Evaluating the Potential to Breed Kura Clover for a Southeastern U.S. Adapted Living Mulch System

Progress report for GS23-293

Project Type: Graduate Student
Funds awarded in 2023: $16,363.00
Projected End Date: 08/31/2025
Grant Recipient: Auburn University
Region: Southern
State: Alabama
Graduate Student:
Major Professor:
Dr. Marnin Wolfe
Auburn University
Expand All

Project Information


Living mulch systems are an emerging technology for sustainable agricultural intensification involving a continuous ground-covering crop into which cash crops like corn are planted seasonally. They have demonstrated the potential to make cropping systems more sustainable by reducing nitrogen fertilizer requirements and weed management costs, increasing water infiltration and conserving water and nutrients for the main crop. Legume living mulches, like one using perennial Kura clover are of strongest interest as they provide nitrogen fixation. Despite their potential, suitable Kura clover and living mulch systems for row-crop production in the southeastern US are yet to be developed. In this project, we are evaluating the potential to breed Kura clover for southeastern US adapted living mulch. We are specifically evaluating a diverse set of Kura clover germplasm for cover and forage quality traits and subsequently develop tools that will help design an efficient selection strategy for Kura improvement. Overall, a seedling evaluation of Kura has been conducted and promising results have been registered. Preliminary analyses have showed large genetic variation (H2 > 0.8) for plant height and groundcover in the Kura seedling population indicating potential for selection. Motivated by these results, a clonal trial with larger plot sizes was successfully planted at EV Smith Research Center in March 2024 to enable further assessment of these traits and biomass quantity and quality associated traits. We are currently exploring opportunities to conduct a multi-environment evaluation of Kura to also assess the role of environment in Kura variety development. These assessments will provide data for initial parental selection and population improvement that will consequently produce new and improved Kura varieties with Southeastern and living mulch system adaptation. 

Project Objectives:

Obj 1: Evaluate the performance of diverse Kura clover germplasm set in a multi-year trial.

Obj 1.1 Identify the best performing genotypes for key morphological, agronomic and     

              physiological traits in this diverse germplasm set. 

Obj 1.2 Assess the genetic correlation structure among evaluated Kura clover traits to  

             understand their inter-relationships. 

Obj 1.3 Assess the heritability and genetic complexity of Kura traits linked to


Obj 2: Develop Near Infrared Spectroscopy equations for rapid analysis of Kura clover

            forage quality traits.

Obj 3: Study the genetic diversity of Kura Clover and the prospect to use genomics to accelerate  



Materials and methods:

Obj 1: Evaluate the performance of diverse Kura clover germplasm set in a multi-

            year trial.

Trial 1: Seedling evaluation of Kura Clover

The first trial was a seedling evaluation of ~ 600 Kura clover accessions that were constituted from 236 half-sib families from USDA National Plant Germplasm System (NPGS, 123 families) and The Land Institute (TLI, 93 families). The NPGS families were Plant Introductions (PI) originating throughout the world whereas the families from TLI’s Kura breeding program were progenies from the PIs. Kura clover being a legume and obligate outcrossing species, the six selected seeds per accession/family were inoculated with Rhizobia and started out in 72-cell trays under greenhouse conditions, with each seed being a unique individual placed in a different tray cell identified by the tray row and column.  Seedlings were raised in 72-cell trays up to two months when seedlings had produced ~ 6 leaflets. Of the 1440 seeds sown, ~ 600 seedlings successfully emerged and these were subsequently transplanted to moist clayey-loam soil at EV Smith Research Center, Tallassee, Alabama in the May of 2023, where they were laid out using an augmented randomized spaced plant trial. TLI2022 Kura Clover, an advanced selection from TLI's breeding program, and Durana White Clover, which is not a Kura clover but a common commercial cultivar with a similar growth habit, served as checks for seedling evaluation trial owing to sufficient seed quantities. Seedlings were spaced 3 feet apart, and each spaced seedling/plant was a genetically unique individual. This seedling evaluation was essentially a rhizome production trial aimed at producing planting material (rhizomes) to enable assessment of cover and forage quality traits of Kura in the clonal trial. However, ~ 70 percent of evaluated germplasm was lost due to severe herbicide damage. Consequently, data including plant height, days to flowering and groundcover was collected on 176 accessions.

Figure 1: Seedling evaluation of Kura clover at EV Smith Research Center, Tallassee, Alabama in 2023. Each spaced plant is a unique Kura genotype.
Figure 1: Seedling evaluation of Kura clover at EV Smith Research Center, Tallassee, Alabama in 2023. Each spaced plant is a unique Kura genotype.











Trial 2: Clonal evaluation of Kura Clover

Following a major loss of the seedling material in Trial 1, a total of 655 Kura clover genotypes from the remnant seed of the 236 families that had been assembled from NPGS and TLI were raised under greenhouse conditions during Fall/Winter season to produce rhizomes for Spring planting. 1416 seeds were started out in 72-cell seed trays in the September of 2023 following the same process as described in Trial 1. After two months (November 2023), the 655 seedlings that had successfully established were transplanted to gallon-size plots filled with potting mix that had been mixed with Oscomote 14-14-14 slow-release fertilizer to enhance rhizome production. The pots were laid out in a greenhouse where they were provided natural lighting, regular watering and room temperature which created a favorable environment for rhizome production.


Kura clover rhizome production under greenhouse conditions
Figure 2: Kura clover rhizome production under greenhouse conditions. A total of 655 genotypes were planted and each pot is a unique Kura genotype.


A major advantage of Kura is that it can be cloned and thus the clones for each accession can be established in row plots to enable assessment of yield-related traits. Of the 655 genotypes, 415 genotypes produced 6 or more rhizomes per plant/genotype/seedling, and these were selected for the clonal evaluation trial that was planted in the March of 2024 at EV Smith Research Center, Tallassee, Alabama. Furthermore, ~ 25 percent of the 415 genotypes produced ~ 12 while the remainder produced 6 but less than 12 rhizomes. Accordingly, a partially replicated design was adopted for the clonal trial with ~ 25 percent of genotypes being replicated twice, thereby making a total of ~ 520 plots. Each plot was a genotype that comprised a single row of 6 rhizomes/plants that were spaced 1.5ft within the row and 3 ft between rows. All rhizomes for all genotypes that were included in the trial were defoliated and dipped in CloneX to enhance root development and uniform establishment. CloneX-dipped rhizomes were wrapped in moist newsprint paper and stored under refrigeration in zipper-lock plastic bags the day prior to planting to minimize desiccation and respiration. The rhizomes were subsequently planted in moist sandy-loam soil and are currently being monitored for emergence/early establishment. NIR spectra and samples for forage quality analysis will be collected from this clonal trial. Evaluated Kura genotypes will also be assessed for biomass weight, plant type, summer hardiness, plant height, days to flowering, chlorophyll content, number of rhizomes per plant, and groundcover measured using canopy area and proximal imaging, among other traits. This trial is expected to be harvested in October 2024.


Kura rhizomes ready for planting
Kura rhizomes ready for planting


Trial 3: Fall 2024 – Mid-October 2025

Initially, germplasm evaluation was planned for a single-site multi-year trial at EV Smith Research Center, Tallassee, Alabama, because of inadequate planting material for studied Kura genotypes. However, the recently conducted spaced plant trial suggested that adequate planting material can be accumulated for two to three fully replicated trials of six-plant-plot sizes in a period of six to eight months. Thus, efforts to explore the opportunity to conduct a multi-environment evaluation of Kura germplasm are ongoing. This type of assessment is especially important to assess the role of environment in Kura growth and establishment which will consequently guide variety development and deployment. Target collaborators include The Land Institute in Kansas, and Donald Danforth Plant Science Center in Missouri, or potentially the University of Florida since the former have previously worked with Kura germplasm. This multi-environment trial is expected to commence in Fall of 2024 and run up to end of Summer 2025.


Obj 2: Develop Near Infrared Spectroscopy equations for rapid analysis of Kura forage quality traits. 

Efforts to purchase a handheld NeoSpectra Near Infrared Reflectance (NIR) analyzer are almost complete, as the equipment is expected before end of May 2024. The NeoSpectra NIR analyzer has been selected for its wide spectral range (1350nm to 2500nm) and versatility under field conditions (https://www.si-ware.com/neospectra-product-pages/neospectra-scanner). This equipment will be used to collect spectra from the recently propagated clonal trial (Trial 2). Spectra collection is expected by end of October. Fresh NeoSpectra-scanned samples will be ground for benchtop NIR analysis at Auburn University Forage Analysis Laboratory, and consequently shipped to the University of Georgia Plant and Soil Analysis Laboratory for wet chemistry analysis. Samples will be analyzed for neutral detergent fiber, acid detergent fiber and crude protein as they are respectively associated with feed intake, digestibility and nutritional content. It is this wet chemistry data that will be used as the reference for development of calibration equations for the NeoSpectra NIR analyzer, and this task is expected to be completed by the end of February 2025.


Obj 3: Study the genetic diversity of Kura Clover and the prospect to use genomics to accelerate breeding prospects.

Tissue samples from a diverse set of 585 Kura genotypes have been collected and preserved under -800C refrigeration in the Auburn University Agro-genomics Lab. Arrangements are being made to ship these samples to the Joint Genome Institute (JGI) and to HudsonAlpha in Huntsville for DNA extraction and subsequent genotyping. The waiting time for genotyping at JGI is roughly six months, and thus genomic data is expected by end of 2024. This genomic data will be used to study genetic diversity of the Kura and subsequently used alongside the field measurements to study inheritance of evaluated cover and forage quality traits and to develop genomic prediction equations for such traits. 

Research results and discussion:

Data including plant height and groundcover were taken from the remaining ~ 30 percent of the seedling population in Trial 1. Plant height was measured using a meter ruler and recorded as the distance from the ground to the tallest leaflet. Groundcover on the other hand was also measured using a meter ruler as the width of the spaced plant. Since the measurements provided herein were recorded on non-replicated single plants, the results presented are tentative and pending confirmation from larger plot-size evaluations in the current clonal trial. For both plant height and groundcover, analysis of variance was performed to determine if evaluated Kura genotypes performed significantly different following the augmented design analysis. Mixed models were used to extract variance components associated with genotype effects via restricted maximum likelihood procedure which was used to compute heritability.

Significant differences (P < 0.05) were observed among evaluated Kura genotypes for both plant height and groundcover. Plant height and groundcover were also moderately correlated (r = 0.45, P < 0.001). Plant height ranged from 2.0 cm to 16.0 cm with a mean of 7.7 cm. Groundcover on the other hand ranged from 5.5 cm to 40.0 cm with a mean of 19.9 cm. Heritability for plant height and groundcover was generally high (H2 > 0.8) indicating large genetic variation for the studied traits in the evaluated Kura population.

groundcover histogram

histogram of plant height in Kura


Participation Summary

Educational & Outreach Activities

Participation Summary:

Education/outreach description:

No educational or outreach activities at this point. A major focus has been to assemble Kura germplasm and multiplying it so that it can be evaluated for groundcover and forage quality traits. 

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.