Progress 09/01/19 to 08/31/22

Outputs
Target Audience:The target audiences for this project are threefold: farmers/ranchers, public and private sector alfalfa breeding programs, and other plant breeders. Our germplasm enhancement project is the first to be conducted systematically within any forage crop (as far as we know) and the results will serve to inform the development of similar programs for other crops. The resulting germplasm pools we are developing as part of that project will be of interest to alfalfa breeding programs as potential germplasm to add to their elite breeding pools as a source of novel genetic variation. Finally, farmers and ranchers will benefit from new cultivars that incorporate these new genetic sources. Because breeding is a long-term endeavor, however, the impact at the farm/ranch level will still be some years away. We have directly interacted with several ranchers regarding this project. We have discussed this project with alfalfa breeders from the major alfalfa breeding companies in the US and they have been very interested in this project. They have written letters of support and at least one of them has been evaluating germplasm developed as part of this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?At all funded locations (CA, NY, WA, and WI) as well as at three unfunded locations (SD, QB, and AB), numerous graduate and undergraduate students were involved with selecting, intercrossing, and measuring plants. The immense variability in these trials gives students and staff (and faculty) an appreciation of the breadth of diversity for many traits that exists in alfalfa germplasm. In addition, discussion of the results of these trials and demonstrating this diversity is very useful to show students in classroom settings as well as fellow scientists in professional settings and farmers and industry representatives at field days the possibilities that exist within the alfalfa germplasm collection. How have the results been disseminated to communities of interest?All locations hosted field days at which farmers, extension personnel, and industry breeders and personnel hear about various projects in the PIs programs, including this one. We also directly discussed this project with industry personnel through private tours. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Although this is a final report, the projects that we conducted or are conducting as part of this grant will continue, provided the PIs can find additional funding. Because we are working with a perennial plant, evaluations are necessarily multi-year. The conceptual idea of the project is that the large alfalfa germplasm collection maintained by the USDA National Plant Germplasm System likely contains alleles and genes that are not present in commercial cultivars but that are of value to alfalfa production in a rapidly changing world. Consequently, we are evaluating accessions from the collection for adaptation to diverse North American environments, selecting plants that have desirable characteristics - vigor, upright growth, freedom from diseases and insect pests, and multi-year survival, intercrossing these selections, and then conducting subsequent cycles of selection to further improve the populations. Objective 1: Continue to improve Northern US adapted germplasm pools a. DORMANT ALFALFA "ROUND 1, CYCLE 2" GERMPLASM TRIAL. We had previously conducted two cycles of selection in four dormant alfalfa populations derived from germplasm that originated in Europe, Central Asia, Siberia, and Turkey. During this grant, we planted evaluation trial in Davis, CA; Tulelake, CA; Madison, WI; Ithaca, NY; Brookings, SD; Ste. Foy, QC; and Lethbridge, AB in autumn 2021 or spring 2022 consisting of 20 Cycle 2 populations we developed in CA, NY, WI, QC, and AB. We collected yield and height data from these trials in summer and fall 2022. All populations have been genotyped by a collaborator in Minnesota using genome-wide genetic markers to assess the genetic similarity of the germplasm to one another and to current cultivars. The C2 selections were grown in cage isolations and seed was produced seed in Prosser, WA of four composite across-location germplasm pools. We are in the process of releasing these germplasms. b. DORMANT ALFALFA "ROUND 2, CYCLE 1" GERMPLASM SCREENING. We created 12 new "R2C1" improved germplasm pools using different starting germplasm. During 2021-2022, we intercrossed selected plants to develop four "Round 2, Cycle 1" populations at each location (CA, NY, and WI). These populations will be evaluated in multi-location trials if we can acquire additional funding for this project. Objective 2: Develop and expand Southern US adapted germplasm pools NON-DORMANT ALFALFA EVALUATION TRIALS. We evaluated replicated trials with 400 germplasm entries established in 2021 at two California locations, in Davis and Five Points (UC Westside Research Station), and in Prosser, WA. The trial Westside was irrigated with saline water beginning in April 2022. Biomass yield was measured in autumn 2021 (Davis and Prosser) and in 2022 (all locations). Plant height was measured multiple times throughout the year at all locations, including in November to assess autumn dormancy. Numerous morphological and phenological traits were measured at one or more locations in 2021 and 2022. Substantial variation in plant biomass and autumn height exists among these accessions. Samples were also taken to assess forage nutritive value; they are still being processed and will be analyzed in 2023. In Prosser, some accessions died due to winter, so in addition to yield, height, and morphological data, we also have a winter hardiness assessment for these accessions. DNA markers has been assayed on all these germplasms and we are in the process of conducting a genome-wide association study (GWAS) comparing marker profiles with phenotypic data to identify genetic markers useful for selecting the various traits. Relationships among these ~400 accessions show that South American germplasm is clearly distinct from non-dormant germplasm derived from North Africa, the Arabian Peninsula, southern Turkey and Iran, and India. Objective 3: Assess the genetic shifts occurring in Northern pools after disease resistance selection. We had initially intended to assess genetic shifts after disease resistance selection. This work was going to be done in conjunction with a private company breeder, who was going to do the disease screening at company expense. However, during the proposed timeframe, he retired and we were not able to complete this aspect of the project at this time.

Publications

Progress 09/01/20 to 08/31/21

Outputs
Target Audience:The target audiences for this project are threefold: farmers/ranchers, public and private sector alfalfa breeding programs, and other plant breeders. Our germplasm enhancement project is the first to be conducted systematically within any forage crop (as far as we know) and the results will serve to inform the development of similar programs for other crops. The resulting germplasm pools we are developing as part of that project will be of interest to alfalfa breeding programs as potential germplasm to add to their elite breeding pools as a source of novel genetic variation. Finally, farmers and ranchers will benefit from new cultivars that incorporate these new genetic sources. Because breeding is a long-term endeavor, however, the impact at the farm/ranch level will still be some years away. We have directly interacted with several ranchers regarding this project. Changes/Problems:There are no major problems. Some trials planned for fall 2021 had to be delayed to spring 2022 due to weather, water, or lingering covid-related labor issues. We had initially planned to compare allele frequency shifts in some pools selected for disease resistance by a colleague at an alfalfa breeding company. He has retired, and the completion of this aspect of the project has been delayed (although should still be feasible to be completed in the coming year). What opportunities for training and professional development has the project provided?At all funded locations (CA, NY, WA, and WI) as well as at four unfunded locations (FL, SD, QB, and AB), numerous graduate and undergraduate students were involved with selecting, intercrossing, and measuring plants. The immense variability in these trials gives students and staff (and faculty) an appreciation of the breadth of diversity for many traits that exists in alfalfa germplasm. In addition, discussion of the results of these trials and demonstrating this diversity is very useful to show students in classroom settings as well as fellow scientists in professional settings and farmers at field days the possibilities that exist within the alfalfa germplasm collection. How have the results been disseminated to communities of interest?All locations host field days at which farmers and extension personnel hear about various projects in the PIs programs, including this one. At a well attended field day in Tulelake in July 2021, we discussed explicitly the value of this project, and demonstrated some of the breadth of germplasm we were evaluating. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will develop four germplasm release applications for the four pools formed across locations. We intend to complete a genetic marker analysis of both northern and southern pools, something that was severely impacted by covid. In a positive twist, we will be able to use a new genotyping array developed by the USDA-ARS, which can serve a a common genotyping platform for all germplasm. This will be very desirable. We will have new trials of northern germplasm in the ground at six locations and will continue evaluating those trials as well as existing southern germplasm trials. Genotyping of southern germplasm will be coupled with our multilocation yield, morphological, and nutritive value phenotyping in a genome-wide association study to identify genomic regions of interest for these traits.

Impacts
What was accomplished under these goals? The overarching goal of the project is to develop pre-breeding germplasm pools derived from National Plant Germplasm System accessions that will be useful for commercial alfalfa breeding programs. There are two sets of pools being developed, one for the northern USA and Canada and one for the southern USA. In each region, four to five populations derived from geographically distinct germplasm accessions are being developed and reselected for multiple generations to stimulate recombination. 1. Improving Northern pools of alfalfa germplasm. a. NORTHERN POOL ALFALFA "ROUND 1, CYCLE 2" GERMPLASM TRIAL. We had previously developed four dormant alfalfa germplasm pools based on geography: Europe, Central Asia, Siberia, and Turkey and completed a second cycle of selection in these populations in six locations (the three funded collaborators on this project in California, Wisconsin and New York as well as three unfunded collaborators in South Dakota, Quebec, Alberta). Seed was produced in the last reporting period at each location and seeds were distributed to all locations in 2021 to plant a trial to evaluate all 24 populations and commercial check cultivars. Some trials were planted in 2021 but others will be planted in spring 2022, including one in Tulelake, CA, due to covid and weather-related delays. These trials will continue for three years to collect data for germplasm releases and to assess comparative performance of germplasm selected from the same base population throughout northern US and Canada. We plan to evaluate all populations with genome-wide genetic markers to assess the genetic similarity of the germplasm. In parallel, co-PI Brian Irish in Prosser, WA grew plants of each of the four populations from each of the six locations. For each of the four germplasm pools, his team planted equal numbers of plants from each of the six selection locations into a bee pollination cage during summer 2021 to produce large quantities of seed of the four populations pooled across selection environments. That seed was harvested in fall 2021 and will be used in new plot trials in 2022. b. NORTHERN POOL ALFALFA "ROUND 2" GERMPLASM SCREENING. The three co-PIs completed a trial of new set of 100 germplasm accessions used to create a second round of improved Northern germplasm pools. During the reporting period, we selected individual plants from trials in Tulelake, CA; Madison, WI; and Ithaca, NY that derived from the four origins discussed above. The plants were moved to the greenhouse for intercrossing during the winter 2021-22. These will be included in new trials in 2022. 2. Improving Southern pools of alfalfa germplasm. NON-DORMANT ALFALFA EVALUATION TRIALS. We successfully established replicated trials with 400 germplasm entries in Davis and Fresno, CA; Prosser, WA; and Gainesville, FL (with unfunded collaborator, Esteban Rios). All trials established successfully. Biomass yield was measured twice in autumn and plant height was measured to infer autumn dormancy at all locations except Fresno because the trial was established in summer due to a lack of water at the research station until then and plants were small throughout the year. Substantial variation in plant biomass and autumn height exists among these accessions. Samples were also taken to assess forage nutritive value at all locations as well; they will be processed and evaluated during 2022.

Publications

Progress 09/01/19 to 08/31/20

Outputs
Target Audience:Our target audience is (1) alfalfa breeding programs in the public and private sectors who will be able to use germplasm from this trial in the future, (2) breeders in other forage crops (and possibly other crops generally) who could use our breeding template or ideas to conduct germplasm pre-breeding programs, and (3) alfalfa growers who will benefit as these improved germplasm resources are incorporated into cultivars. Changes/Problems:We did not receive final approval of the project, and hence, did not receive first year funding, until 22 April 2020. This has affected the initiation of several field trials, but we will establish them in autumn (instead of spring), which won't have a large effect on the project goals. The Covid-19 pandemic has not had a major impact on the fieldwork aspects of the project so far. Because funding was delayed, by the time we received the final approval, we were already dealing with the suspension of laboratory research due to Covid-19 and this has delayed initiation of the genetic marker work in Objective 3. Nevertheless, we should have no problem completing that aspect of the proposal. What opportunities for training and professional development has the project provided?One graduate student, one undergraduate student, one professional staff, and three technical staff were involved in selecting plants in the field, learning about different germplasm sources of alfalfa, and developing new genetic populations. How have the results been disseminated to communities of interest?In the current reporting period, we have primarily interacted with various communities through email or phone calls. Several field days have been cancelled due to Covid-19 and alternatives have not yet been developed by the extension specialists who organize them. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Continue to improve Northern germplasm pools Continuation plans Selections in 2020 from the 2018 germplasm evaluation trials will be identified in autumn 2020 and intercrossed. These populations will subsequently be intercrossed into the exisiting four northern pools already developed above. The goal is to continually expand the genetic diversity present within these pools even as we select for desirable attributes. We will maintain local populations from each selection environment, but also produce a Cycle 2 bulked population incorporating seed from all three locations. These intercross blocks will be at the USDA-ARS station in Prosser, WA under cage isolation in summer 2021; these germplasms will be released and deposited in the NPGS collection curated by co-PI Irish. The new Cycle 2 populations developed in winter 2020 will be replanted into selection nurseries at the three locations in either fall 2020 or spring 2021. This trial will continue for two years before plants are selected. Objective 2. Develop and expand Southern germplasm pools. Continuation plans Due to the late arrival of funding for the project and the subsequent suspension of many activities due to Covid-19, we delayed planting new trials until autumn 2020. This autumn, we will plant two sets of trials. First, we will trial a new set of 200 PI accessions in two California locations (WSREC and Davis) and in Prosser, WA. This germplasm will derive from the four areas of the germplasm pools described above. These trials will be evaluated for two years, with selections made in autumn 2022. Additionally, we will plant new nurseries of the C1 populations at the same three locations, with 500 plants per population. Plots will be evaluated for two years before selections are made. Objective 3. Assess the genetic shifts occurring in Northern pools after disease resistance selection. Continuation plans We will extract DNA and run genome-wide markers and evaluate the genetic diversity of our selected populations with their respective base populations, with the original PI accessions, and with a collection of adapted cultivars. We are interested to know how well our new populations sample variation in the germplasm system, how much variation of new populations is lost during selection, and to what extent our selections differ on a genome-wide basis from current cultivars. Further, some selection for disease resistance has been conducted by Dr. Mark Smith at S&W seed within these populations, and we are interested to see what types of genetic shifts have occurred as a result of that selection. Marker analysis will begin in the second half of 2020 and continue through 2021. As we generate new populations, they will be genotyped to add to our database.

Impacts
What was accomplished under these goals? I am reporting on progress of this project from 1 September 2019 until today, 10 June 2020. We did not receive final approval of the project, and hence, did not receive first year funding, until 22 April 2020. This has affected the initiation of several aspects of the project, as I'll describe below. This project is a continuation of a previously funded project from this same program, Award Number 2017-70005-27117. Because of this, we have made significant progress on this project despite the late receipt of funds, since field trials were already established. The Covid-19 pandemic has not had a major impact on the fieldwork aspects of the project so far. Because funding was delayed, by the time we received the final approval, we were already dealing with the suspension of laboratory research due to Covid-19 and this has delayed initiation of the genetic marker work in Objective 3. Objective 1. Continue to improve Northern germplasm pools Progress to date The three collaborating locations (NY, WI, and CA) all made selections in autumn 2019 of desirable plants from each of four geographically distinct germplasm pools adapted to the northern US. These three pools are Cycle 1 populations developed by co-PI Riday. The selected C1 plants were intercrossed during winter-spring 2020 at each location creating four locally adapted Cycle 2 populations at each location. The same three locations continued to evaluate a trial of 100 germplasm accessions planted in 2018 for performance. Objective 2. Develop and expand Southern germplasm pools. Progress to date In autumn 2019, we selected parental plants for four geographically distinct germplasm pools from an evaluation nursery of 200 Plant Introduction accessions growing at the Westside Research and Extension Center (WSREC) in Fresno County, CA since 2016. The four populations (roughly, India, Arabian Peninsula, Egyptian (NE Africa), and Northwestern Africa) form the basis of a southern germplasm pool pre-breeding program. The selected plants of these four populations were intercrossed in the greenhouse over winter/spring 2020 to form the Cycle 1 (C1) southern germplasm pool populations. Objective 3. Assess the genetic shifts occurring in Northern pools after disease resistance selection. Progress to date We have collected leaf tissue of all selected plants from all locations. Due to Covid-19, our lab activities have been suspended for the past three months, but we are beginning to reopen labs at this point. Hopefully we will be able to move this aspect of the project forward at this point.

Publications