Duration: 10/01/2022 to 09/30/2027

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

The economics of producing food, fiber, and energy products is a major issue in providing food security. Forage crops are the foundation of livestock and dairy enterprises in North America. According to the USDA National Agricultural Statistics Service, in 2019, 30.6 million acres of hay and haylage were harvested, worth more than $20.6 billion. These figures are conservative estimates of forage production since significant acreage is devoted to pastures and rangelands for which no estimates of economic value are readily available.

 

Use of leguminous forages minimizes nitrogen fertilization because they fix atmospheric N, thus reducing inputs and the risk of environmental contamination from fertilizer usage. The fibrous roots of grass species reduce soil erosion and capture environmental contaminants. Because these forage species are perennial, land disturbance is minimized, thus reducing the potential for soil erosion. In addition, they sequester carbon and reduce greenhouse gas emissions.

 

Breeding of perennial forage crops has resulted in improved cultivars that make livestock and dairy production more economical by reducing inputs and increasing outputs. Forage species, such as switchgrass, have potential for energy production. Compared to other types of crop species, perennial forage species enable more sustainable agricultural systems. Improved forage cultivars translate to benefits to agricultural producers of animal and energy products. The seed industry benefits from the production and marketing of improved cultivars. All Americans benefit from food security, reduced costs of food and energy, and protection of the environment by reduced use of pesticides, herbicides, and fertilizers.

 

During the last few decades, the number of forage breeders in North America has been decreasing. The number of forage researchers in the USA decreased by 60% between 1984 and 2009. The number of extension workers declined by 30% (Rouquette et al., 2009). In some state experiment stations, as a forage scientist has left their position, they were not replaced. For example, forage breeding positions have been lost at Iowa State University, Oklahoma State University, and Kansas State University. The number of forage scientists at USDA-ARS also has declined. When the forage breeder at USDA-ARS in Mandan, ND, retired, his position was not replaced. The Noble Foundation, a private research institute, ceased research activities in alfalfa genetics in 2021.

 

There are few private breeding companies, with ongoing consolidations further reducing their numbers. Currently, three main companies constitute the bulk of private alfalfa breeding in the USA (Forage Genetics, Intl., S&W Seed Company, and Corteva Agriscience). These companies work on few perennial forage species, primarily alfalfa with minor efforts on a few grass and clover species. Several major international forage seed companies focus on cool-season grasses and clovers, but most of that breeding is done outside the USA. Many forage species of importance in North America are receiving no attention by private breeders.

 

As budgets and the number of scientists have been reduced, the need for cooperative research is more essential than ever. Most forage breeders work on multiple forage species, thus diluting efforts on individual species. Because these forage species are perennial, establishing fields is less frequent than with annual crops. Therefore, seed is sold less frequently per unit land area compared to that of annual crops. Unless forage cultivars are broadly adapted for use across a large range of environments, seed companies are not interested in new cultivars because of the limited market. All of these factors point to the need for cooperative research to make significant advances in developing improved forage cultivars adapted to a wide range of environments. The current NE-1710 project fosters the interactions necessary to achieve goals with diminishing resources without unnecessary duplication.

 

This project aligns with the following NIFA Priority Science Areas:

1.     Agroclimate science. There is a need for forage crops that will be productive under abiotic stresses, including drought, flooding, cold and warm temperatures, and soil salinity and acidity.
2.     Bioeconomy-Bioenergy-Bioproducts. Cooperative research is needed for developing cultivars of bioenergy crops with improved biomass and quality, while protecting these crops from biotic and abiotic stress conditions.
3.     Human nutrition. Breeding crops with higher forage yield and quality, longevity, and resistance or tolerance to biotic and abiotic stress conditions will ensure an ample supply of good quality feed to animals, an essential step in securing food for consumers.
4.     Sustainable agricultural production systems. Diverse, perennial forage systems and increased cover crop use in annual crop production are important for increasing agricultural sustainability.

 

Without cooperative research through the multistate project, the ability to accomplish these priorities would be severely curtailed. In the absence of a cooperative research network of forage researchers, new forage cultivars would be more narrowly adapted and the scope and scale of research outputs would be more limited. Farmers rely on forage breeders to improve the productivity of these crops, especially when new diseases, insects, and other problems arise, and the impact on providing feed for the livestock industries, especially for beef and dairy production, would be huge. 

 

The impacts of the proposed research will be significant. Germplasm with new traits will be available to private and other public breeders to use in their programs for developing improved cultivars. Improved forage cultivars directly released from the multistate project scientists will make seed and forage production more economical for farmers and seed companies. Development of breeding methods, both traditional and molecular methods, will enhance efficiencies and effectiveness of improving forages for traits of low heritability or from unadapted genetic backgrounds. Data from forage yield trials across multiple locations and years will be available for breeders to use for selecting experimental populations that will be released as cultivars and available for licensing, for the seed industry in advertising seed of the cultivars, and for extension educators and farmers when selecting cultivars for their locations. These data also will help breeders to better understand the broad adaptation and resilience of forage cultivars. Development of forage species as feedstocks for the biofuel industry ultimately will contribute toward more secure and sustainable energy production. Between CO2 driven climate change and rising prices of petroleum products, long-term focus needs to be on renewable, sustainable energy sources. The overall impact will be more economical food and energy production while reducing negative environmental impacts in the agricultural systems.

 

The scientists cooperating in this project have the ability to accomplish the proposed research. The current NE-1710 project consists of most of the North American forage breeders, who have cooperated in research for many years. In addition to scientists at state agricultural experiment stations, the multistate cooperative research among forage scientists has evolved over the years to include more scientists from USDA-ARS, Agriculture and Agri-Food Canada, Canadian universities, and the Land Institute. These forage breeders and scientists have extensive experience in research on forages.

 

Many accomplishments have already been realized in the form of improved germplasm and cultivars; information on breeding methods for improving forage yield; and data on forage yield of multiple species for use by breeders, the seed industry, farmers, and extension educators. Extension presentations and information on the web have informed various stakeholders of the new information and cultivars developed by this project. Other scientists have been informed of the research results through professional publications and presentations at professional conferences. The scientists have the equipment along with field, greenhouse, and laboratory facilities to accomplish the proposed work.

 

Because of the long-term nature of research on perennial forage species, some of the research initiated in the last few years will continue into the next project period. Some of the research, however, will be new as a result of the collaborative efforts and discussions during our technical committee meetings. The research includes both traditional breeding and new molecular genetic technologies, and will enhance adaptation and resilience to changing environments. In addition, emphasis will continue in cooperative research on plant species for biofuel use as well as use for the livestock industry.

 

Funding for these collaborative efforts would be only partially covered by the multistate-Hatch funding. Most of the funding would come from other sources such as the seed industry, royalties from seed sales of cultivars, private sources, and various public funding sources at the state and federal levels (primarily competitive grants). In the past, the existence of the NE-1710 project and its predecessors have been a key factor in helping to secure other grant funds, such as USDA-NIFA grants, for accomplishing the research goals.

 

This proposal continues a long-term, continent-wide research project that has provided multi-location testing and selection environments to a number of forage and biofuel breeding projects. The current multistate Hatch project, NE1710, which ends in 2022, has grown to include forage and cover crop grass and legume breeders and bioenergy researchers throughout the US and Canada. Due to this geographical breadth, we have been able to implement experiments with nation-wide benefit and cannot be accomplished by any breeder individually. Individual participants in the proposal have their own research projects narrowly focused on species adapted to their regions, and the needs of producers at their locations. Our goal with this multi-location research project is to identify several major objectives that complement each location’s individual research projects but that, through the collaborative arrangements provided by the umbrella of this project, provide a larger geographical context in order to ensure new cultivars will have broad adaptability in different geographies, flexibility in a range of different cropping systems or uses, and resilience to shifting weather patterns.

 

In this renewal, we have developed projects related to the major perennial herbaceous crop groups, viz., legumes (alfalfa, birdsfoot trefoil, clovers), annual cover crops, cool-season grasses (tall fescue, orchardgrass, meadow bromegrass, intermediate wheatgrass), and warm-season grasses (switchgrass and giant miscanthus). Our sub-projects vary in size, from ongoing major breeding efforts in alfalfa to smaller projects that are the only coordinated breeding and improvement efforts for certain crops like birdsfoot trefoil. While numerous other forage crops are of importance, particularly in certain regions or specific niches, these crops represent major species of interest across broad regions of the continent. Therefore, we focus on these projects which require multi-location collaboration.

 

Furthermore, acknowledging that funding methods have changed over time, most of the collaborators, even those at state AES, do not receive funding for this regional research. Consequently, these projects are ones that complement existing research and can reasonably be tied with ongoing goals each collaborator has at their location. This also means that expensive objectives (e.g., those involving large-scale DNA sequencing or genomic selection) cannot be proposed within these research projects. However, by combining our programs, we can develop a framework to ask interesting questions about topics of broad interest, e.g., genotype ×environment interaction, that we can then use to attract external funding. 

 

Related, Current and Previous Work

Previous research results from this project (NE1710)

The previous multistate project NE1710 pursued two overarching objectives, to first improve legumes (alfalfa, birds-foot trefoil, and red clover), cool-season grasses (orchardgrass, tall fescue, and meadow bromegrass), and warm season grasses (switchgrass), and second to draw on the power of multi-site trials to understand patterns of genotype by environment variation.

 

Legumes:

Alfalfa:

In NE1710 we developed germplasm pools from NPGS germplasm for multi-site trials. We divided the germplasm into northern and southern pools to evaluate material most likely to be climatically adapted.  The Northern germplasm was divided into four populations based on its provenance: Central Asian, Russian, Ottoman and Siberian. Populations have been kept separate for seed increase. Multi-site trialling of northern material occurred at Cornell, NY Madison WI, Nova Scotia, and Southern Quebec. Southern material was trialed in California’s central valley, Georgia, and Florida. Western sites have experienced extreme drought in recent years, conditions climatic models expect to become more common. Prebreeding activities in NE1710 are essential to meeting these challenges.

 

Birds Foot Trefoil:

With a growing market for grassfed milk and meat, legume forages that do not cause bloat are essential.  The leading candidate for this is birdsfoot trefoil, Lotus corniculatus.  Birdsfoot trefoil from different regions were combined, in a five-location trial with sites in Nova Scotia, New York (Ithaca), Rhode Island (West Kingston), Wisconsin (Madison), and Utah (Logan). The work examined bioactive compounds that can control ruminant intestinal parasites. Although the sites are established, COVID-induced laboratory shutdowns, particularly at USDA ARS labs and at partner labs in Canada, have created a sample backlog.  We propose to expand the multi-site trials in coming work, as COVID delays abate.

Cool Season grasses

Developing resilient cool-season grasses adapted to variable climatic conditions has involved multi-site trials with selections at six locations, including two in Utah, two in Saskatchewan, and two in Quebec.  At each of these sites, selections have been made on several cool season grasses, specifically tall fescue, meadowbrome, timothy, and orchardgrass. At the more northern sites, winter hardiness has been an issue, particularly in Saskatchewan. The Saskatchewan group is looking at sugar accumulation as an indicator of cold tolerance, using RNA-seq during cold acclimation. At more southern sites, trials are needed to develop grasses that thrive in cool seasons, which is essential for some management systems. 

 

Warm Season Grasses

Warm season grasses are of great importance in pasture-based systems across the Southern US, are key biofuel crops, and a 10 site switchgrass trial has been performed by David Lowry (Michigan State University) and Tom Jeunger (University of Texas). Supported by material coming from this trial, work has progressed in characterizing head-smut fungus, a major pathogen of this warm season grass.  As there is no known sexual cycle, evidence may emerge that the disease is caused by a single clone of the fungus.

 

Miscanthus

With climate change bringing more extreme summer heat to many northern locations, there is a need for more work on warm season grasses in locations once considered too cool for them. As part of this need, more work is needed to understand variation in nutrient mobilization and cold adaptation in this species. There are currently three commercial lines of giant miscanthus (Miscanthus x giganteus; Mxg) planted in the U.S.  Two (Freedom and Nagara) are patented and licensed to AGrow Tech.  The third, Illinois clone, exists in the public domain. Giant miscanthus is a tall, perennial, deciduous C4 grass with annual tonnage that makes it valuable as a biomass crop. It is derived from crosses between M. sinensis (2n=2x=38) and M. sacchariflorus (2n=4x=76). Since it is derived from parents of different ploidy levels, and has an odd number of chromosome sets, it is seed sterile (2n=3x=57).  Being seed sterile precludes breeding of any of the Mxg cultivars except for somaclonal manipulations. The only way to generate new Mxg cultivars is by reconstituting the novel Mxg triploids is through crosses of new M. sinensis and new M. sacchariflorus crosses. Several Mxg seedlings have been generated at the University of Illinois and are being tested at various locations in the U.S. 

 

2.0 G X E for Tall Fescue, orchardgrass, Red Clover

Much of the power of a multistate Hatch is the capacity to look at differences in forage cultivar performance at different locations/environments.  NE1710 has examined multisite G*E in tall fescue and orchardgrass. Work with red clover has been delayed due to logistical challenges of moving seed across the US-Canadian border. To date, 14 entries of tall fescue, made by germplasm from Georgia and Nova Scotia have been evaluated. The panel was planted at 3 locations, including the University of Georgia, Cornell University, and Nova Scotia. Two years of data were collected at UGA and Cornell, and one year at Nova Scotia. The objective is to have a minimum of 3 years of field data per location to assess GxE interactions. Analyses will be performed by sub-project lead A. Missaoui in Georgia.  

 

Relevant background for research planned in this project

Although participants in this project individually conduct breeding, pre-breeding, genetics, and agronomic research on various forage and biofuel crops, the specific research projects we are proposing here represent aspects of these improvement programs that can be enhanced through regional and continent-wide collaboration. These projects are not meant to be comprehensive programs covering all, or even a few, forage and biofuel crops. They also reflect areas of continued interest in the group, covering repeated cycles of this multi-state Hatch group.

 

Alfalfa Improvement

Alfalfa breeders have been highly successful at improving alfalfa over the past 80 years, so that most new cultivars today have high levels of resistance to multiple pathogens or pests (e.g., Bouton 2021). However, the variable and extreme weather brought by climate change and the ongoing movement of pests and pathogens creates the need for increased resilience. Further, the improvement of alfalfa yield, other than that resulting from improved resistances, has been slow or non-existent over the past 30 years (Brummer and Casler, 2014). A broad goal for alfalfa improvement is to ensure breeding germplasm is available to assist breeders dealing with these challenges in the future, which can be accomplished by pre-breeding (e.g. Scotti and Brummer, 2010).

 

The U.S. National Plant Germplasm System (NPGS) maintains over 3000 alfalfa (Medicago sativa L.) germplasm accessions (Irish and Greene, 2021). This collection is a rich resource for North American alfalfa breeders. However, insufficient work has been expended in prebreeding the germplasm in this collection to develop new sources of variation for quantitative traits, such as yield and adaptation. Prebreeding can be defined as the process of converting a large number of germplasm accessions into a few adapted breeding pools that could be incorporated into commercial breeding programs (e.g., Egan et al., 2021). 

 

With rapid changes in climate, prebreeding offers the potential of providing breeders with new, semi-improved populations to incorporate into their programs. At its 2010 meeting, the North American Alfalfa Improvement Conference (NAAIC, an international organization of public and private alfalfa researchers) made the systematic germplasm improvement program one of the highest priority research projects. Past examples using non-North American alfalfa germplasm to enhance elite alfalfa germplasm pools include the development of leafhopper resistant alfalfa (Elden and McCaslin, 1997), lodging tolerant alfalfa (Lamb et al., 2000), disease resistance (Elgin et. al, 1988). Numerous studies have also show that genetically distinct alfalfa germplasm could be used as the basis for obtaining yield heterosis in hybrid or semi-hybrid alfalfa breeding schemes (Dudley and Davis, 1966; Busbice and Rawlings, 1974; Riday and Brummer 2006; Bhandari et al., 2007; Parajuli et al., 2021). Over the past decade and a half, a program to pre-breed pure yellow flowered alfalfa germplasm (subsp. falcata) has been underway (e.g., Riday and Wagner, 2012), falling within the NE1710 regional project.

 

Historically, alfalfa germplasm was classified into pools based on region of origin (e.g., Barnes et al. (1977). However, defining pools of extant breeding germplasm in 2021 is more difficult, given the extensive mixing over the past century; if anything, work with genome-wide SNPS suggests cultivars are structured according to fall dormancy classification (e.g., Li et al., 2014; Qiang et al., 2015; Ilhan et al., 2016), possibly reflecting past introgression of M. falcata germplasm. Recently genome assemblies have been published for diploid (Li et al., 2020) and tetraploid (Shen et al., 2020, Chen et al., 2020) alfalfa. Across all these experiments and past marker based assessments (reviewed by Li and Brummer 2012), the differentiation of yellow and purple flowered taxa is well established. Among tetraploids, however, a clear gradation is obvious with the hybrid subsp. xvaria intermediate between the two ends, effectively creating a continuum between true sativa and true falcata types. More fully understanding overall genetic variation would help to structure future breeding programs. For all these reasons this project is necessary to more fully exploit the NPGS germplasm system to pre-breed alfalfa. Work was started under NE1710 to develop a yellow-flowered ‘falcata’ pool. The project described here would expand this work by developing multiple geographic based germplasm pools in subsp. sativa.

 

Winter annual cover crop improvement

In 2017, 15.4 million acres of cover crops were planted in the US, representing a 50% increase in acreage over the previous five years (Census of Agriculture, 2017). The most common  species planted as cover crops include small grains (e.g., cereal rye, oats, winter wheat), legumes (e.g., clovers, hairy vetch, winter pea), and brassicas (e.g., radish, turnip) (CTIC, 2020). To date, cover crop breeding efforts have been quite limited, and few improved varieties are available to farmers. Farmers often purchase seed as “variety not stated” (VNS), and VNS seed has unknown and suboptimal performance for traits of importance to growers, including winter hardiness, maturity, nitrogen fixation, and biomass production (Wayman et al, 2017). Starting in 2015 a national Cover Crop Breeding Network was established, including universities, USDA, and farmers, and currently breeds four winter annual cover crops. Cover crop variety trials have also been conducted at universities and by USDA (USDA-NRCS Plant Materials Centers, 2020) to a limited extent. However, there is not currently a coordinated variety trialing system in the public or private sector with high resolution in the Northeast or in other regions across North America.

 

Root traits are a critical component of the soil organic carbon (SOC) contributions of cover crops (Austin et al., 2017; Mazzilli et al., 2015; Kong and Six, 2010), driving the potential contribution of cover crops to climate change mitigation and provide rotational value (e.g. Marques et al., 2020). However, root traits remain poorly characterized both among and within cover crop species, and the few cover crop breeding and variety characterization efforts have focused on a limited suite of economical traits, for example nitrogen fixation (Moore et al., 2020; Muller et al., 2021). If cover crops are to reach their potential as a tool to fight climate change, a major focus on breeding for root traits is needed.  

 

Switchgrass Improvement

Switchgrass (Panicum virgatum L.) is a native warm-season C4 perennial range grass that is a target

feedstock for U.S. production of sustainable cellulosic biofuels, electricity, and synthetic gas (Boateng et

al, 2007; Schmer et al., 2008). The principal objective of switchgrass breeding programs has been to

develop cultivars with high biomass production (Parrish et al., 2012). However, high yield depends on the cultivar’s ability to tolerate environmental and biotic stresses. Large monoculture biofuel plantings have not yet been sown, but when they are, disease pressure will increase (Crouch et al., 2009; Uppalapati et al., 2013; Zhu et al., 2013; Serba et al., 2015, Sykes et al., 2016, Bowen et al., 2021). Therefore, understanding genetic variation for resistance to the most serious disease pathogens needs to be undertaken now (Stewart and Cromey 2011).

 

Switchgrass harbors substantial natural genetic variation for many traits, and that variation appears to

extend to disease resistance. Much genetic variation within switchgrass is partitioned between upland

and lowland ecotypes, driven by adaptation to habitats differing in soil water availability and other factors

(e.g., Milano et al., 2016; Lowry et al., 2019, 2021). In addition, many genetically-based abiotic stress responses and disease susceptibility vary by latitude (e.g. McMillan, 1964; Meyer et al., 2014; Lovell et al., 2016; Lowry et al., 2019; Lovell et al., 2021; Gustafson et al., 2003; Uppalapati et al., 2013; Serba et al., 2015, VanWallendael et al., 2021). Due to recent work, breeders have a growing understanding of the genetics of resistance to the multiple microbial pathogens of switchgrass (VanWallendael et al., 2021). However, we still require an evaluation of pathogen diversity across geographically distinct switchgrass populations at a national scale. Given that pathogen diversity differs by region, the success of the proposed work will necessitate multistate collaboration.

 

Cool-season Grass Improvement

Cool-season (C3) grasses comprise a major forage source in both the United States and Canada. Cool-season grasses are grown both in monoculture plantings and mixed with legumes and stands are

managed for grazing, hay, and/or silage production. Meadow bromegrass (Bromus riparius Rehm.), tall

fescue (Lolium arundinaceum (Schreb.) Darbys.) and orchardgrass (Dactylis glomerata L.) are three

important cool-season grasses used in temperate regions of North America. Tall fescue and

orchardgrass are widely grown and are well known to farmers, and meadow bromegrass has been

recommended across Canada for some time (Knowles et al., 1993). There is interest in the U., with new cultivars being developed in Montana and Utah. The increased consumer demand for grass-fed and

organic animal products necessitate further improvement of cool-season grasses. Additionally, the effects of ongoing climate change necessitate selection for increased drought, heat, cold, and salinity tolerance. 

 

The former NE1010 and NE1710 project developed a meadow bromegrass breeding population by crossing superior plants selected at four locations across North America. However, this germplasm has not been tested widely. Tall fescue is recommended across North America; however, improving its palatability and nutritive value would expand its use. New soft-leaf tall fescue populations developed in Utah may be useful if they prove to be adapted across the tall fescue growing regions of North America and the soft leaf trait, which improves palatability, is expressed in all environments. Orchardgrass is cultivated in the northern regions of the United States and in all Canadian provinces. In Europe, increased concentrations of water soluble carbohydrates (WSC) in grasses provided increased animal production and decreased environmental impacts (Miller et al., 2001; Lee et al., 2001, Tubritt et al., 2018). Increased WSC may also buffer plants against various abiotic stresses, including drought and freezing (Volaire and Thomas, 1995; Sanada et al., 2007; Robins and Lovatt 2016). No cultivars have been developed in North America for higher WSC, despite the value of the trait. The development of cultivars with increased levels of WSC is a new breeding objective of forage breeding programs in recent years.

 

Birdsfoot trefoil improvement

Birdsfoot trefoil, as a non-bloating legume, is a desirable component of grazing systems. Breeding

programs have focused on improving persistence and seedling germination. One of the characteristics that makes trefoil a useful forage legume is their condensed tannin (CT) content. The quantity of CT present in legumes has been shown to correlate with animal productivity as well as impact gastrointestinal parasites in forage-fed ruminants (Waghorn and McNabb, 2003; Min et al, 2003; Ghelichkhan et al., 2017). Condensed tannin is a term that includes a group of diverse secondary compounds. The specific compounds associated with the above improved animal performance were not identified. Research into the impact of CT on the parasitic gastrointestinal nematodes that are parasitic to livestock has suggested the possibility that higher CT content of forage legumes will reduce the levels of infection (Hoste et al., 2006; Marley et al., 2006; Waghorn and McNabb, 2003). Assessing the CT profiles among currently available birdsfoot trefoil cultivars will aid in estimating available genetic variability for this trait.

 

Relation to other CRIS/REEIS research

The proposed work is the only multi-state umbrella for forage grass and legume breeding.  As such it serves as an umbrella, supporting multi-site work that cannot be done otherwise. For those with Hatch assignments at their home institutions, this project aligns with their local projects. These all show the need for a multistate umbrella to link individual research projects.

1. NE1710 complements several mixed pasture projects, such as NH.W-2020-02151, a northern tier pasture management project, and  TEN2020-05081, Keyser et al, supplementing cool season grasses in warmer Southeastern US.
2. Among warm season grasses, several projects are looking at switchgrass (ALAZ11192020MA (PI Aspinwall, switchgrass improvement and PENW-2018-05922, PI Carlson northern switchgrass improvement) and Miscanthus (ILLU-802-628, PI Sacks on Miscanthus genomics and  8042-13611-029-08S PI McCarthy, focused on Miscanthus in the Delmarva peninsula). A new NIFA award to PI Rios at UF (2022-67013-36252) complements these efforts with molecular breeding in bermudagrass (Cynodon spp.).
3. NE1710 supports several alfalfa breeding and improvement projects, including CA-D-PLS-2557-CG (PI Brummer), WNP03692 (Zhang et al, hybrid breeding with purging), WIS04059 on cutting time web platform development, CA-D-PLS-2482-CG  (Putnam and Hutchmaker, alfalfa tolerance of saline soils).  
4. As we have noted, there has been a multi-decadal decline in the number of forage breeders and agronomists.  Consequently training new breeders is critical.  WISW-2020-11303 (PI Newman) is a training grant on forage breeding and agronomy.
5. Finally, NE1710 is an umbrella for a number of individual Hatch projects. These include the following as examples. SD00R669-18 (Boe, alfalfa and switchgrass), CA-D-PLS-2246-RR (Brummer, alfalfa), NYC-149948  V. Moore Cornell, alfalfa, NYC-149946 (Robbins and Hansen, alfalfa breeding), MIS-162160 (Baldwin on switchgrass and Miscanthus), KY006108 (Phillips, grasses), MAS00557 Nusslein UMASS on cool season grasses, UTA-01480 Miller on forages, UTA-01482 (Creech on alfalfa breeding), FLA-AGR-005761  (Rios, alfalfa), ND06141 on grazing, VT-H02603 (Greenwood, cattle diets), VT-H02501MS (von Wettberg, cover crops), NH00665 (Smith, weeds of pastures), TEX0-1-6324  (Smith and Smith, forage grasses and clovers), ND01514 (Berti, forage management), SD00H740-22 (Wu, alfalfa phenotyping), NM-RAY-17H (Ray, alfalfa), OKL03137 (Wu, warm seasons), MIN-13-117 (Ehkle, cool seasons), NJ12182 (Bonos, switchgrass), TEN00518 (Bhandari, Switchgrass).

Objectives

1. Developing broadly adapted, climate resilient forages for sustainable cropping systems.
   Comments: This objective has six sub-objectives. 1.1. Developing regionally adapted, resilient alfalfa germplasm pools. Cooperating locations: AES: Cornell Univ., Univ. Florida, Univ. Vermont, and Univ. California, Davis [co-lead]; USDA-ARS: Logan, UT and Madison, WI [co-lead]; AAFC: Québec, QC, Saskatoon, SK, 1.2. Evaluating annual cover crops for regional adaptation and climate resilience and mitigation. AES: Cornell Univ. [Lead], Univ. Rhode Island; Univ. Vermont, AAFC: Truro, NS. 1.3. Developing switchgrass germplasm with improved fungal pathogen resistance. Cooperating locations: AES: Cornell Univ. [lead], Mississippi State Univ., Rutgers Univ., South Dakota State Univ.; USDA-ARS: Madison, WI. 1.4. Developing resilient cool-season grasses adapted to variable climatic conditions. Cooperating locations: AES: Cornell Univ.; South Dakota State Univ.; Univ. California, Davis; Univ. Kentucky, and Univ. Minnesota; USDA-ARS: Logan, UT [co-lead] and Madison, WI; AAFC: Québec, QC and Saskatoon, SK [co-lead]. 1.5. Determining the extent of genetic variability of CT among currently available birdsfoot trefoil cultivars and elite lines. Cooperating locations: AES: Cornell Univ., Univ. Rhode Island; Univ. Vermont USDA-ARS: Logan, UT and Madison, WI; AAFC: Truro, NS [lead]. 1.6. Evaluating Miscanthus for forage and bioenergy across warm season locations. AES: Univ. Mississippi [co-lead], Univ. Illinois [co-lead].
2. Understanding genotype by environment interactions across multiple forage species
   Comments: Cooperating locations: AES: Auburn Univ., Cornell Univ., Mississippi State Univ., Rutgers Univ., South Dakota State Univ., Univ. California, Davis, Univ. Florida, Univ. Georgia, Univ. Kentucky, Univ. Minnesota, Univ. Rhode Island, Univ. Tennessee; Univ. Vermont; USDA-ARS: Logan, UT and Madison, WI; AAFC, Lethbridge, AB, Québec, QC, Saskatoon, SK, and Truro, NS.

Methods

1. 1.     Developing broadly adapted, climate resilient forages for sustainable cropping systems.

    

   The six sub-objectives described here are natural outgrowths of the existing breeding programs of the participants. These six projects complement and extend individual programs and could not be conducted without regional (or continent-wide) participation.

   
   

   1.1 Developing regionally adapted, resilient alfalfa germplasm pools

   The overarching goal of this project is to use the NPGS alfalfa germplasm collection to enhance genetic

   diversity in elite North American alfalfa breeding pools, continuing work started in NE1710. Selection from the NPGS pools will be organized into region of origin germplasm pools that can be useful for long-term genetic improvement of alfalfa and potentially valuable for the creation of heterotic groups and hybrid cultivars. The project consists of a series of related experiments that by nature are collaborative across multiple North American locations. 

   
   

   Germplasm pool development. 

   Based largely on accessions that are documented as landraces or cultivars from regions outside North America, we have been developing pools based on Northern (fall dormancy levels from 1-5) and Southern adaptation (fall dormancy 5- 12). These populations are derived from discrete ecogeographic regions, with four Northern and four Southern pools. The Northern pools include Siberia/Mongolia, Central Asia, Balkans-Turkey-Black Sea Region, and North Eastern Europe to the Ural Mountains; Southern pools are from South America, North Africa, Southern Asia (India, Iran), and the Arabian Peninsula. In conjunction with those regional pools, in proposed work we plan to develop two broadly based populations by pooling the four regional pools.

   
   

   Evaluation/selection within new germplasm pools for broad adaptation 

   We expect that multiple cycles of selection will be necessary for these pools to be both broadly adaptable to North American climates and useful in commercial breeding programs. Therefore, we established multi-location breeding nurseries to select plants within populations to develop improved versions of these germplasms in NE1710, and expect to continue them to allow sufficient generations for broad adaptation. In addition to scientists involved at the outset of this project, we will also invite other breeders to participate either by becoming members of the regional project itself or by conducting evaluation trials. Trials will include comparisons of new germplasm against “check” varieties.

   
   

   Diversity evaluation of germplasm pool

   In order to validate our germplasm pool approach we will conduct genomic studies of developing pools using falcata as outgroup to determine how distinctive the eco-geographic regional germplasm pools are in relationship to North American elite alfalfa germplasm. This analysis, led by Rios, Brummer, and colleagues, could help us also define less utilized regional germplasm sources and guide enhancement efforts. 

   
   

   Nondormant cultivars have been released and are widely adopted in the southeastern United States (Bouton, 2021). In Florida, nondormant cultivars have been developed for improved adaptation to the state’s subtropical agroecosystem [‘Florida 66’ (Horner, 1970), ‘Florida 77’ (Horner and Ruelke, 1981), and ‘Florida 99’], but these cultivars are not commercially available. PD Rios resumed the breeding program at UF using Florida-adapted germplasm as the basis for the program. Advanced breeding lines have shown improved yield in Florida, Mississippi and Oklahoma, and a new non-dormant cultivar bred through conventional approaches was released in 2021, and the invitation to negotiate is underway (closes on May 2 2022). . Besides working with existing non-dormant germplasm pools, 121 diverse populations (cultivars, breeding lines and landraces) from various sources showed significant phenotypic diversity for biomass yield across several harvests in Florida. Plant selection was performed within populations and used in crosses to generate a training population consisting of 145 full-sib and 36 half-sib families. These families were established in the field to phenotype for biomass production and nutritive value (Acharya et al., 2020). This breeding population represents the training set for ongoing genomic prediction and high-throughput phenotyping studies for biomass yield and nutritive value (Biswas et al., 2021).

   
   

   Seed increases and yield/performance trials of new germplasm pools 

   Seed of the germplasm pools will be increased to provide sufficient seed quantities for yield evaluation at multiple locations. New germplasms are expected as part of this project that will be freely available to commercial alfalfa breeding interests through the NPGS system. We will characterize the new pools for various traits, including (but not limited to) fall dormancy, insect/disease resistance, salinity, winter hardiness, and waterlogging tolerance. We will work with other US alfalfa scientists in the public and private sectors to evaluate key insect and disease resistances of most importance nationally and within specific regions.

   
   

   Release of germplasm to the public. 

   All prebreeding populations developed through this project will be made publicly available through the National Plant Germplasm System, accessible through the Germplasm Resources Information Network. More advanced material will be published as appropriate in the Journal of Plant of Registrations.

   
   

   1.2: Evaluating annual cover crops for regional adaptation and climate resilience and mitigation. 

   The overarching goal of this project is to evaluate winter annual cover crop varieties for suitability for cropping systems in the Northeast US and Eastern Canada. The project is new to NE2210, and builds on existing cover crop breeding collaborations between researchers both within and beyond this group. The project includes evaluation of cover crop species and varieties within species for regional adaptation and suitability for cover crop and forage uses and potential for climate resilience/mitigation. In the proposed study, a trial including commercial cultivars of hairy vetch, winter pea, and crimson and bersem clover will be planted at each study location, including New York, Vermont, Rhode Island, and Nova Scotia. Advanced breeding material from project collaborators will also be included in the trial as available. Trials will be planted on a fall date appropriate for each location. Experiments will be planted in a randomized complete block design with four replications. Plots will be drilled in multi-row plots, approximately five feet wide and 15 feet long, with exact size and row spacing depending on available equipment at each site, including checks.

   
   

   Plots will be evaluated for key cover crop and forage traits including emergence, fall and spring vigor, winter survival, and maturity at termination. Fall emergence and winter survival will be evaluated on a visual percentage basis. Vigor evaluation will be conducted on a 1-9 visual rating scale. Maturity will be assessed on the scale developed by Kalu and Fick (1983) and modified as appropriate for each crop. Biomass will be harvested, dried, and weighed at a regionally appropriate time in the spring before typical cash crop planting. Data will be analyzed with a focus on G x E and identification of stable, broadly adapted varieties for the Northeast.

   In addition, root traits will be evaluated at a subset of locations (University of Vermont and Cornell) using “shovelomics” methods developed for legumes (Burridge et al., 2016) and adapted to species of interest as needed. Four plants per plot will be excavated, washed, and evaluated for key root system traits using a combination of manual and image-based evaluation methods. These data will provide an assessment of the diversity of root structural traits both within and among cover crop species, and will be used to determine potential to select for cover crops with improved root characteristics and the potential for climate mitigation and adaptation.

   
   

   1.3 Developing switchgrass germplasm with improved fungal pathogen resistance.

   As biofuel production expands, planting of large monocultures of switchgrass will likely result in

   increased disease, especially those caused by fungal pathogens. In order to develop switchgrass

   germplasm and cultivars with resistance to fungal pathogens, breeders first need to understand which

   pathogens are present in potential biofuel growing regions. Because no previous study has evaluated pathogen diversity at the national scale, this project presents an unparalleled opportunity to evaluate germplasm for resistance to these disease agents. Given that pathogen diversity differs by region, the success of the proposed work requires multistate collaboration. This project builds on efforts started in NE1710, but that were disrupted by the pandemic.

   
   

   Quantify important switchgrass pathogens across the US

    We will characterize the geographic distribution and severity of economically important/yield-reducing diseases caused by phytopathogenic fungi in the north-central, eastern, and south-eastern United States through surveys of common cultivars and breeding lines. Fungi can cause a range of mild to severe disease in plant hosts. However, disease symptoms or signs of phytopathogenic fungi may not immediately reveal the specific identity of a pathogen. Thus, we will first continue to quantify the severity of fungal diseases in switchgrass with an annual survey (2022-2025) of all commercially important and promising new lines of switchgrass in yield trials and nurseries in Mississippi (Mississippi State University), New Jersey (Rutgers University), New York State (Cornell University), South Dakota (South Dakota State University), and Wisconsin (USDA). This will build on preliminary data collected from 2017-2019.  Disease surveys will focus on determining the severity of four diseases: 1) rust (Puccinia emaculata and Uromyces graminicola); 2) anthracnose (Colletotrichum navitas); 3) Bipolaris leaf spot; and, 4) head smut (Tilletia species). For each survey, collaborators will score only promising new lines and commercially important cultivars for infection using established, standardized visual disease severity rating systems (e.g., for rust, 0-9 Puccinia emaculata system). From these surveys, we will establish how disease severity varies by geographic location, differs between northern upland and southern lowland populations, and differs among germplasm within ecotypes.

   
   

   Identify and quantify specific host-pathogen relationships

    We will identify patterns of phenotypic variation for switchgrass resistance and, hence, susceptibility to fungal pathogens across common cultivars, breeding lines, and field sites. We have noted previously that varying fertilization regimes (esp. nitrogen application rate) and summer rainfall can significantly influence the incidence and severity of switchgrass disease, particularly anthracnose and head smut (S.C. Kenaley, Cornell Univ. unpub. data). To identify environmental factors associated with the severity of disease(s), we will compile a set of environmental covariates for each field site including: (i) 2022-2025 weather data, obtained from PRISM; (ii) soil fertility (established by collaborators); (iii) proximity of alternate hosts for foliar pathogens of switchgrass; and (iv) extent of vegetation types within 1 km, as determined from field observation and the USDA National Agricultural Statistics Service Cropland Data Layer. To determine how landscape features predict genotype-specific disease severity, we will conduct stepwise general linear model fitting. Here, the response variable will be disease severity and the landscape environmental factors will be predictor variables. Model comparison using Akaike information criterion (AIC) will be executed to identify best-fit models.

   
   

   Pathogen resistance in switchgrass

    We will quantify pathogens across common cultivars and breeding lines to identify switchgrass germplasm possessing tolerance and/or resistance to regional phytopathogenic fungi. We will compare results among field sites to identify regionally adapted switchgrass plants for possible cultivar release and further downstream genetic analyses. We will continue to collect fungi with switchgrass leaves annually in mid-summer to early fall 2022-2025 at all sites, continuing intermittent collections that were made from 2017-2019 and hindered by the pandemic. Culturable fungi will be identified by morphological analysis, whereas for rust and smut fungi, will be identified based on teliospore measurements and DNA marker results. Drs. Shawn Kenaley and Gary Bergstrom (Cornell Univ.) have conducted the only morphological and phylogenetic analyses of switchgrass rust fungi to date and, hence, have the expertise and procedures to successfully complete the aforementioned molecular and morphometric analyses. 

   
   

   New resistant switchgrass germplasm

    Through the evaluations of fungi populations described above, we will be able to identify switchgrass populations and/or genotypes that are resistant or tolerant to one or more fungal pathogens. Resistant germplasm will be evaluated for biofuel characteristics and considered for release, based on the release protocols of the institutions involved in the project. Further selection, intercrossing, and population development will be conducted by the partners to generate highly productive, disease resistant cultivars. New cultivars will be exclusively licensed through Roundstone Native Seed Company.  In addition to release, switchgrass cultivars are being stored/released through GRIN. Recent cultivars ‘Tusca’ (imazapic resistant lowland switchgrass), ‘Espresso” – high germination, high velocity of germination lowland switchgrass, and “Robusto’– high germination, high velocity of germination upland switchgrass.  

   
   

   1.4 Developing resilient cool-season grasses adapted to variable climatic conditions.

   This project will develop cultivars and breeding lines of important cool-season grasses widely used in multiple North American forage breeding programs, including meadow bromegrass, tall fescue, orchardgrass, and timothy. Specific objectives are: 1) Evaluate new elite populations of cool-season grasses for potential cultivar release and commercialization 2) Select individual genotypes from the elite populations to develop new elite germplasm for future genetic improvement. 3) Identification of candidate genes associated with cold acclimation in orchardgrass.  

   
   

   Experimental approach: From 2017-2021 (Phase I ), 10 populations of meadow bromegrass, 7 populations of tall fescue, 9 populations of orchardgrass, and 10 populations of timothy grass were characterized for biomass production, nutritive value, and survival for three production years at six locations. These included sites at: Logan and Pantuitch, Utah, Saskatoon and Swift Current, Saskatchewan, and Normandin and St-Augustin, Québec. Superior genotypes for each species were selected by each test site for new population development in 2021. In the proposed study (Phase II), plots of new elite populations of meadow brome, tall fescue, orchardgrass, and timothy grass developed by Phase I study will be established at the above mentioned experimental sites. The experimental design at each site was randomized complete block design with 4 replications. Plot size will be 1.4 x 2 m, with 4 rows containing 12 plants/row.  Biomass and nutritive value (ADF, NDF and CP) will be collected during the first two production years. Survival data will be collected each year. Based on the multiple year data, the best populations at each site will be identified and selected for seed increase. The elite populations will potentially be released as commercial cultivars (Ampac Seed). In addition, new plant selection will be made for each species to create new elite germplasm for future breeding of the four cool-season grasses.

   
   

   To determine water soluble carbohydrate (WSC) content, orchardgrass cultivar Killarney was cloned and a new nursery was transplanted in early spring 2019 at Saint-Augustin, Quebec and Saskatoon, SK Canada. At each site, the experimental design was a randomized complete block with four replications. The clones will be grouped into three sampling dates of 1-wk before a killing frost, immediately after (day temp. -2oC for more than 5 hours), and 1-wk after a killing frost. At each sampling, clones will be dug out and rinsed off before the stem bases (2-3g) were collected for WSC. The crown samples for RNAseq will also be collected from the field and stored at -80oC in the laboratory. RNA will be extracted from the samples, and RNAseq analysis will be carried out to determine differentially expressed genes and unique alleles for cold tolerance in orchardgrass. 

   
   
   

   1.5 Determining the extent of genetic variability of condensed tannins (CT) among currently available birdsfoot trefoil cultivars and elite lines

   We will assess the CT profile and content of birdsfoot trefoil germplasm across diverse climatic conditions in northern latitudes. This investigation will be initiated by establishing small, replicated plots of birdsfoot trefoil cultivars and elite lines in regions where the investigators are located. In 2019 and 2020, prior to harvesting, the plots were assessed for development of stage. Plots will be sampled for CT profile and content and harvested to determine forage yield. We plan to repeat and expand this work to have a third year, and to bring in a new site in Vermont. Although fairly small in scope, this work is essential as the only continent wide work on this versatile and nutritious forage legume.

   
   

   NEW: 1.6 Miscanthus

   Preliminary trials in Illinois and Mississippi are planned to evaluate Giant miscanthus giant Miscanthus (Miscanthus x giganteus; Mxg).  Several Mxg seedlings have been generated at the University of Illinois and will be tested at these climatically extreme sites.  Evaluation will focus on biomass, agronomic characteristics, and disease. In Mississippi, cool season tolerance will be evaluated.  This work, although preliminary in scope, is essential to scale up activities to a full nationwide scale.

   
   
   

   2.0. Understanding genotype by environment by management interactions across multiple forage species

      To provide the information needed for broadly adapted germplasm suitable to a range of production systems, multi-location and multi-production trials are needed.  The concluding NE1710 has a component of G × E analysis that we intend to expand and deepen with similar methods but expanded datasets and an increased focus on the interacting effects of management decisions (G × E × M).  Multi-location evaluation data for broadly adapted as well as location specific germplasm developed by members of the NE1710 committee will be generated either jointly or independently.  Cooperating locations where testing will be conducted will be identified for each species. An individual from one of the locations will act as the coordinator. Seed from each cooperating location and the core set of cultivars will be distributed by the coordinator to each cooperating location. Seed of each entry will be submitted by each cooperating location to the coordinator of the trial, entries will typically not exceed four to six entries. For each species a core set of check cultivars will be included for testing. 

   
   

   Entries will be established following local practices in small plot trials and will be maintained for two to three years following the establishment year. In some locations this may include multiple management approaches. Each location will collect a core set of data, such as seedling vigor and plant stand in the establishment year, forage yield from a minimum of one harvest in the two to three years following establishment, forage quality data where the local cooperator has that capability and persistence following the last harvest in the second year following establishment. Each location will also collect data unique to the biotic and abiotic stresses for their specific environment. Individual locations may choose to include additional check cultivars when a test is specific to their location. At the conclusion of the final harvest year, raw/rep data will be sent from each cooperating location to the coordinator of that crop. This should occur no later than mid November. The coordinator for that crop will analyze and summarize the data, reporting G × E × M interactions for establishment, forage yield, and persistence. All data, including that unique to specific locations, will be summarized in mean tables, which will then be distributed to all cooperators.

Measurement of Progress and Results

Outputs

• Increase in improved forage cultivars that enhance the economics of livestock producers. Comments: Seed sales of new cultivars, when available.
• Increase in the alfalfa germplasm available to breeders in North America to develop improved cultivars more resilient and better adapted to climate stress in the future. Comments: We will release germplasm to NPGS when appropriate. We are also working on public releases.
• Increase in scientific knowledge through journal papers that will assist other scientists in future plant breeding activities.
• Increase preliminary data to enable future grant proposal development (e.g., alfalfa germplasm structure and use; switchgrass QTL mapping and marker development for pathogen resistance; water soluble carbohydrate analysis in grasses; bioactive compounds in trefoil)

Outcomes or Projected Impacts

• Increased acreage of cultivars with high forage yield and quality will result in enhanced economic vitality of forage and livestock production operations.
• Increase in improved germplasm will be provide material for further breeding and/or immediate commercialization with improved adaptation and resilience for production and persistence under pasture and hay production systems.
• Data that provide producers with options for cultivar selection, planting and harvesting to optimize production.

Milestones

(2022):Establishment of new cover crop sites

(2023):Establishment of Miscanthus trial plots

(2024):Assemble of multi-year G*E data

(2024):Among our milestones, we will include germplasm releases. These releases will include submissions to NPGS. Some of our material is in queue for submission to Journal of Plant of Registrations (JOPR). Several releases from our past project are currently in backlogs, awaiting state Agriculture Experiment Station approval due to COVID issues.

Projected Participation

View Appendix E: Participation

Outreach Plan

All members of the technical committee are involved in outreach to the scientific community, the seed industry, and the farm community in their region. The primary means of outreach to the scientific community include publications in peer reviewed journals and presentations at conferences. Many of the members of the technical committee have active breeding programs that release cultivars, and these members typically have connections with seed companies to market their cultivars. The technical committee will work to enhance communication between scientists and industry colleagues to more effectively transfer results to industry and also to ensure research is being conducted on topics of relevance to the industry. In the current proposal, the alfalfa germplasm project already has S&W Seeds as a collaborating member and will involve other alfalfa companies as the project continues. Similar efforts to engage with seed companies will be made for the other objectives. For all objectives, the ultimate goal is to develop cultivars that can be licensed to seed companies and sold to farmers or other users. Finally, all members of the committee routinely speak at extension or grower meetings in their respective locations and work to ensure that extension personnel know about and are conversant ontheir research programs.

Throughout the life of the project, we will regularly invite other forage breeders, pathologists, entomologists, physiologists, and agronomists in the public and private sectors to collaborate on aspects of the projects, as needed. Any of these participants are also welcome to join the project as official members. Our goal is to be as inclusive as we can be to ensure we reach our objectives.  

Organization/Governance

This project is organized by objective, with each objective having one or more lead scientists. Like the current NE1710 project, the lead scientists will prepare annual summaries of research in their objective (or sub-objective) and lead the discussion at the annual meeting. These scientists are tasked with keeping the objective moving forward, meeting the objectives in a timely manner, and tracking to ensure that each participant is keeping their part of the project going according to plan. All other participants contribute updates on their work.

 

The annual meetings have a chair and a secretary, who typically rotates to chair the succeeding year. The secretary for the next meeting is elected by the membership each year.

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