessment of the population with a principal component analysis. This analysis identified some separation by location across large geographic distances ranging from Vermont to Pennsylvania as well as scattered individuals with no population structure. However, significant clonality does exist within a single field, suggesting relatively high homogeneity among inoculum within a single infected field. To further address within-field pathogen epidemiology, early season rust isolates were collected in 2018, and surrounding shrubs from original collection sites were also samples later in the season to investigate within-field rust spread. Further GBS analyses will be performed on the remaining isolates from 2015, 2016, 2017, and 2018, and genetic differences will be calculated. To address Objective 2, in late summer 2017 we surveyed the progeny, parents, and grandparents in a S. purpurea F2 mapping population planted in a replicated trial in Geneva, NY. Since this population had already been genotyped by GBS, we used these data to map QTL for rust severity. When compared to QTL mapped using survey data from 2015, we detected overlapping QTL. These QTL mapping results will be further analyzed to identify candidate genes for more detailed analysis and for the development of markers for marker-assisted selection. This will be greatly aided by very high quality PacBio genome assemblies being constructed by JGI and Hudson Alpha as a part of a Community Science Program project summitted by collaborator Steve DiFazio with Larry Smart. These are being annotated at JGI and should be released soon. The approach for objective 3 utilized field rust severity surveys collected from the S. purpurea F2 mapping population in 2015 and 2017 to identify candidate 28 resistant and 28 susceptible genotypes. These genotypes along with the parents, 'Fish Creek' and 'Wolcott', and grandparents, '94006' and '94001', were included in a randomized complete block design with two replications of two treatment groups, inoculated and uninoculated (control), and three time points of leaf collections. Six weeks after planting, T0 was collected from both treatments just prior to the brush inoculation of the inoculated treatment group with reference Melampsora leaf rust isolate R15-033-03. Both treatments were placed in separate mist chambers overnight to be removed the next morning. Additional leaf collections were made post inoculation at 42, and 66 hrs. Rust severity ratings were collected seven days post inoculation and correlated to field measurements to validate that our experiment was representative of the field. RNA extractions of leaf discs are currently underway and will be sent to the Cornell Institute of Biotechnology for sequencing. Once competed this data will be used to map eQTL for response to infection by Melampsora leaf rust Our tactic to address objective 4 involves mapping rust resistance genes in eight F1 species hybrid families in which there is a common Salix purpurea reference genotype parent, either female, 94006, or male, 94001. The populations are F1 species hybrids with S. viminalis, S. suchowensis, S. koriyanagi, S. integra, or S. alberti which show differential susceptibility to M. americana, with S. viminalis displaying the greatest resistance. Three of the eight families in the F1 mapping strategy were already established in field trials in 2013 and 2014 with 88, 100, and 100 individuals. In 2016 we planted 150 individuals in each of the remaining five F1 mapping populations in field trials in Geneva, NY. We surveyed rust severity in first-year post-coppice regrowth of all eight common parent F1 hybrid mapping populations in 2017 but due to low field presence we were unable to collect 2018 survey data. These mapping populations have been genotyped by GBS at Univ. of Wisconsin Biotech Center although the Wisconsin lab had significant difficulty extracting DNA from certain willow populations. This task is behind schedule due to the technical failures of the Wisconsin lab, but SNP variants have been called and development of pseudo-test cross maps for each population is underway.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Dustin G. Wilkerson, Craig H. Carlson, Chase R. Crowell, Christine D. Smart, Lawrence B. Smart. Dissecting Genetic Resistance to Willow Leaf Rust (Melampsora spp.) Using Common Parent Mapping Populations of Shrub Willow (Salix spp.), a Biomass Energy Crop . 2018 Plant and Animal Genome Conference, San Diego, CA. January 2018. Poster Presentation.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Crowell, C., Carlson, C., Wilkerson, D., Smart, L., Smart, C. Population structure and genetic diversity of Melampsora spp. collected from Salix purpurea in the Northeast United States. 2018. Phytopathology 108(10)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Smart, L.B., Craig Carlson, Eric Fabio, Michelle Serapiglia, Yongwook Choi, Agnes Chan, Chris Town, Ran Zhou, and Steve DiFazio. Transcriptome analysis of diploid and triploid species hybrids of shrub willow , WATBio Conference, Oxford University, Oxford, UK, Sept. 25, 2017, Invited speaker.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Smart,L.B., Craig Carlson, Fred Gouker, Dustin Wilkerson, Chase Crowell, Chris Smart, Yongwook Choi, Agnes Chan, Chris Town, Ran Zhou, Steve DiFazio. Dissecting the genomic basis for triploid heterosis and disease resistance in willow hybrids , DOE Genomic Science Program Annual Meeting, Tysons Corners, VA, Feb. 27, 2018. Invited plenary talk.
|
Progress 09/01/16 to 08/31/17
Outputs
Target Audience:The target audience is primarily researchers and students involved in the development of woody bioenergy crops in the US and internationally. This work also benefits professionals in agriculture and horticulture and associated businesses that have interest in on-farm production of renewable feedstocks for heat, power, and biofuels. This work is engaging researchers and students in the field of plant pathology, especially those with interest in rust fungi and host resistance mechanisms. It is also engaging students and researchers in plant breeding, especially those interested in woody perennials. Changes/Problems:Several of the project objectives were based on marker analysis using genotyping-by-sequencing originally planned and budgeted to be done at the Cornell Institute of Biotechnology in Ithaca, NY. However, Cornell University lost a law suit brought by KeyGene for patent violation in conducting GBS and has ceased to conduct GBS on a service basis. Instead, we contracted for plant DNA isolation and GBS services through the University of Wisconsin-Madison at higher costs. The DNA isolation method they used was not effective for certain species hybrids of willow, and the GBS failed for a high proportion of samples. The Wisconsin lab is rerunning GBS analysis at no additional cost using DNA isolated in our lab - at additional cost, but these failures have severely delayed the completion of the common parent hybrid maps. What opportunities for training and professional development has the project provided?A Ph.D. student in Plant Pathology, Chase Crowell, is working with co-PI Chris Smart on the pathogen side of the project, and he has gained experience in identifying and isolating Melampsora, propagating single-pustule isolates, collecting spores, extracting DNA, and analysis of GBS data. He has participated in regular project meetings and has gained a strong understanding of project aims and objectives. An undergraduate research intern, Rebecca Crust, worked on the project for nine weeks in summer 2017 in Chris Smart's lab to characterize the genetic diversity of Melampsora isolates. A Ph.D student in Plant Breeding, Dustin Wilkerson, started working in PI Larry Smart's lab in January 2017 to study the plant breeding and genomics side of the project and is gaining experience in statistical analysis of rust survey data, in bioinformatic processing of GBS data, and in genetic mapping and QTL analysis. He has participated in regular Smart lab meetings and is gaining an understanding of willow genetics and genomics. How have the results been disseminated to communities of interest?Results on Melampsora genetic diversity and pathogenicity were presented in poster presentations at the DOE Genomic Science PI Meeting in Crystal City, VA in Feb 2017 and at the Annual JGI Users Meeting in Walnut Creek, CA in March 2017. Project activities have also been disseminated via Twitter through the handles @CornellWillow and @ChristineSmart6. What do you plan to do during the next reporting period to accomplish the goals?In the next year, we plan to complete the following tasks to meet our objectives. For objective 1, we plan to complete the genotyping-by-sequencing marker analysis of at least 95 additional isolates of Melampsora americana and work with JGI to complete the Melampsora americana genome sequence and annotation. For objective 2, we will map QTL from 2017 rust severity data in association and F2 mapping populations and compare with mapping results from 2015 data. With improved PacBio assemblies of S. purpurea we will focus on identifying candidate genes at QTL loci and developing marker-assisted selection strategies. For objective 3, we are characterizing the time line of disease development in order to optimize tissue collection for eQTL analysis of resistant and susceptible willow genotypes. We plan to carry this out using two time points - one very early to detect differential plant responses and one later to characterize changes in fungal gene expression in response to plant defenses. For objective 4, we will complete the GBS marker development of the eight F1 species hybrid families, allowing us to map resistance genes based on the rust severity surveys conducted in 2017. We will complete additional rust severity surveys if disease is present in 2018.
Impacts
What was accomplished under these goals?
To address Objective 1, we collected Melampsora leaf rust from Salix purpurea and commercial hybrids from sites in NY, WV, PA, VT, and MI in the summer/fall of 2015 and purified 184 single-spore isolates through in planta propagation on detached Salix purpurea leaves. Very little rust disease was observed in the 2016 growing season, so only 10 additional collections were made. From July to November 2017, infected leaves from 40 shrubs were collected representing four locations. Those isolates are being purified to single pustule isolates. For 95 of the 2015 single pustule isolates, spores were collected, DNA extracted, and genotyping by sequencing was performed. Raw reads were received September 12, 2017, and the M. larici-populina genome (v2) was used as a reference in the Tassel 5 pipeline for variant calling. Resulting variants were used for a preliminary assessment of the population with a principal component analysis. This analysis identified some separation by location as well as scattered individuals with no population structure. There does not appear to be extensive clonality among the isolates. Because the populations in Portland NY, and Morgantown WV appear to be distinct from other isolates, we collected additional isolates from these locations in 2017 along with isolates from two locations in Geneva NY. Further GBS analyses will be performed on the remaining isolates from 2015, 2016 and 2017 and genetic differences will be calculated. A Community Science Program project submitted by co-PI Chris Smart was approved by the DOE Joint Genome Institute to sequence the genome of our M. americana isolate 033-03. We prepared 40 µg of high quality gDNA, which was used by JGI to construct two 10 kb libraries that were sequenced using PacBio. Additionally, 10 µg of total RNA from leaves of the reference willow genotype 94006 infected with M. americana 033-03 and sampled at 10 different time intervals then pooled was submitted to generate RNA-Seq data to aid in gene annotation of the M. americana genome assembly. This reference genome of M. americana will be much better for GBS variant calling than the M. larici-populina genome and can be a future resource for characterizing effectors. To address Objective 2, in late summer 2017 we surveyed rust severity in Salix purpurea association populations planted on three sites, in Geneva, NY, Portland, NY, and Morgantown, WV. We also surveyed the progeny, parents, and grandparents in a S. purpurea F2 mapping population planted in a replicated trial in Geneva, NY. Since both of these populations were already genotyped by GBS, we can use them to map QTL for rust severity and compare these QTL with those mapped using survey data from 2015. These QTL mapping results will be further analyzed to identify candidate genes for more detailed analysis and for the development of markers for marker-assisted selection. This will be greatly aided by very high quality PacBio genome assemblies being constructed by JGI and Hudson Alpha as a part of a Community Science Program project summitted by collaborator Steve DiFazio with Larry Smart. These are being annotated at JGI and should be released soon. Our tactic to address objective 4 involves mapping rust resistance genes in eight F1 species hybrid families in which there is a common Salix purpurea reference genotype parent, either female 94006 or male 94001. The populations are F1 species hybrids with S. viminalis, S. suchowensis, S. koriyanagi, S. integra, or S. alberti which show differential susceptibility to M. americana, with S. viminalis displaying the greatest resistance. Three of the eight families in the F1 mapping strategy were already established in field trials in 2013 and 2014 with 88, 100, and 100 individuals. In 2016 we planted 150 individuals in each of the remaining five F1 mapping populations that were not already established in field trials in Geneva, NY. We surveyed rust severity in first-year post-coppice regrowth of all eight common parent F1 hybrid mapping populations. These mapping populations are being genotyped by GBS at Univ. of Wisconsin Biotech Center, since the Cornell Institute of Biotechnology has stopped performing GBS when it lost a law suit from KeyGene. The Wisconsin lab had significant difficulty extracting DNA from certain willow populations, so we have had to isolate DNA in our lab in order to complete the GBS. This task is behind schedule due to the technical failures of the Wisconsin lab, but once the genotyping is finished, we will call SNP variants and develop pseudo-test cross maps for each population.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Lawrence B. Smart, Chase R. Crowell, Mariami M. Bekauri, Craig H. Carlson, Fred E. Gouker, Ali Cala, and Christine D. Smart. Genomics-Assisted Breeding for Leaf Rust (Melampsora) Resistance in Shrub Willow (Salix) Bioenergy Crops DOE Genomic Science PI Meeting, Crystal City, VA, Feb 6, 2017. Poster presentation.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Chase R. Crowell, Mariami Bekauri, Craig H. Carlson, Lawrence B. Smart, and Christine D. Smart Genomic resources to study the genetic diversity of the shrub willow pathogen, Melampsora americana JGI Users Meeting, Walnut Creek, CA, March 20-23, 2017, Poster presentation.
|
Progress 09/01/15 to 08/31/16
Outputs
Target Audience:The target audience is primarily researchers and students involved in the development of woody bioenergy crops in the US and internationally. This work also benefits professionals in agriculture and horticulture and associated businesses that have interest in on-farm production of renewable feedstocks for heat, power, and biofuels. This work is engaging researchers and students in the field of plant pathology, especially those with interest in rust fungi and host resistance mechanisms. It is also engaging students and researchers in plant breeding, especially those interested in woody perennials. Changes/Problems:Several of the project objectives were based on marker analysis using genotyping-by-sequencing originally planned and budgeted to be done at the Cornell Institute of Biotechnology in Ithaca, NY. However, Cornell University lost a law suit brought by KeyGene for patent violation in conducting GBS and has ceased to conduct GBS on a service basis. Instead, we will contract for GBS services through the University of Wisconsin-Madison at a slightly higher cost. What opportunities for training and professional development has the project provided?A Ph.D. student in Plant Pathology, Chase Crowell, was recruited to work with co-PI Chris Smart on the pathogen side of the project, and he has gained experience in identifying and isolating Melampsora, propagating single-pustule isolates, collecting spores, extracting DNA, amplifying sequences by PCR, and analyzing those sequences. He has participated in regular project meetings and has gained a strong understanding of project aims and objectives. Two summer undergraduate research interns, Ali Cala and Anna Agloro, were recruited to work on the project for nine weeks in 2016, Ali working with Chris Smart to characterize pathogenicity of Melampsora isolates and Anna working with Larry Smart to collect tissue from F1 hybrid families for genotyping and to characterize growth of the hybrid families. A Ph.D candidate, Dustin Wilkerson, has been recruited to join PI Larry Smart's lab to study the plant breeding and genomics side of the project starting in Jan. 2017. How have the results been disseminated to communities of interest?Initial results on Melampsora genetic diversity and pathogenicity were presented in a poster presentation at the Annual Meeting of the American Phytopathological Society in Tampa, FL in August 2016. Project activities have also been disseminated via Twitter through the handles @CornellWillow and @ChristineSmart6. What do you plan to do during the next reporting period to accomplish the goals?In the next year, we plan to complete the following tasks to meet our objectives. For objective 1, we plan to complete the genotyping-by-sequencing marker analysis of at least 95 isolates of Melampsora americana. For objective 2, we will cut back the willow plants in all mapping populations and survey rust incidence in the late summer and fall of 2017 to replicate mapping results of 2016. We will also further analyze the QTL identified in 2016. For objective 3, we will begin to characterize the time line of disease development in order to optimize tissue collection for eQTL analysis of resistant and susceptible willow genotypes. For objective 4, we will complete the GBS marker development of the eight F1 species hybrid families, allowing us to map resistance genes based on rust severity surveys to be conducted in 2017.
Impacts
What was accomplished under these goals?
To address Objective 1, we collected Melampsora leaf rust from Salix purpurea and commercial hybrids from sites in NY, WV, PA, VT, and MI in the summer/fall of 2015. Single pustule isolates were purified from the infected leaves and were cultured on clean leaves of Salix purpurea under controlled environment conditions. We have developed approximately 300 single-spore isolates through in planta propagation on detached Salix purpurea leaves on water agar in Petri dishes that are kept in growth chambers at 100% humidity (16 h light) for at least three cycles of single pustule inoculations to ensure that the ensuing collected spores are of a single genotype. Each sample is contained it its own Petri dish to prevent cross contamination. Cultures have been preserved in a -20C freezer. We have demonstrated that we can use these frozen cultures to initiate new infection on clean willow leaves. Spores were collected using a "cyclone spore collector" made by Geoff Harms at the University of Minnesota, thus avoiding the inclusion of plant tissue. Collected spores were frozen until ready for DNA extraction. DNA isolations are performed using a chloroform-phenol extraction method developed by Vanesa Segovia of ICARDA for rust uredospores. This method includes lysing the uredospores with silica beads and an extraction buffer, followed by separation and precipitation of nucleic acids. Resulting DNA was treated with ribonuclease and cleaned using a Zymo DNA Concentrator-5 kit. We have isolated high-quality DNA from the spores of a number of isolates. PCR amplification of the ITS region was performed and sequenced. Resulting sequences were aligned using ClustalW and a maximum likelihood phylogeny was generated with the inclusion of known M. americana and M. paradoxa ITS sequences for species identification. Our data show that both M. americana and M. paradoxa are present in our isolate collections. Interestingly, we see that all isolates clustering with M. paradoxa were collected from the same location in New York State in early to mid-June, much earlier than rust emergence in all other sites from which rust was collected., amplified the ITS region and sequenced those products to confirm that we have isolates of both M. americana and M. paradoxa. After species identification, virulence assays were performed using a single rust isolate (R15-033-03) in order to assess effector interactions on diverse Salix spp. taxa currently utilized in breeding efforts in our program. Approximately 5 mg of newly collected spores were inoculated onto a leaf of each taxa and grown in the growth chamber for 10 days. Five biological replicates were utilized in each of three technical replicates. Percent disease was assessed daily and area under the disease progress curve (AUDPC) was calculated. Results show a wide range of resistance and susceptibility among the 12 selected host genotypes with this rust isolate. These genotypes represent the parents of F1 mapping populations developed in our program. The range of resistance and susceptibility among the diverse parents gives us confidence that we will be able to map segregation of different resistance loci in the F1 mapping populations. To address Objective 2, we surveyed rust incidence in Salix purpurea association populations planted on two sites, in Geneva, NY and in Portland, NY in September 2015. We also surveyed the progeny, parents, and grandparents in an F2 mapping population planted in a replicated trial in Geneva, NY. Since both of these populations were already genotyped by GBS, we were able to quickly map QTL for rust severity. In the association population a GWAS analysis revealed significant hits on Chromosomes 01 and 02. Further analysis of the allelic effects of the QTL on Chr 01 indicated that the heterozygote was more susceptible than the homozygotes for either allele. The mapping results from the F2 population indicated a significant QTL on Chr 01 that was only 40 kb from the significant hit in the association population. There were also significant QTL on Chr 05 and Chr 10. There was significant sex dimorphism for rust severity in both the association population in Geneva (n=107) and the F2 population (n=485), with males being more susceptible to rust than females. This held true for the parents and grandparents of the F2 population, where the male parent was more susceptible than the female parent, and the male grandparent more susceptible than the female grandparent. In the association trial, three of the blocks were fertilized and showed significantly higher SPAD readings as a surrogate for leaf N accumulation. There was also sex dimorphism for SPAD, with males showing higher readings and a positive interaction between SPAD and rust severity, which might be expected since Melampsora americana is an obligate biotroph and might be expected to proliferate more rapidly in leaves with higher SPAD and thus more N. These QTL mapping results will be further analyzed to identify candidate genes for more detailed analysis and for the development of marker for marker-assisted selection. Our tactic to address objective 4 involves mapping rust resistance genes in eight F1 species hybrid families in which there is a common Salix purpurea reference genotype parent, either female 94006 or male 94001. Three of the eight families in the F1 mapping strategy were already established in field trials in 2013 and 2014. In 2016 we planted the remaining five F1 mapping populations that were not already established in field trials in Geneva, NY. For each of the five families, cuttings of 150 progeny were collected in March 2016 and those were planted in three-plant plots in each of four replicate blocks in a randomized complete block design together with the parents a several commercial cultivars checks in June 2016. Since there was severe drought in Geneva in 2016, irrigation was applied throughout the growing season to ensure the best possible establishment. Shoot tips were collected from the genotypes planted in these trials for the purpose of isolating DNA and conducting GBS genotyping to support mapping of resistance genes in these F1 species hybrid families.
Publications
- Type:
Websites
Status:
Published
Year Published:
2016
Citation:
http://willow.cals.cornell.edu
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Chase R. Crowell, Mariami Bekauri, Craig H. Carlson, Fred E. Gouker, Lawrence B. Smart and Christine D. Smart. 2016 Melampsora rust running amuck: Exploring willow rust population diversity in the Northeast United States. Annual Meeting of the American Phytopathological Society, Tampa, FL, July 30-Aug. 3, 2016, Poster presentation.
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