Progress 12/01/18 to 11/30/22
Outputs
Target Audience:This project focused on providing information and materials for plant breeders working on improvement of switchgrass and other perennial grasses as well as researchers in the USDA, DOE, and other government agencies involved in renewable energy research & development and potential producers of bioenergy feedstocks and products. Changes/Problems: Delays in laboratory research and in obtaining data from service facilities was encountered due to the covid pandemic. The No Cost Extension year permitted remaining funds to be used to accomplish all of the goals and objectives of the project, although several publications are still in preparation. Dr. Donald Viands, the lead Co-PD at Cornell University for this project, retired in June 2021. Dr. Julie Hansen was chosen by Cornell University to assume Dr. Viands role as lead co-PD at Cornell on July 1, 2021, because of her long-standing participation in the project. A new faculty member at Cornell, Dr. Virginia Moore, replaced who replaced Dr. Viands. also actively participating in the project, This personnel change did not delay the project. What opportunities for training and professional development has the project provided?This project provided graduate research assistantships to support Jeremy Sutherland, a PhD student in the Bioinformatics and Genomics graduate program at Pennsylvania State University and to Christopher Tkach, a PhD student in Plant Biology at Rutgers University. Jeremy and Chris played key roles in conducting field work (collecting samples and phenotypic data), laboratory research (DNA sequencing, sequence analysis, and phenotypic data analysis), and preparation of manuscripts. Furthermore, several undergraduate assistants gained hands-on experience, assisting in both field and lab project activities. How have the results been disseminated to communities of interest?The results are being distributed by the students, staff and PIs in this project to the research and biomass energy communities through peer-reviewed publications and presentations at agronomy, genomics, and bioenergy conferences and workshops, including annual of the DOE/USDA Genomic Sciences Program Annual Contractor-Grantee meetings. An online presentation of the project by PhD student Christopher Tkach can be viewed at https://www.youtube.com/watch?v=UhhFueIR_v0. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
IMPACTS This project advanced the breeding of improved disease-resistant switchgrass (Panicum virgatum L.) for cultivation at diverse growing sites in the Northeastern US. Genetic and environmental effects on yield, disease susceptibility, and soil root-zone microbes were also elucidated. Objective 1 A range-wide provenance collection (panel) of 552 genotypes were replicated 3 times at 3 sites in NJ, NY, and PA. Height, circumference, overall productivity, calculated volume, ratings for anthracnose and smut disease severity were measured for over 3 years. Little bipolaris infection was observed, while smut and anthracnose occurred at all 3 locations. Top growth biomass was harvested from 128 selected genotypes in each replication for percent dry matter and dry yield data. Across all genotypes, mean yield was highest at the NY field site, followed by NJ field site, and lowest at the PA mine reclamation site due to disturbed soil, slope and proximity to trees. Seeded cultivar yield trials were evaluated for disease incidence, plant heights, biomass yield, and biomass composition over 3 years at the 3 trial sites (NY, NJ, and PA). Nurseries consisting of 180 switchgrass half-siblings were planted in NJ and NY selected from 4 populations from our previous collaboration: 1) Upland ecotypes selected from NY and NJ sites; 2) Upland ecotypes selected from PA site; 3) Lowland ecotypes selected from NY and NJ sites; 4) Lowland ecotypes selected from PA site. Each population consisted of 45 half-siblings, replicated four times at each location in a randomized complete block design. Selections were made based on vigor, anthracnose damage, leaf height, and qualitative identification of the best plants for each half-sibling. The resulting population consisted of 63 genotypes each for upland and lowland populations, including plants from NJ and NY breeding nurseries, and promising individuals from the provenance panel. Ramets were collected and exchanged between the breeding programs, from which seed production nurseries were established in NJ and NY. Objective 2 Anthracnose disease severity ratings were collected for 4 years in a mapping population of 240 individuals segregating for anthracnose disease severity obtained by crossing an anthracnose tolerant upland switchgrass plant with an anthracnose susceptible upland switchgrass plant. Bipolaris leaf spot did not appear. A genetic map was constructed of 4,129 DNA markers covering 1907.6cM. The map contains 19 linkage groups, each corresponding to a single chromosome in the switchgrass version 5 reference genome, with 2 linkage groups for arms of chromosome 8N. QTL analysis identified a single major locus for anthracnose resistance on chromosome 8K and a putative locus on chromosome 1K. The 8K locus explained ~20% of the anthracnose severity variation while the putative locus explained an additional ~16% of variation. Disease-resistance candidate genes in the QTL regions will be published. Candidate genes were also identified by comparing gene expression between anthracnose tolerant and anthracnose susceptible plants after infection with Colletotrichum navitas conidial suspension vs. control plants inoculated with sterile broth. RNA sequence was collected from leaf samples at 0, 1, 5, and 14 days post inoculation (dpi). Differentially expressed genes were detected by comparing infected vs control plants at each time point. The RGA3-like, RGA4-like and RMP1-like resistance genes were differentially expressed in the tolerant plant only. RGA2-like resistance genes were activated in both tolerant and susceptible plants, with the tolerant plants activating the genes within 1 dpi, while the susceptible plants did not activate the genes until 5 dpi. This suggests the timing of activation of disease resistance genes may be important in the severity of anthracnose disease. Objective 3 A genome-wide study identified associations of DNA markers in the 552 genotypes of the provenance panel with biomass-related traits and disease ratings from the 3 field sites over 3 years. A total of 143,967 high-quality polyploid DNA markers in the switchgrass version 5 reference genome were used, among which 8 significant associations of DNA sequences were identified for height, 2 for circumference, 65 for calculated volume, 1 for vigor ratings, 5 for anthracnose disease severity, and a single putative association just below significance level on chromosome 7N for smut incidence. Candidate genes in the chromosome regions associated with the traits will be published. Objective 4 Composition of soil microbiomes was investigated for associations with plant genotype, yield and disease resistance. Fungal (ITS) and bacterial (16S) genes were amplified and sequenced from soil DNA samples from the root zones (rhizosphere) of all 552 genotypes of the provenance panel. We first compared the rhizosphere microbiome compositions for 383 of the panel genotypes at the time the panel was sampled from the original common garden site at Ithaca, NY, prior to transplanting into the 3 field sites. Rhizosphere bacterial diversity in the common garden was affected by the ploidy and the biogeographical and evolutionary histories of the source populations of the genotypes in the panel. The rhizosphere microbial diversity at the time was also correlated with variation in economically important traits within the panel. We then examined bacterial and fungal diversity in soil rhizosphere samples collected from 128 selected switchgrass genotypes after 3 years of growth at the 3 sites. The 16S rRNA gene amplicon sequences for rhizosphere bacteria revealed large differences in rhizosphere microbiome composition across the 3 field sites. There were no obvious differences in microbiome composition between ecotypes, cytotypes, and genetically related groups of plants within each site, but broad-sense heritability of taxons varied among the sites. Deeper analysis showed that specific host plant genotype had a strong effect on microbiome composition. The abundance of 154 taxa of bacteria were correlated with anthracnose disease severity, biomass yield, and specific DNA sequences in the switchgrass genome. Our findings suggest that host genome influence on the rhizosphere microbiome in switchgrass varies across both microbial taxonomic level and local environmental conditions. From sequence variations in the internal transcribed spacer (ITS) gene, we found that fungal species diversity was significantly different between field sites and switchgrass ecotypes. The PA mineland site, which is highly degraded, exhibited the least fungal rhizosphere species diversity. Upland ecotypes exhibited the greatest fungal rhizosphere diversity, on average. Similar taxa were found among sites, but each site was enriched for a unique set of taxa, suggesting a strong influence of local environment. Across the sites, the switchgrass rhizosphere was dominated by six fungal phyla: Ascomycota, Basidiomycota, Glomermycota, Mortierellomycota, Zoopangomycota, and Unidentified variants. Glomeromycota forms symbiotic arbuscular mycorrhizas with the roots of more than 80% of vascular land plants, including switchgrass. A higher relative abundance of Glomeromycota was found at the degraded PA site compared to NJ and NY sites. Conversely, we saw a lower relative abundance of Glomeromycota at the NJ site, where nitrogen fertilizer was applied during plant establishment and is a preferred, well-drained, soil type for switchgrass cultivation. We also observed less environmental turnover for Glomermycota, compared to other phyla in the switchgrass rhizosphere. This suggests that Glomermycota species persisted after transplantation to the new sites, while other phyla in the rhizosphere were more susceptible to competition with fungi in the new environments, possibly due to local adaptation. We found only a single positive relationship between Glomeromycota and switchgrass biomass yield at the NY site.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Sutherland J, Bell T, Trexler RV, Carlson JE, Lasky JR. 2022. Host genomic influence on bacterial composition in the switchgrass rhizosphere. Molecular Ecology, 31(14): 3934-3950, https://doi.org/10.1111/mec.16549.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Songsomboon, K., Crawford, R., Crawford, J., Hansen, J., Cummings, J., Mattson, N., Bergstrom, G.C. and Viands, D.R., 2022. Genome-Wide Associations with Resistance to Bipolaris Leaf Spot (Bipolaris oryzae (Breda de Haan) Shoemaker) in a Northern Switchgrass Population (Panicum virgatum L.). Plants, 11(10), p.1362.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Christopher Tkach, Jeremy Sutherland, John E. Carlson, Terrence H. Bell, Jesse R. Lasky, Julie L. Hansen, Ryan V. Crawford, Virginia Moore and Stacy Bonos, 2022, Biomass Yield Trial of Experimental Switchgrass Breeding Selections, In: Proceedings of the Thirty-First Annual Rutgers Turfgrass Symposium, page 58, poster, Rutgers 2022 Turfgrass Symposium, March 17, 2022, oral presentation and poster.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Ryan V Crawford, Terrence Bell, Stacy A. Bonos, Jesse R. Lasky, Marvin H. Hall, Julie L. Hansen, Virginia Moore, Jeremy Sutherland, Christopher Tkach, Donald R. Viands and John Carlson, 2022, Experimental Switchgrass Cultivars Outperformed Commercial Checks at Prime and Reclaimed Mine Sites, Abstract, ASA, CSSA, SSSA International Annual Meeting.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Christopher Tkach, Christine Kubik, Jennifer Vaiciunas, Josh A. Honig, John Carlson and Stacy A. Bonos, 2022, Transcriptome Analysis of Anthracnose Resistance in Switchgrass Using RNA-Seq, ASA, CSSA, SSSA International Annual Meeting.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Christopher Tkach, Jeremy Sutherland, John Carlson, Terrence Bell, Jesse R. Lasky, Julie L. Hansen, Ryan V Crawford, Virginia Moore and Stacy A. Bonos, 2022, Switchgrass Mapping Population Segregating for Anthracnose Resistance, ASA, CSSA, SSSA International Annual Meeting.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Christopher Tkach, Jeremy Sutherland, Stacy A. Bonos, John E. Carlson, Terrence H. Bell, Jesse R. Lasky, Julie L. Hansen, Ryan V. Crawford, and Donald Viands, 2021, Genome wide association study of anthracnose disease in switchgrass, In: Proceedings of the Thirty-First Annual Rutgers Turfgrass Symposium, page 44, Rutgers 2021 Turfgrass Symposium - Advances in Turfgrass Science: Looking to the Future, March 17, 2021.
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Progress 12/01/20 to 11/30/21
Outputs
Target Audience:This project focuses on providing information and materials for plant breeders working on improvement of switchgrass and other perennial grasses as well as researchers in the USDA, DOE, and other government agencies involved in renewable energy research & development and potential producers of bioenergy feedstocks and products. Changes/Problems:Delays in laboratory research and in obtaining data from service facilities continued to be encountered in project year 3 due to the covid pandemic, although to less extant than the previous year. The approved one year No Cost Extension permits us to use remaining funds to continue to accomplish all remaining goals and objectives of the project, including publications Dr. Donald Viands, the lead Co-PD at Cornell University for this project, retired in June 2021. Dr. Julie Hansen was chosen by Cornell University to assume Dr. Viands role as lead co-PD at Cornell on July 1, 2021, because of her long-standing participation in the project. A new faculty member at Cornell, Dr. Virginia Moore, replaced who replaced Dr. Viands. also actively participating in the project, What opportunities for training and professional development has the project provided?The project has provided graduate research assistantships to support Jeremy Sutherland, a PhD student in the Bioinformatics and Genomics graduate program at Pennsylvania State University and to Christopher Tkach, a Graduate Assistant in Plant Biology at Rutgers University. Jeremy and Chris played key roles again this year in both conducting field work (collecting samples and phenotypic data) as well as laboratory research (preparing DNAs for sequencing, and sequence analysis, and analysis of the phenotypic data). Furthermore 3 undergraduate assistants gained experience in field and lab research as part of the Cornell team's project activities. How have the results been disseminated to communities of interest?A major publication from the project, prepared by PhD student Jeremy Sutherland and titled "Host genomic influence on bacterial composition in the switchgrass rhizosphere", was submitted for peer review to Molecular Ecology on September 2, 2021 (Manuscript ID MEC-21-1028). To more quickly deliver the manuscript to the research community, we also published a preprint of the paper in the journal BioRxiv on September 2, 2021. The results from the project were also disseminated to the bioenergy and genomics research communities through a talk and poster presentation by the PI at the annual meeting of the DOE/USDA Genomic Sciences Program Annual Contractor-Grantee Meeting What do you plan to do during the next reporting period to accomplish the goals?Objective 1 a. Collections of data from the field trials and nurseries for disease resistance and yield were completed in year 3. b. In year 4 the Cornel group will analyze biomass composition of the biomass samples collected from the 13 cultivars in the seeded yield trials at the 3 sites. All data obtained will be shared among project collaborators. c. Final selections will be made from the NY breeding nurseries and likely combined with material from the NJ breeding nurseries for isolation and seed production in the 2022 growing season. d. Make final selections of superior, anthracnose-tolerant genotypes in the GWAS panel to incorporate into the Cornell and Rutgers breeding programs for advanced cultivar development. e. Manuscripts on advances in cultivar development for Anthracnose resistance and yield during the project will be developed. Objective 2 a. Map order of SNPs in the linkage groups will be finalized and QTL analysis will be performed using MapQTL v6.0. b. Candidate genes for disease resistance and growth within the QTL will identified by reference to the switchgrass reference genome V5. c. RNA will be isolated from the Anthracnose-treated parent plant leaves (stored at -80C) using the Qiagen RNeasy Plant Mini Kit and sequenced on an Illumina platform. Transcriptome analysis will be conducted to validate underlying genes discovered from the QTL mapping family and to identify additional candidate genes for anthracnose resistance at other loci not captured by the QTL analysis. d. Manuscripts on the genetic linkage map, QTL, transcriptome results, and candidate genes for disease resistance will be developed. Objective 3 a. Conduct GWAS analyses to identify genes containing SNPs associated with disease resistance and vigor using the phenotypic data collected across the multiple trial sites in years 1, 2 and 3. b. Prepare manuscript reporting findings on switchgrass genetic associations with disease resistance/susceptibility and growth variation in switchgrass Objective 4 a. Complete analyses of microbiome composition among the selected 128 switchgrass genotypes in the GWAS panel after establishment at the 3 field trials sites. Publish manuscript on results of microbiome composition and associations of specific microbiome taxa with switchgrass genotypes. b. Search for SNP alleles associated with GxE interactions of genotypes , phenotypes, microbiomes and site parameters for the 128 switchgrass genotypes in the GWAs panel. Prepare manuscript(s) describing the GxExM interactions observed, and provide recommendations for future research and for applications of the results in future, genome-enabled advanced breeding.
Impacts
What was accomplished under these goals?
IMPACTS This project focuses on accelerating the breeding of superior, disease-resistant, climate-resilient switchgrass (Panicum virgatum L.) cultivars. Diseases such anthracnose (caused by Colletotrichum navitas) and Bipolaris leaf spot (caused by Bipolaris oryzae), along with environmental stresses from climate and marginal growing sites can cause severe losses in yield, especially in the Northeast. We have established new cultivar yield trials and planted a GWAS panel at 3 agronomically distinct field trial sites in NY, PA, and NJ, as well as expanding existing seed production nurseries at Cornell and Rutgers. The GWAS panel trials have provided new genome-wide data to better understand the sources of genetic and environmental variation affecting yield and disease susceptibility in switchgrass, including the roles of soil microbes. The GWAS panel trials have also provided new genotypes with greater Anthracnose resistance and productivity even on the mine reclamation site in PA. In these ways, through a combination of novel basic research and traditional breeding efforts our results show promise for expanding the range of switchgrass biomass cultivation in the Northeast. Objective 1 1. A second evaluation of the seeded yield trials was completed, including disease incidence, heights of plants, biomass yield, and sampling of plants for biomass composition analysis by NIR to be conducted during first quarter of NCE year 4. Vigor of plants at the NY and NJ sites was in general excellent. Vigor of plants at the mine reclamation site in PA was again poor in comparison, however improved relative to 2019. 2. The Ithaca NY plant breeding nursery of 178 half-siblings was evaluated for vigor and leaf disease incidence. Final selections for propagation and seed production in 2022 will be determined during the NCE period from most the promising genotypes identified in project years 2 and 3. 3: A third and final year of phenotypic data collection for plant growth, plant vigor and disease severity ratings was completed from all 552 genotypes in the 3 replicates of the GWAS panel at the 3 trial sites. This included measurements of height, circumference, overall plant productivity, and ratings of anthracnose and smut disease severity. In an addition, for this final year of data collection, in October the 128 genotypes selected in year 1 for intensive analyses were harvested individually by hand and their biomass weights determined. Samples from the 128 genotypes were also taken for determination of dry matter biomass composition. An initial comparison of performances among the 3 trial sites identified 22 accessions and cultivars that were in either the top 25% of genotype in heights or the top 40 % of genotypes in circumferences in at least 2 of the locations. 4. At Cornell, 929 superior genotypes were propagated to seed production blocks and evaluated for Anthracnose and smut severity. Seed was harvested in fall, 2021 from the "best" genotypes (the tallest, most vigorous and upright plants, with the least incidence of disease). 5. At Rutgers, Anthracnose disease severity and heights data were taken for individual plants. A high degree of a suspected viral disease was observed on many of the lowland ecotype plants at the NJ site. The presence of this viral disease acted as a confounding factor for anthracnose incidence ratings on the lowland ecotypes. The Ithaca NY nursery, the same 180 half-sibling families were also scored for vigor and leaf disease incidence and plant heights. Objective 2 1. At Rutgers, Illumina RADseq reads were produced for each of the 240 full siblings and parents of the genetic mapping family. A total of 6,942 SNP markers were identified within 90% of the individuals in the population. Linkage groups corresponding to the 18 chromosomes of tetraploid switchgrass were constructed. Map optimization continues. 2. Four years of phenotypic data have been collected in the field on growth and Anthracnose severity from the 240 individuals in the mapping family which can be used to identify QTL map locations for Anthracnose resistance after optimization of the genetic linkage map. 3. Replicates of the Anthracnose-resistant and Anthracnose-susceptible parent plants of the full sibling family were inoculated with a mixture of 5 different isolates of Colletotrichum navitas in a controlled growth chamber. Tissue samples were collected at 0, 24, 48, 72, 120 and 336 hours post inoculation and flash frozen in liquid nitrogen, which are being stored at -80°C until RNA can be isolated for sequencing. Foliar disease symptoms were first observed 120 hours post inoculation, and Colletotrichum infection confirmed by the Rutgers Plant Diagnostic Laboratory. Objective 3 1. A third and final year of data was collected for height, circumference, vigor, and anthracnose incidence (and for a second year for smut incidence) for each plant in the GWAS panel. In addition, 128 genotypes were harvested and weighed for biomass yield. The phenotypic data trended along the same lines as in years 1 and 2. No cases of bipolaris infection were observed. 2. Preliminary GWAS analyses for SNP-trait associations with disease ratings and growth data were conducted with phenotypic data from years 1 and 2 (2019 and 2020), initially using GBS genotypes (Lu et al 2013) and exome sequence capture. More conclusive results are anticipated using the 3 years of phenotypic data now available, to be conducted in the NCE project year 4. Objective 4 1. DNAs were isolated from the rhizosphere soil core samples collected near the base of plants of 128 selected switchgrass genotypes at all 3 trial sites in year 1 (2019). Amplicons were generated from each DNA sample for both ITS (fungal) and 16S (bacterial) loci and sequenced as Illumina libraries, which due to the covid pandemic was delayed until this project year. Initial analyses of the 16S data indicated that overall bacterial alpha diversity did not appear to be significantly different between switchgrass genotypes at each site. An initial analyses of ITS data also did not show much difference in alpha diversity of fungal taxa among genotypes. Principle component analysis for core taxa by relative abundance across reps however produced distinct clusters based on site, explaining about ~14% to ~17% of variance. Some highly prevalent fungal taxa, such as Mortierella which has species known to promote growth in plants, will be investigated at more depth during NCE year 4. 2. We submitted a manuscript for publication which describes and compares the rhizosphere microbiome compositions among 383 of the switchgrass GWAS panel genotypes at the time that the panel was sampled from the Ithaca common garden site, and prior to transplanting into the 3 PA, NJ, and NY field sites for this study. We found that rhizosphere bacterial diversity was affected by the ploidy and the biogeographical and evolutionary histories of the source populations of the switchgrass genotypes within the GWAs panel. The level of rhizosphere diversity was also found to be correlated with variation in economically important traits within the GWAs panel. 3. Analyses conducted in years 2 and 3 focused on associations of microbes with SNPs in coding switchgrass sequences using a set of exome-capture baits for the switchgrass genome (Evans et al., 2018). Moving forward, the most recent, updated genome reference (v5) will be employed.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
1. Sutherland, J., Bell, T., Trexler, R.V., Carlson, J. and Lasky, J., 2021. Host genomic influence on bacterial composition in the switchgrass rhizosphere. BioRxiv September 2, 2021, doi: https://doi.org/10.1101/2021.09.01.458593
- Type:
Journal Articles
Status:
Submitted
Year Published:
2021
Citation:
Sutherland, Jeremy; Bell, Terrence; Trexler, Ryan; Carlson, John; Lasky, Jesse, "Host genomic influence on bacterial composition in the switchgrass rhizosphere" submitted to Molecular Ecology September 2, 2021, Manuscript ID MEC-21-1028.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
3. Jeremy Sutherland, Ryan Crawford, Ryan Trexler, Christopher Tkach, Terrence Bell, Stacy Bonos, Marvin Hall, Julie Hansen, Jesse Lasky, Donald Viands, John Carlson, 2021. Breeding resilient, disease-resistant switchgrass cultivars for marginal lands, DOE/USDA Genomic Sciences Program Annual Contractor-Grantee Meeting, Feb 22-24, 2021, Poster presentation.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
4. John Carlson, Ryan Crawford, Jeremy Sutherland, Christopher Tkach, Terrence Bell, Stacy Bonos, Marvin Hall, Julie Hansen, Jesse Lasky, and Donald Viands, 2021, Breeding Resilient, Disease-Resistant Switchgrass Cultivars for Marginal Lands, Proceedings of the DOE/USDA Genomic Sciences Program Annual Contractor-Grantee Meeting, Feb 22-24, 2021, page 134.
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Progress 12/01/19 to 11/30/20
Outputs
Target Audience:1. Plant breeders working on improvement of switchgrass and other perennial grasses. 2. USDA, DOE, and other government agencies involved in renewable energy research & development. Changes/Problems:1. Although the DNA extractions from the rhizosphere soil samples collected in 2019 were completed early in project year 2, only the 16S loci amplifications and library constructions were completed prior to the advent of the covid pandemic. University safety guidelines to address the pandemic restricted lab occupancy to a single researcher in most cases, and core facilities at our institutions, as well as most commercial facilities, were closed for an extended period. This resulted in what should have been weeks of lab work now taking months to complete. We originally planned to have all data collected, including metagenomic DNA sequence data, by the end of year 2, with year 3 focused on data analysis. However, the collection of sequence data will now stretch well into project year 3, before full-scale GxE analyses will commence. 2. The delays in laboratory research and in obtaining sequence data from service facilities encountered in 2020 due to the covid pandemic also resulted in delays in expenditures for lab supplies, DNA library construction, and sequencing fees. Thus, in year 2 our expenditures fell short of the approved project budget, as was also the case in year 1. The total approved budget for years 1 and 2 together was $687,645. Although the complete financial statement for yeas 2 will not be available until January, 2021, our best estimate of expenses from account ledgers for years 1 and 2 will total app. $462,400. We request approval for carry over of all unspent funds at the end of project year 2 to year 3, both for the main grant and any surplus in the subawards to co-PDs at Rutgers and Cornell universities. This will permit us to continue to work towards accomplishing all of the goals and objectives of the project. 3. Dr. Donald Viands, the Co-PD at Cornell University for this project, recently announced that he is retiring in early 2021 and that new faculty member Dr. Virginia Moore will replace him, starting February 1, 2021. Dr. Moore is much interested in participating in this project, and Professor Viands recommends that Dr. Moore is highly qualified for assuming his responsibilities and role as Co-PD. The PD Dr. Carlson, and the participants in the project, support this change which will provide continuity of leadership for project activities at Cornell. Thus, we request that the cognizant NIFA officer for this program please formally replace Dr. Viands with Dr. Virginia Moore as Co-PD at Cornell for project year 3, beginning Feb. 1, 2021. What opportunities for training and professional development has the project provided?The project has provided graduate research assistantships to Jeremy Sutherland, a PhD student in the Bioinformatics and Genomics graduate program at Pennsylvania State University, and to Christopher Tkach, a Graduate Assistant in Plant Biology at Rutgers University. Jeremy and Chris this year conducted both field work (collecting phenotypic data) as well as laboratory research (preparing DNAs for sequencing, and sequence analysis, and analysis of the phenotypic data). Furthermore 3 undergraduate assistants gained experience in field and lab work as part of the Rutgers and Penn State project activities. How have the results been disseminated to communities of interest?In project year 2, the results from the project were disseminated to the research community through talks and poster presentations to the annual meeting of the American Society of Agronomy, the Crop Science Society of America, and the Soil Science Society of America; DOE/USDA Genomic Sciences Program Annual Contractor-Grantee Meeting; and at the Future of Bioenergy and Biorenewables Workshop held at Penn State University. What do you plan to do during the next reporting period to accomplish the goals? For Objective 1 "Expand selection and testing of superior, disease-resistant cultivars" in year 3 we will: Complete comparisons of yield and disease incidence in 13 advanced cultivars in replicated seeded plots at the MY, PA, and NJ field sites. Collect a final year of growth, vigor, and disease tolerance data in established nurseries of superior breeding lines. Collect a final year of growth, vigor, and disease tolerance data in the replicated plantings of the association panel. At the end of year 3, biomass weights well be taken as well. Identify superior, anthracnose-tolerant genotypes in the GWAS panel to transfer into the breeding program crossing blocks to advance cultivar development. Screen nursery established from 180 breeding lines for improved disease resistance and biomass yield. For Objective 2 "QTL and candidate gene discovery for disease resistance/susceptibility and yield", in year 3 we will: Construct an SNP-based genetic linkage map for the full sib family of 240 plants segregating for anthracnose resistance using RADseq sequence data. Identify QTL genetic map locations for anthracnose resistance and growth using phenotypic data collected in the field from the mapping family. If possible, identify putative candidate genes for anthracnose resistance and growth by reference to annotated genes in the switchgrass genome assembly. For Objective 3 "Identify associations of SNPs and candidate genes with anthracnose and Bipolaris disease ratings", in year 3 we will: Complete collection of growth measurements and disease ratings for core set of 128 genotypes selected from the association panel. Conduct stepwise GWAS analyses for SNPs associated with disease and vigor phenotypes in the 128 genotypes. For Objective 4 "Identify genome- and metagenome-wide Genotype-by-Environment associations" Complete collection of amplicon sequence data and analysis of rhizosphere microbiome composition established on the 128 core genotypes at the 3 trial sites. Compare microbiome compositions among the 128 switchgrass genotypes, both prior to transplanting from Ithaca common garden and after establishment of the plants at the 3 field trials sites. Search for SNP alleles associated with GxE interactions of plant genotypes, phenotypes, microbiomes and site parameters.
Impacts
What was accomplished under these goals?
Objective1 A seeded yield trial was established in year 1 at the NY, NJ, and PA field sites for 13 cultivar selections. In project year 2, we conducted an initial evaluation of the seeded yield trials for disease incidence (little anthracnose was observed with exception of the NJ site) and biomass yield. Vigor of plants at the mine reclamation site in PA was poor compared to the NY and NJ sites. In NY, plot heights were measured and harvested with a Carter forage harvester at one meter cutting width and 20 centimeter cutting height on 9/28/20. In NJ, plots were harvested by hand in a 1m x1m area at a height of 20 cm on 10/21/20. In PA, 48 inches of 5 rows (one-tenth of plot area) were harvested and weighed on 11/19/20. Samples are being evaluated for biomass composition. The Ithaca NY mature spaced-plant breeding nursery, planted in 2018, was evaluated for vigor and leaf disease incidence in mid-August and for height in late October. The nursery consisted of replicated eight plant half-sibling progeny rows from two populations (178 half-siblings in total), one population selected from upland ecotype switchgrass and one population selected from lowland ecotype switchgrass. Four nights of unseasonably early frost caused premature leaf senescence, particularly on lowland ecotypes, and made a second leaf disease evaluation impractical. A second year of phenotypic data collection for plant growth, plant vigor and disease severity ratings was completed from all 552 genotypes in the 3 replicates of the association panel. Measurements were taken late in the growing season for height, circumference, and plant vigor. Ratings were taken in mid-July for anthracnose severity (from 1 to 5) and smut severity (1 to 3). BiPolaris infections were not observed. Volume and vigor values were lowest at the PA mine reclamation site, although some genotypes performed well. In NY, the plant or plants of superior breeding lines with best phenotypes were identified in late October in the 2019 breeding nursery and in August 2019 in the 2018 breeding nursery. Nine hundred and twenty-two plants crosses of the plants were identified for crossing with to superior genotypes in the association panel in 2021. At Rutgers, the nursery of half-sibling families from superior breeding lines was scored for anthracnose in mid-August on a 1-10 scale (1 highest disease incidence). From the four groups (MLB, PLB, MUB, and PUB) 558 plants were identified as having low disease severity and substantially greater heights. Staff shortages due to Covid-19 restrictions made individual plant height measurements in 2020 impractical. The Ithaca nursery, consisting of the same 180 half-sibling families, was scored for vigor and leaf disease incidence in early August, 2020. Plant heights were evaluated in mid-October. At Ithaca, an unseasonably early frost made a second leaf disease evaluation impractical. Objective 2 At Rutgers the QTL mapping population of 240 full-sib switchgrass plants segregating for anthracnose disease were transferred from the field site to the greenhouse after the growing season in year 1, when 3 years of phenotypic data had been collected. The greenhouse conditions provided fresh, healthy tissue samples from the mapping population plants in year 2 for DNA isolation. The ddRADseq sequencing of the samples were delayed due to the pandemic, but is currently (November 2020) underway. The data will be available early in project year 3 for map construction and QTL analysis. Three years of phenotypic data collection in the field had been completed by the end of project year 1. To continue with anthracnose disease evaluation, the mapping population was replanted in the field in spring 2020, after tissues were harvested in the greenhouse. This year, the population was evaluated for smut on 7/14/20 and only a few individual plants expressed smut symptoms. Anthracnose was evaluated on 8/14/20. A relatively normal distribution of anthracnose disease severity was observed with a skew towards more disease susceptibility within the population. Objective 3 Data for height, circumference, vigor, and anthracnose incidence were obtained for each plant, as in year 1 (see Objective 1). In addition, this year we also collected smut incidence and disease ratings for all plants at all sites. Values for plant volume were computed from height and circumference to serve as a proxy for biomass yield. In general, normal distributions of data were observed, with similar means in both years. A preliminary GWAS analysis for SNP-trait associations was conducted with year 1 volume results and anthracnose ratings for the 540 association panel genotypes from all replicates and sites. Tassel v5.0 software was employed under the Mixed Linear Model, with a significance threshold set at -Log10 P-value of 5x10-8. Only 90,861 SNPs present in more than 25% of the individuals were retained, form the original 700,000 SNPs pre genotype (Lu et al 2013). Six SNP markers were identified with -Log10 P-value scores at or above the significance level for the association with anthracnose disease. For association tests of SNPs with calculated plant volumes, results with p-values below 1x10-3 were removed prior to producing a Manhattan plot which revealed two SNP markers above the genome wide threshold of significance level. Four additional SNP markers fell just below the significance threshold and could also be considered for subsequent analyses. This initial result confirms that further GWAS for discovery of SNP markers for biomass yield and disease resistance/susceptibility will be productive. More conclusive results are anticipated using multiple years of phenotypic data. The QTL mapping project being conducted in parallel will also provide strong evidence in support of SNP marker and candidate gene selections. Objective 4 Analysis of rhizosphere microbiomes for the association family genotypes at the original common garden site near Ithaca, NY (Lu et al. 2013) was completed. The 16S Amplicon sequence data, from 382 rhizosphere soil samples from the association population plants in year 1, revealed a rich pre-transplanting rhizosphere microbial community of at least 493 bacterial genera and 57 fungal genera. Alpha diversity analyses revealed an influence of host switchgrass genotypes on rhizosphere bacterial diversity. Rhizosphere diversity differed primarily along three modes of switchgrass stratification - ploidy, ecotype, and populations. In addition, we isolated the DNAs from the "post-establishment" rhizosphere soil samples collected in year 1 for replicates of the 128 set of core switchgrass genotypes. Amplicons for the bacterial 16S locus were generated, however the covid pandemic then resulted in the sequencing facilities being closed until late in project year 2. The 16S amplicon libraries and ITS locus amplicon generation and library construction are now underway. Sequencing and analyses should be completed early in year 3.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2020
Citation:
Tkach, C., J. Sutherland, S. Bonos, J. E. Carlson, T. H. Bell, J. R. Lasky, J. L. Hansen, R. V. Crawford, D. Viands. 2020. Analysis of anthracnose disease in an association panel of switchgrass on marginal land. In agronomy abstracts ASA, CSSA, SSSA Annual Meeting Nov 7, 2020 (Talk at Virtual Meeting).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2020
Citation:
2. Jeremy Sutherland, Ryan Crawford, Ryan Trexler, Christopher Tkach, Terrence Bell, Stacy Bonos, Marvin Hall, Julie Hansen, Jesse Lasky, Donald Viands, John Carlson. 2020. Breeding resilient, disease-resistant switchgrass cultivars for marginal lands, DOE/USDA Genomic Sciences Program Annual Contractor-Grantee Meeting, Tyson⿿s Corner, VA, Feb 24-26, 2020 (poster presentation).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2020
Citation:
3. Jeremy Sutherland, Ryan Crawford, Ryan Trexler, Christopher Tkach, Terrence Bell, Stacy Bonos, Marvin Hall, Julie Hansen, Jesse Lasky, Donald Viands, John Carlson. 2020. The Future of Bioenergy and Biorenewables Workshop, The Nittany Lion Inn, State College, Pennsylvania, November 10, 2020 (poster presentation).
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Progress 12/01/18 to 11/30/19
Outputs
Target Audience:1. Plant breeders working on improvement of switchgrass and other perennial grasses. 2. USDA, DOE, and other government agencies involved in renewable energy research & development. Changes/Problems:CHANGES TO PROPOSED RESEARCH PLANS 1. Due to interruptions caused by severe weather, collection of soil samples at the Rutgers field site in NJ was incomplete. We attempted to complete the full sampling on at least three occasions, but after 5 weeks decided the window of opportunity had passed, as we did not want risk conducting the analyses for GxE interactions and microbiome-host plant associations with samples collected at distant times in the growing season. We did obtain soil core samples at the NJ site in July from at least one replicate for all 128 genotypes, and from 2 replicates for 60 genotypes. The Rutgers soil samples will still be sequenced to look for trends in the data and to validate the results of microbiome and microbiome-host phenotype interaction obtained from the complete replicated sets of soil sequence data from the Cornell and Penn State sites. The growth, yield, and disease rating data sets are complete for all 3 field sites including Rutgers. So GWAS of phenotypic traits in the switchgrass association panel will still be conducted as planned. 2. Although most of the DNA extractions from the rhizosphere soil samples collected in July 2019 will be conducted by the end on year 1, as scheduled, the DNA extractions may not be entirely completed until January, 2020. This only sets back the 16S and ITS library constructions and sequencing by 2 months which should not affect overall progress. 3. Some delays were encountered in setting up the financial accounts at Penn State and the subawards after the project official start date on December 1, 2018. Unfortunately the subawards were not fully executed and available to the Co-PDs at Rutgers and Cornell until May, 2019, which delayed staff recruitment for the project until after the 2019 field season was already underway. Staff and students already in place at Rutgers and Cornell were shifted to the project for 2019 and fortunately the graduate student at Penn State (Sutherland) was able to start in the project in January, which helped keep the project on schedule. However the Rutgers, Cornell and PSU groups all have funds remaining from year 1 budgets that will need to be carried over to year 2. We request that the unused funds from year 1 be carried over to year 2, especially the app. 25% of salary and wages funds that could not be spent in year 1 at Rutgers and Cornell. What opportunities for training and professional development has the project provided?The project has provided a graduate research assistantship to Jeremy Sutherland who is a PhD student in the Bioinformatics and Genomics graduate program at Pennsylvania State University. Jeremy started January 1, 2019, only one month after the commencemcnte of year one of the project. Jeremy has been fully engaged this year in conducting field work (collecting phenotypic data at the PSU site), colelcting soil samples at all 3 field sites, and preparing the soil DNAs for sequencing. During this time he has also learned methodologies for metagenomics and genetic analyses of phenotypic data. In addition the project has supported 3 undergraduate students as part-time field and lab asistants providing them with hands-on experience in genetics and genomics research. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?PLANS FOR PROJECT YEAR TWO (12-01-2019 - 11-30-2020): For objective 1.1 ("Comparison of seeded plots for disease tolerance") In year 2 we will continue to collect disease ratings among advanced experimental cultivars from the USDA NEWBio project, and standard switchgrass cultivars as checks, in field trials at Cornell and Rutgers, towards the development of superior disease-resistant cultivars. For Objective 1.2 ("Evaluate vigor and disease tolerance in mature nurseries and in the replicated plantings of the association panel to identify superior breeding lines") In year 2 we will collect an additional year of phenotypic data using uniform metrics vigor and disease tolerance in field trials at Cornell, Rutgers, and Penn State. In addition, we will begin to select superior genotypes to move into crossing blocks to develop new cultivars. For Objective 2.3 ("Construct genetic linkage map and identify QTL locations for anthracnose resistance") In year 2 we will use RADseq data to develop an SNP-based genetic linkage map of the switchgrass mapping population at Rutgers that is segregating for anthracnose resistance/susceptibility. We may also collect a final (4th) year of anthracnose resistance/susceptibility data from the mapping family prior to identifying QTL locations and candidate genes. For Objective 3.1 ("Evaluate association panels for vigor and disease tolerance to identify SNP-trait associations at all 3 sites") In year 2 we will take measurements for vigor and disease tolerance. For Objective 3.2 ("GWAS analyses for SNPs with disease and vigor phenotypes in 128 genotypes selected from the association panel") In year 2 we will run initial tests for associations of SNPs with disease and vigor phenotypes in the 128 core genotypes in the association panel, using phenotypic data gathered in year 1 from al 3 sites. For Objective 4.1 ("Analysis of rhizosphere microbiome composition after establishment of the 3 trial sites"). In year 2 we will complete the analysis of the pre-planting rhizosphere microbiomes and proceed to comparative taxonomic analyses of rhizosphere microbiome compositions of the 128 core genotypes in the association at the 3 trial sites panel post-establishment. For Objective 4.2 ("Identify GxE interactions of plant genotypes (SNP alleles), phenotypes, microbiomes and site parameters"). In year 2 we will begin the step-wise analysis of Genotype x Environment interactions among 3 trial sites and the 128 core genotypes in the association panel a these sites, using previously published GBS data and phenotypic data collected in year 1.
Impacts
What was accomplished under these goals?
PROGRESS REPORT FOR YEAR 1: Objective 1. Seed from two previously established switchgrass selections and crossing blocks was sent from Rutgers to Cornell, where the seed was cleaned, weighed and tested for germination. Seed was also processed for planting from another two crossing blocks at Cornell. The seed was distributed to all project teams and yield trials were established. Germination and seedling establishment was high at all sites. Uniform methods for phenotyping and disease ratings across the project were agreed upon. Ryan Crawford from Cornell held a training session on phenotyping and disease rating for students and staff. . Mature cultivar nurseries at Cornell University and Rutgers University grew well and were evaluated for growth and disease incidence. Superior breeding lines were identified. Switchgrass forage was removed from the trials in the fall. The association panels are well established at all 3 sites. Growth was abundant at the Cornell, Rutgers and PSU field sites. Fields were sprayed with roundup for weed control prior to switchgrass greening in the spring. The 128 plants (3 replicates of each) selected for detailed analyses of microbes and phenotypes were marked. Soil samples were collected at all 3 sites for metagenomics. Plants at Cornell, Rutgers and PSU sites were rated for height, vigor, circumference, and disease incidence. Switchgrass forage was removed in the fall. Good results for seed germination were obtained from the 180 advanced lines, 32 seedlings per line,planted at the Rutgers trial site. At Cornell, a new switchgrass breeding nursery was started with seed from four crossing blocks from the previous NEWBio project. The seed was germinated, transplanted to greenhouse flats, and later transplanted to a field nursery in spring 2019. Objective 2. A 3rd and final year of data was collected in the QTL mapping population for disease severity and overall plant vigor, for genetic linkage mapping and identification of QTL. The plants in the QTL mapping population were then cut-back and brought into the greenhouse to provide high quality and contamination-free DNA from fresh healthy tissue. Objective 3. Phenotypic data was collected for plant growth (height and diameter), plant vigor and disease severity ratings from all 3 replicates for all 552 genotypes in the association panel at the 3 trial sites. Measurements were taken for height in cm, circumference in cm, anthracnose severity from 1 to 5, and general plant vigor from 1 to 5. BiPolaris infections were not observed in 2019. Values for plant volume were computed in cubic-cm from height and circumference using the formula: Volume=(PI()*((Circumference/(2*PI()))^2)*Height). Heritability estimates were also calculated, which averaged 0.393, 0.352, 0.337, 0.326, and 0.410 for vigor, Anthracnose resistance, volume, height and circumference respectively. Substantial normally-distributed quantitative phenotypic variation was observed between sites and among genotypes for all of the traits being studied. A site-productivity trend of Rutgers>Cornell> PSU was observd for height, circumference and biomass volume. In contrast, the Cornell site outperformed the other two sites in overall plant vigor, while Anthracnose incidence was severe overall at all 3 sites. Heritability levels were high for all traits at all 3 sites.The phenotypic variation and heritability will be sufficient for GWAS and GxE analyses to uncover genes and SNP alleles important in biomass productivity, disease-resistance, adaptation, and as breeding/selection tools. Objective 4. In pre-award work, triplicate soil and root samples were collected from all of the association population plants within 2 days after we collected samples ("plugs") of each genotype in triplicate from Mike Casler's switchgrass provenance trial common garden site at Cornell University near Ithaca, NY (Lu et al. 2013). DNA was isolated from all of the soil sample and quantified. The 382 samples yielding highest DNA quality and representing all of the wild population groups in the Lu et al. (2013) study were selected for the initial "pre-planting" soil metagenome sequencing. Amplicons for the 16S and ITS loci were generated from all 382 samples using consensus bacterial and fungal PCR primers. Separate NGS libraries were constructed and barcoded robotically for each of the 382 DNAs. Pools of 96 libraries were sequenced using an Illumina HiSeq 2000 to a depth of app. 15X (app. 60Gb in total). The metagenome amplicon sequence data revealed a pre-transplanting rhizosphere microbial community with a rich composition of at least 493 bacterial genera and 57 fungal genera. Knowledge of the composition of rhizosphere microbiomes associated with these plants provides the starting point for our 3 trials, representing the past interactions between the switchgrass genotypes and both aboveground and belowground environmental conditions during establishment at the original nursery at Cornell. We selected 128 association panel genotypes from among the 382 sampled at the transplant stage in which to conduct detailed monitoring of changes in rhizosphere microbiome composition that may occur as the association population becomes re-established in the 3 field trials in PA, NJ, and NY. The 128 genotypes were selected to maximize the range of switchgrass source populations represented. Soil core samples were taken from the 128 selected switchgrass genotypes at the 3 trial sites in Ithaca NY, Freehold NJ, and Philipsburg PA, over a period of 13 days, from July 12th through July 24th, 2019. For each genotype, two to three cores were collected from within 10 cm from the base of the plant at a depth of ~15 cm. Soil cores were kept in coolers for transport to a -20C freezer at the end of each day. Soil core samples were collected from the rhizosphere of each of the 128 genotypes from all 3 replicate blocks at the Cornell and Penn State trial sites. However, heavy rains and windstorms prevented completion of collection of samples at the Rutgers trial site, even though multiple attempts were made. For the Rutgers trial site, soil samples were collected from one rep' of all of the 128 genotypes during the first visit in July. After 5 weeks of failed return attempts, we decided that it was then too late in the season and too long after the initial collections to try again. Any additional soil samples collected at a later point in the season could have biased our down-stream comparisons among of microbiomes among the different soil types among the sites.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Carlson, JE, 2019, Breeding resilient, disease-resistant switchgrass cultivars for marginal lands , DOE/USDA Genomic Sciences Program Annual Contractor-Grantee Meeting, Tysons Corner, VA, Feb 25-27, 2019.
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