Progress report for GNE24-326
Project Information
Cereal rye (Secale cereale) is the most popular and
winter hardy cover crop. Alternating cover crop species in a crop
rotation diversifies the ecosystem services from cover crops.
Winter pea (Pisum sativum L.) is a popular winter annual
legume cover crop due to its ability to fix nitrogen. However,
current cultivars exhibit inconsistent winter survival in the
northeast US. This study will conduct genome-wide association
studies on winter peas to discover pea germplasm with cold
temperature and freeze-thaw tolerance and locate genetic markers
for cold tolerance. The identified germplasm and genetic markers
can be utilized for accelerated breeding of winter hardy peas
adapted to northeast US in the future.
In the northeast US, corn and other major cash crops are
harvested late in the fall, leaving a small planting window for
winter cover crops. When planted late, winter cover crops,
including cereal rye, often experience below-freezing
temperatures on most nights during germination and in their early
growth stages. This can limit the establishment, ground cover,
and biomass production of cereal rye. Selecting the cereal rye
accessions for cold temperature germination and vigor can lead to
the development of a cultivar with a higher level of winter
hardiness than current cultivars. Such cultivars can be planted
later in the fall without reducing their cover and overall
benefits. These projects will benefit northeast farmers of the US
who are planting winter peas and cereal rye, either for cover
crops or any other purposes.
- Discover pea germplasm with cold temperature and freeze-thaw
tolerance for breeding winter annual cover crop varieties. - Locate genetic markers for cold temperature tolerance in
winter pea to accelerate breeding of winter hardy pea cultivars. - Identify efficient methods to breed cereal rye for cold
temperature germination rate and vigor and develop rye cultivars
with improved growth at late fall planting dates in the Northeast
US.
The purpose of this project is to improve the winter hardiness in two popular winter cover crops, winter peas, and cereal rye. Winter pea (Pisum sativum L.) is one of the widely planted annual legume cover crops because of its potential to fix nitrogen (N) and effective weed suppression (Clark, 2007). Peas can return approximately 25 lb/acre of N to the soil, thus reducing soil nutrient depletion and N application in the following crop (Oelke, et al., 2022). However, the Northeast US has very harsh winter conditions leading to very low and inconsistent winter survival of most cover crops including winter peas. Cold temperature stress reduces nodulation and nitrogen fixation in winter annual legume cover crops (Thurston et al., 2022). Moreover, winter killed cover crops produce less biomass (Florence et al., 2019), suppress weed less (Ranaldo et al., 2020), lead to high N leaching (Gollner et al., 2020), and provide a lower total amount of ecosystem services due to their shorter lifespan compared to winter hardy cover crops (Kaye et al., 2019; White et al., 2016). Despite efforts to breed winter hardy peas and identify genomic regions associated with winter hardiness across the globe, consistent winter survival has not yet been achieved. In addition, frequent freeze-thaw cycles caused by climate change have further reduced the cold tolerance of peas. Therefore, more efforts are necessary to improve the winter hardiness of peas.
Genome wide association studies on the cold tolerance of winter peas in a new population will help to identify new sources of winter hardiness and genetic markers associated with winter hardiness (Liu et al., 2017). The personnel in this project are part of the Cover Crop Breeding (CCB) Network, a group of plant breeders, agronomists, and farmers that have been breeding winter peas and other cover crops for adaptation to the northern US. Winter hardy material and markers/genomic regions identified through this project can be used by CCB network collaborators and other researchers in the marker-assisted selection and breeding of winter hardy peas. Marker assisted breeding shortens the breeding cycle, allowing for the earlier release of new regionally adapted varieties. Development and adoption of winter hardy peas can reduce the N cost for the following cash crop, increasing the profitability of Northeast farmers. Planting winter peas before corn or a non-legume cash crop can mitigate the risk of transferring pests and pathogens from rye (the most widely used winter hardy cover crop) to corn or other cash crops in the Poaceae family (Dawadi et al., 2019; Ha & Hart, 2020; Snapp et al., 2005). It will ensure sustainability and resilience, and foster conditions where farmers have high profit, high quality of life, and communities can thrive.
Cereal rye is a commonly used winter annual cover crop because of its ability to withstand cold temperatures and can be planted late in the fall (Wayman et al., 2017). It offers many benefits over other cover crops, such as rapid production of ground cover, prevention of soil compaction, high biomass production, and the highest level of winter hardiness (Li et al., 2011). It has become a staple winter cover crop in corn-soybeans production systems. Cereal rye can germinate in temperatures as low as 1oC (Clark, 2007) and can tolerate temperatures as low as -30°F once it is well established (Grubinger, 2021).
However, most cereal rye cultivars are open-pollinated and there is a large variation in performance within and among cultivars (Gailans, 2021). In the Northeast US, corn is harvested between October 20 and November 20 (USDA, 1997). When farmers plant rye after corn harvest, cereal rye would experience below freezing temperatures most of the nights during germination and early growth stages, which can limit its establishment, ground cover, biomass production, and nitrogen scavenging (Farsad et al., 2011; Mirsky et al., 2009; Szuleta et al., 2022). Selecting cereal rye for cold temperature germination and vigor can lead to the development of a cereal rye cultivar with a higher level of winter hardiness than current cultivars. Such cultivar can be planted later in the fall without reducing its benefits. This will increase the profitability of farms in the Northeast in the long term by contributing to soil health and protection, ensuring sustainability and resilience, and fostering conditions where farmers have high profit, high quality of life and communities can thrive. The development of cereal rye cultivar with high cold germination and vigor allows farmers to fit cereal rye into common crop rotations.
Research
Project I. Genomewide association studies for cold tolerance in winter peas
This study started in November 2024 and will end by May 2025.
1. Material
The study is being conducted using USDA Pea Single Plant Plus Collection (PSPPC) which constitutes 431 accessions, some USDA accessions identified as winter hardy by CCB collaborators (50 accessions), and CCB network selected pea materials (10 accessions). These materials were readily available to us through our collaborators and the USDA Germplasm Resources Information Network (GRIN).
2. Genotyping
The PSPPC has already been genotyped, and the information is publicly available (Holdsworth et al., 2017). For pea accessions and populations that are not already genotyped, tissue samples will be harvested and sent to UW-Madison Biotechnology Center for genotyping-by-sequencing (GBS). GBS is not offered at the Cornell genomics facility.
3. Phenotyping
Phenotyping for cold temperature tolerance in all populations are being performed in a controlled environment cold temperature growth chamber. The chambers have been operational since October 2024. Approximately 500 pea populations (described above) will be tested for freezing tolerance at two final (minimum) temperatures ( -12oC and -15oC) in separate experiments.
3.1 Protocol development
Starting in the first week of November, five commercial cultivars (2 susceptible, 1 moderately hardy, and 2 hardy cultivars) were used to identify optimal protocols in a controlled-environment growth chamber. We tested various acclimation periods, durations of exposure at the final temperature, and final temperatures. A protocol that can differentiate between winter hardy and susceptible germplasm was developed. During the protocol development process, we discovered that even spring-type peas can survive as low as 2°C. As a result, our initial phenotyping experiment, which proposed 2°C as the final temperature, has been excluded.
3.2. First Phenotyping Experiment
The first replication of this experiment was planted on January 8. The experiment will be repeated four times to produce four biological replicates. This experiment will reach a final minimum temperature of -10°C.
Plants will be grown under normal conditions (19°C/12°C day/night temperature) until they reach the two-leaf stage. At this stage, they will be acclimated for 7 days at 8°C/2°C day/night temperature with a 10-hour photoperiod. Following the acclimation period, the cold treatment will begin during the night. The temperature will be gradually reduced at a rate of 2°C per hour until it reaches -10°C. Plants will be exposed to -10°C for 10 hours before being allowed to recover at normal room temperature for up to 14 days.
The phenotypic data will be recorded based on visual estimation of winter damage, winter survival, and ion leakage. The percentage survival will be calculated by dividing the number of plants that survived by the total number of plants. To determine the level of membrane damage under cold temperatures, the relative leakage assay will be used (Hatsugai & Katagiri, 2018), which involves analyzing the leaves using the methods described by Hatsugai & Katagiri (2018). Briefly, the leaf tissues will be washed three times in deionized water and then immersed in 25 ml of deionized water at 26°C for 12 hours. The initial conductivity (C1) will be measured using a Horiba LAQUAtwin EC-33 compact conductivity meter (Horiba LAQUAtwin, Woodland, TX, USA). The leaf samples in deionized water will then be boiled for 15 minutes before cooling to room temperature, and the total conductivity (C2) will be measured. The relative ion leakage will be calculated as:
C1/C2 × 100%.
The median lethal temperature (LT50) will also be calculated for the top-performing accessions.
3.3. Second Phenotyping Experiment
This experiment will reach a final minimum temperature of -15°C. Accessions with complete death or major damage in the first experiment will not be used in this experiment. A randomized complete block design (RCBD) will be used with four biological replicates and 10 plants per accession. The cold temperature treatment and cold acclimation procedures will be similar to the first experiment. In this experiment, after plants are left in the final temperature for 10 hours, half of the plants in each accession will be taken out of the chamber to recover at room temperature, while the other half will be exposed to additional sudden freeze/thaw conditions. For freeze/thaw conditions, the temperature will be increased to 10°C at the rate of 5°C per hour, maintained at 10°C for 6 hours, and then decreased again to -15°C at the rate of 2°C per hour. Phenotypic data on visual estimation of winter damage rating, winter survival, and ion leakage will be collected as in the first experiment. The phenotypic information on the accessions will be used for analysis.
4. Data analysis
Phenotypic data, such as winter survival, electrolyte leakage, damage, LT50, and regeneration potential will be used in the analysis. GWAS will be performed in R Studio. Briefly, principal component analysis will be conducted to find groupings of populations. The kinship analysis will be conducted to identify the relationship between the accessions and the genetic diversity in the population. Association analysis will be conducted using the genotype data and phenotype data with two models including GLM (generalized linear model) +Q (population structure) and MLM (mixed linear model) + Q + K (relative kinship). Common markers that will be found as significant in both models will be revealed to have the most reliable association with cold temperature tolerance in peas.
Project II: Evaluating cereal rye population and methods of selection for cold temperature germination and vigor
1. Material:
The original material for the study was obtained from CCB collaborators at NC State University. At NC State University, 10 bulked full-sibling families from 10 different crosses (Table 1) were open pollinated in a field to obtain 10 half-sibling families (C0). Highly allelopathic lines were included as male parents and northern region adapted commercial rye cultivars were included as females (Table 1). Since fall 2023, these families have been grown in both a field and controlled environments to select the best 5% of plants for cold germination and vigor. The selection process will involve one cycle in the field and two cycles in a controlled environment. The field selection (C1F) was done at Willsboro Farm in Willsboro Point, NY, and the harvesting will be completed by July 2024. Funding for this Willsboro field selection is provided by the Northern New York Agricultural Development Program (NNYADP) until December 2024. The controlled environment selection was done using a gusseted thermogradient table. The first cycle (C1C) of controlled environment selection took place from fall 2023 to late spring 2024, and the second cycle (C2C) will run from late spring to early fall 2024. The study will use bulked seeds from the original NC state population, the common cultivar ND Gardener as a control, and bulked seeds from selected populations from each selection cycle, totaling four genotypes (C0, C1F, C1C, C2C).
|
Table 1. Cereal rye population obtained from NC State University |
|||
|
SN |
Female |
Male |
|
|
1 |
NDGardner |
X |
NC20-A122-2 |
|
2 |
NDGardner |
X |
NC20-R103-2 |
|
3 |
NDGardner |
X |
NC20-A117-1 |
|
4 |
NDGardner |
X |
NC20-A133 |
|
5 |
NDGardner |
X |
NC20-R114 |
|
6 |
NDGardner |
X |
NC20-A129-2 |
|
7 |
NDGardner |
X |
NC20-R101-3 |
|
8 |
NDGardner |
X |
NC20-A130-2 |
|
9 |
Aroostook |
X |
NC20-R114 |
|
10 |
Aroostook |
X |
NC20-A122-3 |
2. Methods
This study is being conducted in both field and controlled environment settings. It started in November 2024 and will end in May 2025.
2.1 Field evaluation
The study was planted in November 2024 at two field locations: Cornell Willsboro Research Station, Willsboro, NY, and Homer C. Thompson Vegetable Research Farm, Ithaca, NY (Figure 1). Willsboro, NY is located further north compared to Ithaca, NY, and experiences more severe winter temperatures. The above-mentioned material was planted in a RCBD with four replications at each location. Each plot consisted of one row of treatment planted at a seeding rate of 60 lbs/acre, bordered by a common rye cultivar. Fall stand count and fall vigor data were collected at the Ithaca location. However, due to late emergence and snow-covered fields, fall stand count and fall vigor data were not collected at the Willsboro location. This data will be collected as soon as the snow clears.
In spring 2025, data on spring stand count, fall vigor, spring vigor, and biomass will be collected at both locations. Percentage survival will be calculated as the number of plants survived per total plants. Percentage survival will be calculated as the number of plants survived per total plants. Vigor will be rated 3 times on a 1 to 9 scale, with the plot with the highest, medium, and lowest vigor being referenced as 9, 5, and 1, respectively. Before collecting the actual data, the entire field will be walked through to make references for the vigor ratings. Plant biomass will be harvested when the plants are at or right before the boot stage, and the samples will be oven-dried to obtain the dry biomass weight. A total of three trips to Willsboro will be made in spring 2025 to collect data.
2.2 Controlled environment evaluation
The controlled environment evaluation was planted in a gusseted thermogradient table on January 10th. A randomized complete block design was used with four replications. The seeding rate matched field conditions (60 lbs/acre). The table is equipped with two circulating baths to regulate the temperature, creating a gradient ranging from -1°C to 3°C across the gussets. We will collect data on the first day of emergence, the average number of days of emergence per family, and vigor.
3. Data analysis
Data analysis will be conducted in fall 2025 in R studio. Response to selection (R) in the controlled environment vs in field and across generations will be calculated as; R = mean emergence of all individuals in that cycle- mean emergence of the base population. The effect of genotype/population, location, and genotype*environment interaction on cold germination, winter survival, vigor, and biomass will be estimated using R Studio. The most efficient and effective way to select for cold temperature germination and vigor will be identified. If populations with improved cold temperature germination and vigor are identified, they will undergo further selection, testing, and released as new cultivars.
Results will be provided in 2026 report.
Education & Outreach Activities and Participation Summary
Participation Summary:
The findings from this study will be shared through a variety of channels to reach a diverse audience across the US. For the past three years, the Moore Lab has been organizing spring field walks to highlight our cover crop breeding efforts, both within the lab and throughout the broader CCB Network. Our participants typically include farmers, gardeners, seed company representatives, extension agents, and other agriculture professionals from Upstate New York. During the 2025 field walks, the project team will present a brief talk describing the project and distribute extension handouts detailing the project objectives, methods, and results. We will also create an interactive station to showcase the effects of cold damage on overwintering cover crops. Susceptible and tolerant plants grown in our growth chambers with visible signs of cold damage and tolerance will be displayed to the participants. Participants will also have the opportunity to explore the cover crop evaluation trials, ask questions, and interact with researchers. Depending on the participant's interest, we will also offer tours of growth chambers and thermogradient table experiments at a later date.
Cornell experimental stations also host an annual ‘Cornell Seed Growers Field Day’ and ‘Field Crops Field Day’. We will conduct a similar demonstration, handouts, and brief oral presentations in the field for these events. To ensure that the research insights reach the farming community, the study will be presented at various farmer meetings, including the New York Certified Organic Field Crops (NYCO) Annual Meeting, Northeast Organic Farmers Association of New York (NOFA-NY) Annual Meeting, and Northeast Cover Crop Council (NECCC) Annual Meeting. These platforms will allow the project team to directly engage with farmers, share the findings, and gather valuable feedback to further refine the research and its practical applications. In addition to the outreach efforts targeting the farming community, the research findings will also be shared with the broader scientific community. The team will present the study at scientific conferences and meetings, such as American Society of Agronomy, Crop Science Society of America, Soil Science Society of America (Tri-Societies) Annual Meeting, National Association of Plant Breeders (NAPB) Annual Meeting, and Cover Crop Breeding Network Annual Meeting.
The findings of both studies will be submitted to relevant and high-impact journals such as Crop Science or G3, which will ensure that the research findings are widely disseminated and accessible to the crop science and genetics community. By leveraging this diverse set of dissemination channels, the research team can effectively reach a broad audience, including researchers, industry professionals, and farmers. This comprehensive outreach plan will ensure that the findings from this study have a significant impact on the advancement of cover crop research and adoption, ultimately contributing to the development of more resilient and sustainable agricultural systems.