Brief Summary of Minutes of Annual Meeting

Attendees: Charlie Brummer, Heathcliffe Riday, Joe Robins, Bill Biligetu, Ali Missaoui, Shawn Kenaley, Maria Monteros, Patricio Munoz, Nancy Elhke, Don Viands and Jose Gonzalez Skype: Yousef Papadoupulus Chair: Charlie Brummer Secretary: Maria Monteros NE1010 background Don Viands: This is a multi-State cooperative research project. State experiment stations get funding for research (HATCH funds), but it is mainly spent for faculty salaries. Funding for travel and support staff is also available at most universities. Funding for NE1010 itself only goes to experiment stations in the US, but scientists not in the experiment stations have been involved in the project (USDA-ARS, Noble, and Ag and Agrifood Canada). With fewer breeders as time advances, cooperative research is the only way to do research and make an impact. Perennial species have the challenge to being economical because they are only planted every 3 or 4 years. It has been a benefit to have this project even though not everyone receives funding. The goal of the project is to encourage cooperative research among forage breeders and pathologists. The current project was written 15 years ago and consisted only of cooperative research in the proposal. ECB: At some states, like CA, the PI does not receive funding directly for research, but needs to have an experiment station project. Therefore, ECB will lead the effort to writing this project. DV: Multi state project associated with different regions. Next project number will be NE-XXXX. Need at least two experiment stations and Don Viands is the only one in the North East. Need someone else from the NE or shift to another region. Need to decide on a region and work with that experiment station to do that. If need to go to another region, need to coordinate with the person in that region. Possibly go through the North Central because Nancy has more history with the project (vs. other regions). Nancy will inquire about the experiment station deadlines since the projects are approved by summer. This project will last until 2017. Even though it is a 15-year project, Cornell requires a project every 5 years. It needs to get approved this year to avoid having a gap; target to have it ready for submission, need to have it ready by the fall. Potential suggested reviewers include: Ken Vogel, Deb Samac, JoAnn Lamb, Arvid Boe, Ian Ray, Ken Quesenberry, John Forster. The project is intended to be different from an information exchange program where everyone just reports what they are doing by focusing on discussion of collaborative research. A strength of this project is the development of collaborative projects between at least two or more researchers. Issue last time is that it had too many projects, different species, and different regions with different targets. Main limitation to working together is the need for funds to do the work in the absence of Hatch discretionary funding. Some proposed projects may not get going, or may need to be changed over time depending on funding. Aim is to accomplish as much of what is proposed to do as possible with each member coming up with funds to do cooperative research. Examples of successful research include: 1. Paper on Genomic Selection (Brummer, Viands and Annie). 2. Trefoil selection with grass vs. no grass (Brummer and Riday). Proposed examples: 1. Alfalfa evaluation of germplasm would fit well with this project. The objectives for the project have largely stayed the same for a few years: Objective 1: Evaluate germplasm and selecting for various traits Objective 2: Breeding forages more effectively Objective 3: Evaluate in yield trials that would eventually lead to release Project report is due in 60 days. Goal: write two or three objectives as a complement to the titles described in the NE-1010 Multistate cooperative research project. Marker research: Break out itself or integrated with the other objectives. Consensus feedback is that it should be a separate objective. Jose indicated it should be together; address a need and a question to use tools from different angles or perspectives to address an issue. Make sure the genomics component comes together with the other two sections. YP: keep the genomics work separate and traits interested in and link them together. Waterlogging and drought tolerance, assess which genes are involved and so on. If keep it separate, need to have a mechanism to link it with the traits. If it is focused on new methodology, then need to keep it separate, but if focused on the trait, need to integrate it so that it makes sense. Another possibility is to use a hybrid approach since have many different species (alfalfa and switchgrass). Some traits would be together across traits or species in different areas. AM: Alfalfa, need standard resources, a set of markers, applicable to all the traits, each time you tackle a trait, need to generate genomic resources, GBS. Collaborative effort to tackle the subject (500-600 set of standard markers) to use for genomic scan. Breeding toolbox type of resource. Expressed the need for organized datasets and community efforts for every trait. HR: grant opportunity to build collaborative infrastructure with breeders; need a collaborative infrastructure to help each other out, beyond the specific traits. Prominence of keeping it separate is to build an organized thing. MM: provided information on the Initiative of the Alfalfa Breeder’s toolbox at Noble Foundation and how that could help advance collaborative efforts to benefit the community. YP: Genomics for red clover but it never took off as part of NE1010 (Heathcliff would lead this). If there is a project for gene expression for red clover, are using M. truncatula. If keep it separately, need funding to get this initiative moving forward. DV: Make sure the molecular technologies are part of this program. NE1010: need to get approval, not competing with other projects. Similar criteria used for selection of funding as other proposals. Once the panel of 6, then it goes further to review of the whole process. Then to all the Crop Sci and Agronomy needs. History of the project will help us but need some tangible projects and significant accomplishments by the end of 5 years. Need milestones. Outline of ideas 1. Adaptation to variable and changing environments 2. Forage and biomass productivity 3. Evaluate new molecular tools to facilitate breeding for improving various traits in perennial forage species 4. Evaluate experimental populations through a testing network; understand genotype x environment interaction in forages. Test materials across broad areas of adaptation. Issue is that many of the variety testing programs are with extension personnel, not with breeders. Breeders often run experimental evaluation trials, however. This is focused on breeding with implicit connections for each researcher and their local group. This thematic topic would demonstrate connection to farmers by testing products and potential products for the producer. Final consumer is the grower; people going directly or through extension, but for alfalfa, output may be useful for industry. What is the goal of this project? Collaborate and/or develop new products? It would be great to get products out, but also the methods, upstream selection that may or may not lead to a product. Output can be a product that a farmer can use (nice to have it) but does not need to happen; can generate scientific knowledge (especially since not have funding), establish collaboration. Get group of folks together to be competitive based on what talked about here. Alfalfa RFP; loose requirements of three locations, overhead for three different institutions, perhaps relax requirements for locations and increase the funding amount. Trend a different way with the three-state requirement that served as a handicap. DV: Long-Xi (Washington) got an increase in his budget from what he requested. Industry support: not ‘need’ it although it would be useful to have it. Need to have ‘stakeholder input’. Regarding stakeholder industry or a few prominent growers, it would be good to have a letter of support with at least a skeletal proposal. Pitch the idea of developing markers for a range of insects or diseases. This could be similar to the work on Verticilium wilt done collaboratively with Long-xi, Debby, Maria and industry partners. Species: Consider species in addition to alfalfa. Common interest in the group with alfalfa; red clover some interest from various researchers. Jose, has resources for cordgrass; develop markers for each species is expensive and time consuming; consider a hybrid approach; cordgrass using GBS and set of SNP markers (KASP) assays for the bulk of markers can be GBS and have known markers at known locations to tie different studies together. Traits: salt tolerance is a common trait in many different species; methodologies across many different grasses are the same; transfer from one species to another. How to link across species without making it six different projects? If one methodology works for one species, then can leverage it to another species. Therefore consider developing standard tools within a species so that multiple people can use in their project. ‘Consensus resources’. Grant: 15 page limit; need to be brief for each of the sections. Don Viands already has a three page justification of the proposal written. Need a literature review also for the grant, Charlie to take the lead for the lit review. Goal: goals enumerated, methodology described and outputs. Possible projects: Alfalfa - Flooding and drought: (Yousef lead) Ali to identify sources of flood tolerance in 900+ entries. - Aluminum: screen 1700 accessions in the field; establish plants that do well in the field, work your way back to establish assays; GH assay with cones, almost are selecting for root elongation; in cones; the ones that succeed are the ones that get out of the cones; start in the field and those should do well in any test; five or six accessions do well in the field; try to look at as many accessions in the field as Ali can. - Don, LongXi: funded project on drought tolerance and salinity. LongXi has established a drought tolerance assay. - Define objectives, methodology and assign leads for each component. - What is the best way to evaluate those traits? - Include things that we can do collaboratively. - Yousef asked Charlie to find someone to lead the drought tolerance study since it was split from the flooding tolerance. Dan has a deficit irrigation program. - Drought lead: Mike Peel with LongXi, and Ian Ray already has a project on drought tolerance. - Salinity: Jose will write up a brief description and solicit additional feedback. - Acid tolerance and aluminum: new populations (Yousef) with Don and Surya. Genetic and selection gain; populations evaluated for productivity under sward conditions in acidic soils. Not sure if go to next phase of acid tolerance until analyze data and make their decision. They made good selection gain for seedling vigor under acid soil and calculated broad sense heritability (0.4 to 0.6 – Don’s location), so procedures used to select are working and have good selection gains. But not sure about getting the max yield potential or go for one more year of selection. Low pH – 5.0 and 5.5. Below pH 5 things get tough for alfalfa to grow. - 2004 trial with Surya, one rep at pH 4.5 and all plants died in that area. - Ali: tested 1780 and ended up with less than 12 that performed well at 4.8 pH. - Yousef: had seedling vigor improvement from 5 – 5.5 even if lime subsurface of the soil (6.5) the subsoil impact will reduce it to below 6. - Aluminum: Ali and Maria (develop new populations and understand Al tolerance mechanism). Biotic - Snout beetle: not much cooperative other than testing material (Don). Getting moderately resistance. Don has made progress from selection but it has been slow. - Root knot nematodes: Ali for alfalfa looking at mapping the three nematodes. Move it to the genomic tools/mapping section of the proposal. - PLH: have linkage drag from introgressing sources of tolerance; Don Viands making progress. Finding sources of resistance to be followed by looking at how it works in different backgrounds. - Switchgrass smut does not infect through the flowers but rather through the roots; have it in the fields now. Good because can ask a lot of questions about smut but cannot get it out. Ali is looking for nematode resistance in switchgrass. - Jose: consider symbiosis with rhizobia and mycorhyza. Don for his PhD work, the higher N fixation, the more fibrous the roots and thus more sites for nodules. - Disease diagnostic: survive flood vs. associated diseases in that environment. - Bergstrom lab has capacity for diagnostic testing of forage pathogens and diseases for at least a core set of them. OBJECTIVE 2. Germplasm enhancement and pre-breeding - Heathcliffe (Wisconsin – Northern germplasm): stakeholder desire for germplasm evaluation of alfalfa; started with landraces and cultivars = 4 pools to manage germplasm. In central Asia, had alfalfa (south of Siberia, greater Turkey, the Balkans and a eastern European pool: Ukraine and Baltics and Siberian north of China. 100 plants per pool for the initial pools. Some of these had disease problem. In Washington, S&W produced seed to generate over 100 half-sib families. Looking for people who will commit to put 1000 – 2000 plant nursery (small trial). Forage biomass productivity: general agronomics for their climate. Standard hay cutting as possible; at the end of evaluation, want to pick the best 5% of material out of each nursery. Companies are doing rapid selection cycles. Don Viands does this all the time. Year 1 establish, its not until Year 3 that can separate things out. Want to access NPGS for pre-breeding and generate material to breed for commercial varieties. Transplants that came in from the field went through GH screen for diseases (companies). - Explore the use of industry to generate seed (Mark and S&W, ex-Pioneer); they can do a seed increase of up to five pools. - USDA winterhardiness zones 7 or lower (5 or 6 was the target); everything they have survived in Wisconsin. - Long-Xi generated 6 to 8 pools of plants for drought - Southern climates (200 accessions in Davis and El Centro; Brian Motes at Noble and Patricio (FL) going in this year. Information on FD and select to develop populations similar to what Heathcliff did in the North. Look at it and then figure out how to composite populations. Look at G x E with multiple locations. Would like to get markers on that population. - Study: validate pools that are in fact distinct and cluster away from commercially available material. - Need strategy to phenotype and genotype the PI collection in alfalfa (how to address the heterozygosity of the populations). - Ali: already has three years of data from NPGS collection. - Shawn: relationship between the species diversity of the species and the pathogens associated with them. 2b Nutritive value - Joe: project with Aberystwyth (Wales); Utah plants produced half the carbohydrates they did. - Ali; evaluate the collection for stem sugar and lignin. In the South, alfalfa for haylage and therefore need to increase stem sugar (NIRS). Heathcliff suggest him to sequence the lignin genes and find variability in those genes. Don, working with forage quality in alfalfa for year. Ali would continue with potential interest from Don. Ali has mainly been looking for variation of the traits. 2c. Seed production - Seed quality interest in gamagrass (Seed Technology by A. Taylor in Geneva Station near Cornell). - Any correlation with seed production in your area and see how the production compares with other areas. Consistency of seed production could be an issue. Seed production will not shift from its current location. Move up to Canada for seed production or bring it down to Idaho depending on pricing and market. - Ali, modified soybean combines to harvest clover seed. Action: participants should describe what plan to do in a paragraph vs. solicit interest (working document). Describe what is the collaborative nature. 4. New molecular tools a. Marker tool development – Maria and others Nematodes (Ali), fall dormancy (Charlie), salinity/drought, (Mike P.), cold tolerance (Ali), Discussed a low cost genotyping assay including a miniscule PCR machine for cheap assay for only a few markers (idea with only a few markers for disease resistance). Heathcliff: 40 microsatellite markers without DNA extraction ($1.50 per assay). SNP-based systems need more markers. Heathcliff will be pursuing anyway. The regions of interest to him are always the same. He knows in red clover the regions in which variation is associated with quantitative traits. PCR-based systems have the advantage that you do not need a genotype number commitment, only the chip restricts you for the number. Only few species have markers on QTL narrowed down to finalize which ones to use. Pursue developing the assays (cheapest assay for about 50 markers); when people come out with markers of value, then they have the assay ready to go. Patricio, target traits are complex and quantitative and thus may need lots of markers. In wild populations and breeding populations LD is nothing but in forage breeding program, may extend longer. Want to develop markers using a cheap assay, and companies would phenotype hundreds of plants. Patricio, develop breeding population to test genomic selection using sequence capture (need more markers than what GBS can provide). To find out which ones to use, start with millions of markers and narrow it down. Heathcliff: has 50,000 red clover entries with phenotypes and tissue in storage; waiting for cheap technology to go in there to pull the trigger and mine the genotyping information. Patricio is also already working in red clover. Heathcliff: use SNPs to develop a SNP assay targeting GBS-based assay for ‘informative’ markers, jointly with Charlie Brummer (QTL). Use KASPr based technology and they do it themselves. Take sequence that validate as a SNP and use it in any other platform without any proprietary platform. Could attach any fluorescent label and now contracting the service. $3 per sample could get 50 markers (contract service). Have robot for DNA extraction. Patricio: doing sequence capture in alfalfa as well as in red clover. People are working on methodologies for genotyping to make it less expensive. Sequenome mass array: cost is $2,800 per run (384 samples per run). Cost per data point is cheaper if have the machine. GBS, have 10 or so markers as an outcome. Breeding: dig up plants in October and need instant access for genotyping and make decisions for Jan 1 to figure out what to advance. Can’t do it with Cornell Univ. to take longer (bioinformatics piece also needed). b. Genomics on grasses: Shawn was leading that (ryegrass as base genome to compare others too). Ryegrass info from the GenomeZipper (Torbin in Denmark vs. NZ, and Ag Victoria). Could develop collaborative projects here if interest. Patricio is doing genomic selection in ryegrass. Utah: association studies with GBS. ACTION: describe which traits are looking at, develop markers, integrate with the breeder’s toolbox (coordination of efforts and project of what are doing). Debby S. include aphanomyces work on markers but perhaps would have more value as a reviewer of the program. Alfalfa breeder’s toolbox: Jose is generating information on forage grasses and would like for it to be part of a future toolbox. Examples of datasets include RNA sequencing data, microarray and others for cordgrass and switchgrass (Jose). Patricio: working on methodologies for genomic selection in autotetraploids (alfalfa, blueberry and potato) considering double reduction, bulk genotyping for genomic selection strategies using data available for tress so now extend it to forages. Toolbox: include genome sequence, transcriptome, mapping, markers associated with traits. Lab work: need to coordinate for a contract-service system. Would like to see more environments to test germplasm and evaluate them phenotypically at multiple locations. Do GWAS in the population, do selections in a population and the subset is what is evaluated at multiple locations. Strategy: used breeding populations through cycles of selection (working jointly with breeders). Fall dormancy: using markers to select for it more broadly (which marker alleles give you the target FD). Charlie mapped in several biparental, in GWAS and differential populations L. Teuber put together from very dormant to non-dormant looking at allele shifts and in a FGI breeding population. Develop additional recombined populations. Break up fall dormancy and other traits (grazing tolerance). To what extent is dormancy linked to other traits. FD is a key trait. FD is easy to phenotype the extremes but the middle is more difficult. Ali: Take Bulldog805 and cross extremes to it (early and late); take 100 plants from each, clone and validate. Develop F2 so have another cycle of recombination to create 600 individuals for association population. Jose: collection of four-way crosses in wheat; collection of small populations of 20 to 30 individuals connected because they share parents. Need to inventory what is available. Patricio, use the evaluation of germplasm from North and South to start jointly rather than working on populations that people are committed or invested on. Patricio suggested starting with testing the joint germplasm as a common mapping resource. Clonal evaluation: take two or three applications, intermate them and then evaluate progenies at other locations (similar to what Don and Charlie had previously done); do it within Northern vs. Southern US region. NECS-141 (from the first cycle clonal selection) from the first evaluation of when had it in Iowa. Charlie selected it, possibly for high yield. Don, the population constructed was high yielding (dozen years ago). GENOTYPING: proposed use of chips but not likely, consider sequence capture 4. Testing new experimental populations/germplasm to understand GxE Trefoil: growth is not very good; most of the yield is the first harvest they have. Consider an evaluation trial under hay vs. simulated grazing (4 or 5 cuts vs. 3 cuts). Same project with Mike Peel (Yousef) for the 2017 proposal, put in new sets of the population. Propose to look at long-term persistence beyond the 3 years, for example 2 vs. 4 or 5 harvest to evaluate a genotype x management interaction. Heathcliff requested a National Red Clover Variety Testing Committee: advertise that are cohesive group, these are all the places that could test. People come to HR for testing. Shawn: single spore smut and cycling them through the greenhouse. These are now available to test for susceptibility instead of waiting for 3 years in the field. It is also possible to screen some pre-commercial or elite lines in switchgrass (place in section of smut inoculation of switchgrass). Overall considerations - Use North Central region due to the history if NE not viable. - ECB: go through North Central - 2016: meet in Madison before the NAAIC meeting on July 12-14, 2016. - 2017: meet in Halifax (Yousef hosts) Next project meeting in Madison, WI (2016) the day before the NAAIC meeting. 2016: Maria Chair of the NE1010 meeting (because Secretary). 2016 Secretary: Jose Gonzalez. Secretary should email minutes to Don Viands and Charlie Brummer. Requested a synopsis statement from meeting participants and about four sentences describing the approaches and plans.

Objective 1: (1) Genomic selection model for biomass yield shown to be effective in alfalfa. (2) Potato leafhopper resistant alfalfa has been developed and will be commercialized. (3) N-R-Gee alfalfa selected for lower pectin is in commercial production. (4) Populations tolerant to Al based on field evaluations have been developed. (5) Snout beetle tolerant alfalfa cultivar released last year and in commercial production. Continued selection has improved resistance further. (6) New birdsfoot trefoil germplasm is being evaluated for potential release. Objective 2: (1) Comparison of selection methods for biomass yield is underway in multiple location trials. (2) Populations were selected using a genomic prediction model. These will be increased in 2016 and tested in field trials in 2017. (3) Red clover populations with improved persistence, general adaptation, and biofuel use are being increased for evaluation. (4) A model to identify desirable reed canarygrass cultivars for biofuel use was developed. (5) Mixture trials documented the value of including legumes with grasses. Objective 3: (1) Evaluations of multiple species of cool and warm season perennial forages continue to be evaluated for forage yield, persistence, and other agronomic traits at multiple locations in North America.

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