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 was 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 ended in October 2025.
1. Material
The study was conducted using 312 accessios from USDA Pea Single Plant Plus Collection (PSPPC), 50 USDA accessions identified as winter hardy by CCB collaborators, and 10 CCB network selected pea materials. 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 were not already genotyped, tissue samples were harvested and sent to UW-Madison Biotechnology Center for genotyping-by-sequencing (GBS).
3. Phenotyping
Phenotyping for cold temperature tolerance in all populations were conducted in controlled environment cold temperature growth chambers. Pea populations (described above) were tested for freezing tolerance at -12oC.
3.1 Protocol development
In fall 2024, 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, and that the duration of cold teperature exposure has a greater effect than the minimum final temperature reached. As a result, only one experiment with -12°C final temperature, was conducted.
3.2. First Phenotyping Experiment
The accessions were divided into two groups based on germination timing. The phenotyping cycle was conducted eight times across two growth chambers, providing four biological replicates. In each cycle, nine flats were planted, with 50 cells per flat. Accessions were randomly assigned to cell positions, and each accession was planted in two cells per cycle.
Plants were grown under normal room conditions (19oC/12oC day/night temperature with a 12-h photoperiod) until they reached the two-leaf stage. At this stage, plants were acclimated for 7 days at 6oC/0oC day/night temperature with a 10-h photoperiod. On the eighth day, the day/night cycle was shortened to a few hours, after which the temperature was gradually reduced at a rate of 2oC per hour until it reached −12oC. Plants were maintained at −12oC for 16 h and then allowed to recover at room temperature for up to 14 days. Lights remained off during the temperature decrease and throughout the cold treatment. A relative humidity of 60% and light intensity of 400 µmol m⁻² s⁻¹ was maintained when the lights were on throughout the experiment.
The phenotypic data was recorded based on visual estimation of winter damage, winter survival, growth scoring (Table 1), and ion leakage. The percentage survival was 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 was used (Hatsugai & Katagiri, 2018), which involved analyzing the leaves using the methods described by Hatsugai & Katagiri (2018). Briefly, the leaf tissues were washed three times in deionized water and then immersed in 2 ml of deionized water at 26°C for 12 hours. The initial conductivity (C1) was measured using a Horiba LAQUAtwin EC-33 compact conductivity meter (Horiba LAQUAtwin, Woodland, TX, USA). The leaf samples in deionized water were then be boiled for 15 minutes before cooling to room temperature, and the total conductivity (C2) was measured. The relative ion leakage was calculated as:
C1/C2 × 100%.
The median lethal temperature (LT50) will be calculated for the top-performing accessions.
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Table 1. Growth score used in the study. |
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|
Score |
Scoring details |
|
0 |
Complete death |
|
1 |
New shoots starting to grow |
|
2 |
Two new open leaves on new shoot or old shoot |
|
3 |
Height of a new shoot 5 cm |
|
4 |
Height of a new shoot 10 cm |
|
5 |
Height of a new shoot 15 cm, or old stems have no signs of damage |
|
6 |
Height of a new shoot 15 cm, and a second small shoot appeared/ total height between 15-30 cm |
|
7 |
Whole plant grown well, height of a new shoot/plant equal to 30 cm |
|
8 |
Height of 2 shoots more than 30 cm/a single shoot 60 cm |
|
9 |
Height of 3 or more shoots more than 30 cm/a single shoot 90 cm |
4. Data analysis
Data analysis will be conducted in spring 2026. Genotypic data will be processed for quality control and SNP calling. Phenotypic data, such as winter survival, electrolyte leakage, damage, LT50, and growth score will be used in the analysis. GWAS will be conducted using TASSEL software. 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 2) 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). These families were grown in both a field and controlled environments from Fall 2023 to May 2025 to select the best 5% of plants for cold germination and vigor. The selection process involved 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 was completed in May 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) ran from late spring to early fall 2024.
The evaluation study funded by this grant used 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).
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Table 2. 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 was conducted in both field and controlled environment settings. It started in November 2024 and ended 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.
In spring 2025, data on spring stand count, fall vigor, spring vigor, and biomass were collected at both locations. Percentage survival was calculated as the number of plants survived per total plants. Percentage survival was calculated as the number of plants survived per total plants. Vigor was 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 was walked through to make references for the vigor ratings. Plant biomass was harvested on first week of May, and the samples wiere oven-dried to obtain the dry biomass weight.
2.2 Controlled environment evaluation
The controlled environment evaluation was conducted in a gusseted thermogradient table from Jan 10 to Feb 10 in 2025. A randomized complete block design was used with four replications. The seeding rate matched field conditions (60 lbs/acre). The table was equipped with two circulating baths to regulate the temperature, creating a gradient ranging from -1°C to 3°C across the gussets. Data was collected on the first day of emergence, percent of plants germinated per family, and vigor.
3. Data analysis
Data analysis will be conducted in spring 2026 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 the final report.
Education & outreach activities and participation summary
Participation summary:
The preliminary findings from this study were shared in American Society of Agronomy, Crop Science Society of America, Soil Science Society of America (Tri-Societies) Annual Meeting, Cornell University Cover Crop Field Day, and Cornell Willsboro Research Farm Field Day in 2025.
Cornell experimental stations host an annual ‘Cornell Seed Growers Field Day’ and ‘Field Crops Field Day’. We will conduct a similar demonstration, handouts, and brief oral presentations for these events in 2026. 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, 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, such as Cover Crop Breeding Network Annual Meeting.
The findings of both studies will be submitted to relevant and high-impact journals such as Crop Science, 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.