Introduction

Inspired by Wes Jackson’s ideas (1980) for a diverse, herbaceous perennial polyculture, the Rodale Research Center (now known as Rodale Institute [RI]) in the early 1980s evaluated approximately 300 different species for their utility in perennial agriculture similar to natural ecosystems. Following evaluation of more than 100 different grass species, intermediate wheatgrass (IWG, Thinopyrum intermedium (Host) Barkworth & D.R. Dewey) was selected in 1985 as having the highest potential to be developed into a perennial grain crop based on its plant structure, seed production characteristics, perennialism, food use potential (Wagoner 1990a), and the fact that it is related to important Triticeae grain crops (Wagoner 1990b) likely indicating a good nutritional profile and lack of anti-nutritive compounds. Prior to selection for domestication, IWG had a history within the United States for erosion control, forage production (Asay and Jensen 1996), and utilization as a tertiary gene pool for annual wheat (Triticum aestivum) improvement (Li and Wang 2009; Pototskaya et al. 2022).

Since the identification of IWG as a target of domestication, a dedicated and expanding research effort has been conducted to bring the idea of perennial grains to fruition. Research on IWG for perennial grain use has focused on all facets of plant breeding, agronomic practices, and potential consumer utilization. Some of this research has shown the environmental benefits posited by perennial crops such as reduced nitrate leaching (Culman et al. 2013; Jungers et al. 2019; Huddell et al. 2023), net carbon accumulation (de Oliveira et al. 2018), increased soil particulate organic matter (van der Pol et al. 2022), and reduced surface runoff of particulate matter (poultry litter) (Katuwal et al. 2023) compared to annual crops. Supplementing positive environmental benefits of perennial grain, agronomic research has led to a better understanding of plant development (Duchene et al. 2021), management (Jungers et al. 2017, 2022), and harvesting (Heineck et al. 2022; Tautges et al. 2023) of IWG as a perennial grain and forage crop. A comprehensive grower guide that details many planting, management, and harvesting methods used for IWG was released in 2023 with particular emphasis on the differences between perennial IWG management and annual cereals (Tautges et al. 2023). Finally, many food applications have been evaluated including flour quality and processing methods (Marti et al. 2015, 2016; Zhang et al. 2015; Rahardjo et al. 2018; Banjade et al. 2019) as well as research looking at malting applications (Marcus and Fox 2023). While current IWG yields are estimated to be about 25–30% of annual wheat yields (Cassman and Connor 2022; Bajgain et al. 2023), IWG grain quality has higher protein, bran, and essential amino acid percentages than annual wheat (Becker et al. 1991). Additionally, research has shown that equal mixtures of IWG and wheat flour can maintain baking quality while increasing nutritional content (Marti et al. 2015).

A central tenet to the environmental, agronomic, and end use research has been that plant breeding will play a pivotal role in developing higher yielding cultivars. Bolstered by early breeding successes in IWG that showed up to 77% increase in grain yield in two cycles of selection (DeHaan et al. 2014, TLI Cycle 0–2, see Discussion) and molecular breeding methods (Moose and Mumm 2008), plant breeding programs are working to develop better performing cultivars with a consequential moment in 2019 with the release of ‘MN-Clearwater’ as the world’s first IWG developed for food consumption (Bajgain et al. 2020b). Kernza refers to the grain that is marketed from IWG cultivars that have been developed for human consumption of the grain, such as MN-Clearwater, and currently there are a number of commercial Kernza products available to consumers. We use “improved IWG” to signify Thinopyrum intermedium germplasm bred for human consumption that could produce Kernza grain to clearly distinguish it from seeds of forage-type IWG. Following the early breeding efforts of RI, there are now breeding programs in Canada, Ukraine, Sweden, and the United States actively breeding for improved IWG. In an effort to speed genetic gain, some of these programs have actively utilized genomic selection (GS) (Zhang et al. 2016; Crain et al. 2021a) to reduce breeding cycle time. Implementation of GS has required molecular methods like genotyping-by-sequencing (GBS) (Elshire et al. 2011; Poland et al. 2012) that have also provided data needed to dissect genetic architecture through biparental quantitative trait loci (QTL) mapping (Zhang et al. 2017; Larson et al. 2019) and association mapping (Bajgain et al. 2019; Altendorf et al. 2021a, b; Bajgain and Anderson 2021; Crain et al. 2022) Taken together, breeding programs have leveraged these newer tools and methodologies to both enhance the agronomic performance of IWG as well as identify causal variants of traits to both assist in breeding and increase our scientific understanding of IWG genetics.

The basic evaluation and development of improved IWG since the 1980s was previously reported (Cox et al. 2002; Zhang et al. 2016) as well as the development of breeding programs and selection methods in programs established after 2001 (DeHaan et al. 2018; Bajgain et al. 2023). As many of the breeding programs have a combination of pedigree or molecular data along with laboratory records, most improved IWG lineages can be traced back to their programs’ beginning material which subsequently traces back to RI selections. For reference, IWG was first introduced into the United States in 1932 (Musil 1948). It is also known that RI evaluated many of the publicly available plant introduction (PI) accessions from the United States Department of Agriculture (USDA) National Plant Germplasm System (NPGS) gene bank (Wagoner 1990a), so there is a clear connection between improved IWG (Kernza) cultivars and PIs although specific PIs have never been published. As most of the IWG PI accessions in the gene bank have been characterized phenotypically and genotypically (Crain et al. 2023), and include at least some source documentation, there is an opportunity to fill in the missing link between the geographic origin of the PI founders and improved IWG cultivars. We strive to identify the likely 20 PI accessions used to initiate the first cycle of the RI breeding program (Polycross-1) along with the potential origins of additional germplasm included in the second cycle (Polycross-2). Selections from Polycross-2 formed the basis of subsequent IWG breeding programs (DeHaan et al. 2018; Bajgain et al. 2023). Using molecular data, remnant seed and plant material, and recently recovered field and laboratory documents, our objective is to solidify the reported early breeding lineages (Zhang et al. 2016; Bajgain et al. 2023) to the NPGS PI material used to develop improved, food-grade IWG.

Materials and methods

Historical records and experiments

In an effort to trace the origins of improved IWG for Kernza grain production, we consulted a number of historical records. Listed in Supplementary File 1, these records included field and laboratory notebooks, program reports, principal investigators, and presentations that helped document IWG evaluation and breeding activities conducted by RI. These records, detailed in the results section, provide an overview of the first three cycles of phenotypic recurrent selection by RI in collaboration with Big Flats Plant Materials Center (BFPMC), which we refer to as Polycross-1, Polycross-2, and Polycross-3.

Genomic analysis

Plant material

To complement historical records, we leveraged existing genotype data from The Land Institute (TLI) IWG breeding program (DeHaan et al. 2018; Crain et al. 2021a, b), the NPGS IWG PI collection (Crain et al. 2023), 306 genotyped genets from remnant seed of RI Polycross-2, and remnant stems, leaves, and inflorescence material from the 14 selected Polycross-2 parents (collectively referred to as “Remnant Rodale”) for a total of 10,456 unique genotyped genets. As each individual genet will have its own unique genetic makeup due to IWG’s outcrossing nature (Jensen et al. 1990), we use the term genet (Zhang et al. 2016) to refer to individual plants of an IWG accession. Using the terminology of Zhang et al. (2016) a single genet can be cloned into multiple ramets (plants) having the same genetic makeup. Within the TLI breeding program, we sampled several germplasm pools including TLI Cycle-6 (n = 3072), TLI Cycle-12 (n = 4032), and Breeding Parents from TLI Cycle-6–13 (n = 803, approximately 100 each cycle) as they represent a direct link from TLI Cycle-6 to TLI Cycle-12. Cycle-6 from TLI represents six generations (genetic recombination and selection) between germplasm that was shared with TLI from RI (Polycross-2 and Selection Nursery 2, see Results). Likewise, another six generations beyond TLI Cycle-6 were represented by TLI Cycle-12. We used TLI Cycle-12 and the breeding parents to increase the number of source assignments to NPGS PI accessions. As this is a mostly closed breeding program (Bajgain et al. 2023), TLI Cycle-6 & 12, and the breeding parents also represent a check that PI assignments were not vastly different between germplasm pools, a potential issue that would likely indicate incorrect model choice. A total of 2329 unique genets representing 371 NPGS accessions (approximately 6 genets per accession) were genotyped previously (Crain et al. 2023) and received from NPGS web request 23,159 (October 26, 2017). Known off-types and PI accessions that did not appear to morphologically resemble IWG were removed from further analysis (Crain et al. 2023), leaving a total of 338 NPGS PI accessions available for further analysis.

Genomic profiling and bioinformatics

DNA was extracted using a range of products including MagMAX (ThermoFisher Scientific, Waltham, MA), BioSprint (QIAGEN, Venlo, The Netherlands), and CTAB (for older tissues from Polycross-2). Across all genets, we used a two-enzyme restriction digest genotyping-by-sequencing (GBS) protocol following the methods of Poland et al. (2012). Multiplexed libraries ranging from 96 to 384 plexing were sequenced on Illumina sequencing platforms. As the samples represent a range of time and locations, sequencing platforms and output increased as technology improved. To call single nucleotide polymorphism (SNP) markers, we used the TASSEL-GBSv2 pipeline (Glaubitz et al. 2014) using the IWG genome V3.1. We filtered the marker data for strictly biallelic SNPs, a minor allele frequency greater than 0.05, and for SNPs to be called in a minimum of 30% of the genets (up to 70% missing data). Individual genets with more than 95% missing data were removed from further analysis. We required a minimum read depth of four to call a homozygote, otherwise the SNP call was set to missing if there were less than four identical reads. Heterozygous calls were allowed with a read depth of two contrasting reads. For the NPGS PI accessions where multiple genets were genotyped from each accession (Crain et al. 2023), we called SNPs on single genets as well as combining genets of a single accession to create a composite genomic profile. After filtering, a total of 25,773 SNPs and 8242 genets were used for further analysis.

We created a pairwise genetic relationship matrix using the stats package (R Core Team 2022), where smaller distance values represented more SNPs in common between individuals and larger values indicated fewer SNPs in common between compared individuals. From this matrix, the first and second most closely related PI accessions were determined for each improved IWG genet from the TLI Cycle-6, TLI Cycle-12, breeding parents, and Remnant Rodale evaluations (n = 7904 of which 97% of these were from advanced generations of the TLI breeding materials). Of the 139 accessions evaluated by RI and available for use in Polycross-1, a subset of 114 have been genotyped. Using the genetic relationship matrix to ascertain population membership (Manel et al. 2005), we first assigned the 7904 improved IWG genets to a potential 114 NPGS source populations (PI accessions). To identify other potential founders that did not have any known genotypic or historic context, namely RI accession #31, we repeated the population assignment by expanding from 114 to 338 possible germplasm sources that represented the majority of the IWG NPGS collection (Crain et al. 2023). We excluded PIs that were collected (not donated to NPGS) ex situ after 1990 as they would have been unavailable to RI, for a total 331 PI accessions. Because written records were quite specific that 20 accessions were used to form Polycross-1 (although accession names were not provided), we chose the 20 NPGS PI accessions that accounted for the most founder assignments of the improved IWG genets (or total number of PI accessions if less than 20) as the most likely genomic progenitors of current improved IWG germplasm for Kernza grain production. To evaluate our ability to discriminate between relationships within and among the NPGS accessions, we tested genetic assignments of 1,997 individuals representing 338 NPGS accessions used in this study. The ggplot2 (Wickham 2016) and VennDiagram (Chen 2022) R packages were used for data visualization.

Results

Historical records

Original material evaluation

Over the course of nearly two decades, RI was actively involved in all aspects of perennial grain development including species evaluation and selection, germplasm enhancements, agronomic practices, and product utilization. Beginning in 1983, an evaluation nursery (hereafter referred to as RI Herbary) of nearly 300 perennial species was established at RI to evaluate plant accessions for their potential use in perennial herbaceous polyculture. Each accession was grown in a single, one-meter-long row separated from other accessions by mowed grass alleyways. Once IWG was selected as the most promising candidate for domestication in 1985, RI obtained more accessions of IWG from a variety of sources including the USDA NPGS gene bank in Pullman, WA, researchers in the United States, seed companies, and from foreign seed banks. Each evaluation row consisted of ten genets from the same PI accession. By 1987, there were 43 unique IWG accessions in the RI Herbary and each accession was evaluated for plant structure and vigor, as well as seed production characteristics including ease of threshing, synchrony of maturity, and shatter resistance. In addition, 100-seed weight, as a yield component trait, was determined for each accession by averaging the weight of 3 randomly selected sets of 100 seeds dried to 12% moisture. From 1988 to 1993, a larger number of IWG accessions were more intensively evaluated annually as described above with the inclusion of two more yield components including seed set rating (Trupp and Slinkard 1965) and seed yield (g) per 10 heads. The seed set rating trait was calculated as the weight (g) of clean, naked seed from 10 seed heads divided by the weight (g) of the unthreshed seed heads. Naked seed was obtained by using a fabricated threshing board and manually processing samples (Supplementary Fig. 1). Evaluations at RI emphasized seed size (as measured by 100-seed weight) and seed head fertility (measured by seed set rating) as these measurements were both highly heritable in IWG and a direct correlation had been established between seed yield and seed head fertility (Slinkard 1965).

Polycross-1

In the fall of 1987, another 99 IWG accessions consisting of 10 genets each were added to the RI Herbary for a total of 139 unique IWG accessions completing the panel of potential candidates for the first polycross (Polycross-1) breeding cycle. Of the evaluated material, 116 accessions were PIs from the USDA NPGS gene bank in Pullman, WA. Selection of parents from the 139 accessions evaluated both in 1988 and 1989 was based on favorable seed yield characteristics including 100-seed weight, seed set rating, and seed yield per 10 heads. This multi-year evaluation included both a drought (1988) and above average precipitation (1989). Twenty accessions which represented favorable phenotypic traits were selected. Segments of each selected accession row were dug from the RI Herbary in November 1989. Each segment was divided into three subsegments, which were placed into pots and vernalized during December 1989. It is possible that subsegments from each accession were clonally derived from one plant or from multiple genets as the rhizomes of individual genets (10 per row) would be intermingled and indistinguishable from each other within these selected accession rows. Thus, we deduce that the minimum number of selected genets was 20 (one per accession) and the maximum number of selected genets was 60 (three per accession) for Polycross-1. In January 1990, the pots were placed in the RI greenhouse for intercrossing to produce Syn-0 seeds. Pots were rearranged frequently to ensure that pollination could occur between all accessions. Syn-0 seeds, produced by this intercrossing, were bulked and then planted into market packs, one seed per cell in June 1990. A total of 360 seedlings (genets) were transplanted in September 1990 at the USDA Natural Resource Conservation Service (NRCS) BFPMC, Big Flats, NY, establishing Selection Nursery-1. The collaboration with BFPMC resulted from both an institutional reorganization of RI and the expertise of BFPMC in selection and varietal development of undomesticated species.

Polycross-2

From 1991 to 1994, Selection Nursery-1 was evaluated jointly by RI and BFPMC for the same traits that were emphasized in initial RI Herbary accession evaluations. The 360 genets grown from Syn-0 seed produced in Polycross-1 were maintained as individual genets with bare ground around each plant. Additionally, seed heads were removed from each plant at maturity to prevent field contamination by shattered seed. Of the 360 genets, 11 genets were chosen to be included in Polycross-2.

While evaluations of Selection Nursery-1 were occurring, RI was actively continuing evaluations and expanding their IWG germplasm collection. In the fall 1988, another 111 IWG accessions were added to the RI Herbary, planted in rows (as described in Original Material Evaluations). Of these 111 accessions, 102 were comprised of half-sib families planted from seeds collected from each of 102 open-pollinated plants selected by RI researchers in 1987 from John Berdahl’s breeding nurseries at the USDA Northern Great Plains Research Laboratory (NGPRL) in Mandan, ND. From 1989 to 1994, all of these new NGPRL-derived RI accessions were evaluated in RI Herbary rows, each containing four half-sib plants. Along with the 11 genets from Selection Nursery-1, three RI accessions originating from NGPRL were selected in 1994 for inclusion in Polycross-2. From each of the three selected accession rows, a segment of plant material was dug up with each selection originating from at least one genet and up to three genets due to the possible intermingling of rhizomes among the half-sib progenies within each RI Herbary accession row.

Polycross-2 consisted of 11 Selection Nursery-1 genets that were cloned to produce 3 ramets of each individual genet and at least one targeted genet (up to a maximum of 3 potential genets) from each of the subsegments (clones) of three NGPRL-derived RI accessions. The 3 clones of each of 14 selections were planted in the field at BFPMC in a pattern to allow maximum cross pollination among all selections in the crossing block. From a breeding point of view, we deduce that Polycross-2 contained at least 14 distinct parental genotypes and a maximum of 20 distinct parental genotypes. Intermating of clones in the field occurred in 1996 producing Syn-1 seeds which would include genetic material from the original 20 selected accessions used in Polycross-1 and the introduced genetic material from the 3 RI Herbary accessions that were originally collected from NGPRL. In fall 1997, 400 individual genets grown from Syn-1 seeds were planted at BFPMC, Big Flats, NY to form Selection Nursery-2. Selection Nursery-2 was evaluated jointly by RI and BFPMC from 1998 to 2001 and only by BFPMC from 2002 to 2005. Seed and materials from Polycross-2 and Selection Nursery-2 were distributed to several scientists and institutions (Fig. 1) which formed the basis of the TLI breeding program in the early 2000’s. Subsequent work by BFPMC selected a total of 16 genets from Selection Nursery-2 to form Polycross-3. Each genet was cloned four times and planted into a crossing block in 2006. Even though BFPMC evaluated Polycross-3 between 2007 and 2016, seed produced from Polycross-3 was not used in successive breeding efforts by TLI.

Fig. 1
figure 1

Rodale Institutes (RI) and Big Flats Plant Material Center intermediate wheatgrass breeding program and germplasm flow, including germplasm distributions, from RI Polycross-1 to Polycross-3

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Recreating polycross-1 from historical records

Although the identity of the original 20 accessions selected for use in Polycross-1 would have been recorded at the time, this information is no longer available. To reconstruct the most likely selected accessions, recently recovered field and laboratory records have been reviewed to determine the most likely accessions used in Polycross-1 (Supplementary File 1). These records provide details of yield component traits in 1988 and 1989 and specifically state that 20 accessions with favorable yield characteristics were selected for Polycross-1; however, the identities of these accessions are now obscure. There was evidence of genotype-by-environment interaction as a list of 22 accessions that had high yield in 1988 (drought year) were often not the highest yielding in 1989 (wet year). Additionally, the average values combined across the two years for the three yield components of the 20 selected accessions were given as 100-seed weight (0.495 g), seed set rating (45%), and yield per 10 heads (2.5g). These averages provided a target value in searching the records to identify the most likely combination of selected accessions. Using this information, we have identified the most likely accessions for these 20 parents (Table 1). Recovered records of the 139 accessions evaluated in 1988 and 1989 show 100-seed weight and seed set rating for both years but yield per 10 head data are limited to 1989 and only a few accessions from 1988. Therefore, calculated values for yield per 10 heads do not perfectly align with the target values. The estimated yield per 10 heads for the most likely selected accessions is 2.67 g compared to a target value of 2.5 g. As there were genotype-by-environment interactions, inclusion of missing data would most likely lower the calculated value closer to the target. Based on historical records, 19 of the 20 selected accessions were from NPGS. The one accession not from NPGS was given to RI in 1982 by Wes Jackson and TLI, but no other information is available about this accession. In terms of geographical origin, USDA records indicate that 13 accessions were from Russia with the majority of these collected between the Black Sea and Caspian Sea (Caspian-Pontic Steppe, Stavropol and Svetlograd regions). Passport data from NPGS suggest that three of the accessions were cultivated when collected, raising the possibility that they may all come from the same cultivar “Rostov(sky) 31” (Table 1).

Table 1 National Plant Germplasm System intermediate wheatgrass plant introductions (PI) inferred to be in Rodale Institute Polycross-1 from historical documents
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Genomic analysis

Genomic profiling of plant material

We compared genomic data from several germplasm pools including TLI Cycle-6, TLI Cycle-12, breeding parents, Remnant Rodale, and NPGS PIs. As genotyping was conducted on different sequencing platforms and times, we evaluated the number of reads and number of SNPs called per genet. The germplasm pool affected the number of SNPs with an overall average of 21,698 SNPs called per genet. Single genets from the NPGS PIs had the fewest called SNPs with an average of 9867 SNPs followed by TLI Cycle-6 with an average of 19,597 SNPs per genet while TLI Cycle-12 have the highest average of 23,043 SNPs per genet. When the NPGS PI accessions were pooled (approximately six genets per accession), the average number of SNPs called per accession increased to 23,086. Along with number of SNPs called, we also examined the read depth of each SNP to verify that there was no apparent bias due to sequencing differences in the data sets. Cycle-6 from TLI had the lowest read depth per SNP with an average of 2.6 reads per SNP. Cycle-12 from TLI had a mean read depth of 7.6, the breeding parents were higher at 8.4, and both Remnant Rodale and NPGS PI accessions had a mean read depth of 11. When individual genets of the NPGS PIs are considered, the average read depth per genet was 1.9 (approximately 6 genets were combined per accession). These values most likely reflect older sequencing technology in TLI Cycle-6 along with different program objectives that targeted higher read depths in the breeding parents, Remnant Rodale and NPGS accessions to ensure a greater amount of data compared to the TLI breeding program that balances practical objectives with cost.

Validation test of genet assignments

To test the sensitivity of our assignments using the relationship matrix, we used the 1997 genets that represented 338 unique NPGS PI IWG accessions. For each individual genet, we tested whether it was most related to other individuals of the same accession based strictly on DNA genotypes. Across 1997 iterations, only one genet was assigned to the incorrect PI accession (99% accuracy).

Likely founders of improved IWG from genomic analysis

Genomic comparison to RI evaluated material

Leveraging the genetic resources that have been developed for implementing genomics assisted breeding in IWG, we investigated the most likely founders of improved IWG using empirical molecular genetic data. Based on historical knowledge of 114 genotyped accessions evaluated by RI, a total of 30 NPGS PI accessions were identified by genomic analysis as the most likely founders across 7904 improved IWG genets (Fig. 2). Of these potential matches, there was a very skewed distribution where 10 NPGS PI accessions accounted for over 98% of the assigned individuals, and 11 NPGS PI accessions had 10 or fewer descendants (Fig. 2). In assessing model fit, we looked at the average increase in distance between the top two NPGS PI candidates (Supplementary Table 1). Across all tested genets, there was an average distance increase of 0.45 between the first and second most related NPGS accession compared to an average distance increase of 0.21 between any other comparisons. The 14 Polycross-2 parents (11 of which were only one generation away from original selected PI accessions) had an average distance increase of 3.8 between the first and second most related NPGS accessions. TLI Cycle-6 and 12 had much smaller average increases of 0.53 and 0.37 between the first and second most likely NPGS accessions. This result is likely based on breeding progress and genetic recombination across cycles obscuring the original founder haplotypes as well as providing evidence to support these assignments.

Fig. 2
figure 2

Bar graph of the number of times each National Plant Germplasm System plant introduction (NPGS PI) intermediate wheatgrass (IWG) accessions were assigned as a founder of 7904 improved IWG genets (from Remnant Rodale, TLI Cycle-6 & 12 and Breeding Parents) for Kernza production. Assignment was based on the 114 genotyped PI accessions originally evaluated by RI

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When considering NPGS PI assignment by test subject (Remnant Rodale, TLI Cycle-6, Breeding Parents, TLI Cycle-12) the assignments of the newer Breeding Parents and TLI Cycle-12 test subjects were a subset of assignments identified in the older TLI Cycle-6 test subjects. This result might be expected as both the Breeding Parents and TLI Cycle-12 were developed from a mostly closed population from TLI Cycle-6. Thus, further analyses will be focused on the older and more diverse Remnant Rodale and TLI Cycle-6 test subjects.

When comparing the 20 most likely selections used in RI Polycross-1 inferred from historical records to the genetic assignments made using molecular data from Remnant Rodale and TLI Cycle-6 test subjects, seven NPGS PIs were identified in all data sources (Fig. 3 and Tables 1 and 2). These seven NPGS PIs (PI 273732, PI 286118, PI 314054, PI 315353, PI 316122, PI 440004, PI 440015) were the closest NPGS PI accession for over 40% of the tested genets indicating they are quite likely founders of current IWG germplasm being improved for Kernza grain production. Ten NPGS PI accessions that were identified as possible selections based on historical records for Polycross-1 did not show any descendants based on genetic assignments; however, these accessions were often part of a series of adjacent PI accessions (i.e. PI440004-18).

Fig. 3
figure 3

National Plant Germplasm System plant introduction (NPGS PI) source assignments (maximum 20) for Rodale Institute (RI) Polycross-1 historical records, Remnant Rodale DNA genotypes, and The Land Institute (TLI) Cycle-6 DNA genotypes. Remnant Rodale is 227 unique genets resulting from RI Polycross-2 which includes genetic contributions from RI Polycross-1 and additional RI selections originating from the Northern Great Plains Research Laboratory breeding program. TLI Cycle-6 is 3072 unique genets from TLI IWG breeding program

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Table 2 National Plant Germplasm System intermediate wheatgrass plant introductions (PI) inferred to be in Rodale Institute Polycross-2 from molecular analysis of Remnant Rodale and The Land Institute breeding program in addition to bolded accessions in Table 1
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Genomic comparison to the broader IWG NPGS collection

As not all the initial RI accessions were genotyped (i.e. RI Accession 31 and 3 accessions selected for inclusion in Polycross-2 which would be included as founders of improved IWG), we expanded the search of potential founders to the broader 331 NPGS IWG accessions that were collected prior to 1990. This broader search identified 47 possible NPGS sources of improved IWG germplasm. However, only 10 potential NPGS accessions accounted for 95% of all assignments as the most likely founders of the tested germplasm, and just 20 NPGS PI accessions were considered founders to more than 10 IWG genets. From written records, an upper limit of 20 NPGS PI founders in Polycross-1 was placed on all analysis (historical records, assignment to NPGS PI evaluated by RI, and assignment to broader NPGS accessions), and a total of 38 NPGS PI accessions were identified as being the potential founders of improved IWG that is currently being used for Kernza grain production (Tables 1, 2, 3).

Table 3 National Plant Germplasm System intermediate wheatgrass (IWG) plant introductions that were not evaluated by Rodale Institute but share similarity with improved IWG germplasm for Kernza grain production in current breeding programs
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Repeating the founder assignment to a broader set of 331 NPGS PI accessions, also allowed us to evaluate the strength of assignment by comparing assignments to the 114 RI accessions to the expanded set. Of the 7904 genets assigned to a source, over 53% were assigned to the same source. One NPGS accession, PI 502351, accounted for another 37% of assignments. According to NPGS Germplasm Resources Information Network passport data, this accession was cultivated material and from Elista, Russia. This region, between the Black Sea and Caspian Sea, is also the origin of other accessions identified as sources of improved IWG. If this material was cultivated, it would likely have superior agronomic traits, like other cultivated material in Table 1, and share large genetic similarities leading to the high rate of assignment to this PI accession.

Inferred geographic origin of improved IWG

Finally, we investigated the most likely geographic origin of germplasm used to develop improved IWG cultivars used for Kernza grain production using NPGS Germpasm Resources Information Network passport data. A total of 27 NPGS PI accessions identified in the recreated RI Polycross-1 and molecular data had reliable location data from which 20 were from Russia, two each from Turkey and Kazakhstan, and one each from Afghanistan, Iran, and Uzbekistan. Seven other accessions either had missing data or location data that was deemed insufficient. For example, PI 286118 was listed with a country of origin of Denmark, yet the sample came from a botanical garden. Given this information, it is very likely this accession had been collected elsewhere before arriving in Denmark, but records of its natural origin are not available to our knowledge. The overlapping NPGS PIs identified as possible sources of improved IWG from our historical and genetic analyses, originated from the Pontic-Caspian steppe between the Black Sea and the Caspian Sea (Fig. 4) likely indicating a primary geographic origin of improved IWG germplasm currently used for Kernza grain production.

Fig. 4
figure 4

Geographic location of 27 National Plant Germplasm System intermediate wheatgrass (IWG) plant introduction (PI) accessions that are inferred to be the founders of improved IWG germplasm used for current Kernza grain production. Orange circles represent common PIs inferred through historical and molecular methods. Green diamonds are PI accessions inferred through historic records only, and purple triangles are PI inferred through molecular methods only

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Discussion

Rodale research activities

In the 1980s, RI initiated an effort to identify potential perennial grains and develop them into a crop. In many ways, RI developed some of the first empirical blueprints of pipeline perennial grain development that has been expanded on by later research (Wagoner 1990b; Cox et al. 2002; DeHaan et al. 2016, 2023). Additionally, the whole systems approach (breeding, agronomy, economics, end use) used by RI is still used today. Some examples of this integrated approach are the Kernza coordinated agriculture project (KernzaCAP, https://kernza.org/kernzacap/), the reflective plant breeding paradigm (Runck et al. 2014), and organized efforts through TLI (https://landintitute.org).

Using two years of phenotypic data RI launched a phenotypic recurrent selection program that would result in the improved germplasm used in modern Kernza grain production. Even though RI would have preferred to have had more years of evaluation for a perennial crop with an anticipated five-year crop lifetime, breeding efforts were balanced between time and institutional support. Seed shared with TLI came from Polycross-2 and Selection Nursery-2, which have also been referred to as BFPMC Cycle 1 and 2 (Fig. 1), served as the basis of improved IWG (DeHaan et al. 2018; Bajgain et al. 2023). Much like Cox et al. suggested in 2002 that new molecular tools would aid in perennial grain development, the advent of genomic selection (Meuwissen et al. 2001) and subsequent next generation sequencing methods have allowed plant (IWG) breeding programs to harness molecular technology for improved plant breeding (Zhang et al. 2016; Bajgain et al. 2020a; Crain et al. 2021a, b). Current breeding programs harnessing genomic selection can make yearly breeding selection with numerous years of phenotypic data informing the models (Crain et al. 2021a, b). Emerging technologies like speed breeding (Watson et al. 2018) have the potential to further reduce breeding cycle time and increase the rate of genetic gain. Much like the initial RI selections that included extreme drought and wet years, genomic selection models can utilize data across multiples years and cycles (Crain et al. 2020) incorporating a range of climatic conditions into the selection information.

Linkage between PI accessions and Kernza Grain production

Overall, the genomic data provide strong support to clarify the partial historical records, which indicate that improved IWG that is currently used for Kernza grain production is primarily descended from a limited number of NPGS PI accessions mainly originating between the Black Sea and Caspian Sea. Using the genomic data, we inferred the most likely source of the 20 accessions in RI Polycross-1; however, most of the material we sampled was after Polycross-2 which had another severe bottleneck of only 14 accessions comprised of at least 14 and not more than 20 genets, Fig. 1. The bottleneck in Polycross-2 included additional genetic material of unknown origin from the three RI selections that had originally been collected from NGPRL.

Potential confounding factors

While the historical records and genomic data had large areas of overlap, there was not complete consistency between the methods. This should not be surprising given the number of accessions evaluated, time span of the programs, and even the different institutions and staff that have been involved. Within the TLI breeding program, most of the germplasm was obtained directly from RI (Polycross-2 and Selection Nursery-2) and was initially referred to as TLI Cycle-0 (DeHaan et al. 2014). However, there was evaluation and incorporation of other NPGS PI accessions before TLI Cycle-6 (Bajgain et al. 2023). Within our analysis, assignment of single NPGS PI genets to their respective source NPGS PI accession was very high demonstrating our ability to discriminate NPGS accessions by DNA genotype. Even though assignments of improved IWG genets to NPGS PI accessions appears plausible, it does not appear to be as precise as evidenced by up to 47 NPGS PI founders for improved IWG genets. Even though this number is higher than reported records, the skewed distribution suggests a number complementary with the recorded data as the source for improved IWG. Both the nature and structure of the data could be influencing these results. Perhaps the most obvious reason is that intermating and genetic recombination has blended the original NPGS accessions in such a way that it is more difficult to match the improved IWG genets to any one specific NPGS accession. As we tried to identify the most likely founders in Polycross-1, most of the material genotyped had at least two cycles of genetic recombination (Remnant Rodale) and up to 14 cycles of genetic recombination (TLI Cycle-12) from the original founders as well as contributions from additional genetic material included in Polycross-2 from the NGPRL breeding program. As each recombination occurred there would have been reshuffling and breaking of the original haplotypes, and additionally there would have been selection pressure applied to obtain agronomically superior plants.

In previous analysis of IWG, we have noted that more than 70% of the genetic variation observed is within accessions (Crain et al. 2023), suggesting that random sampling of seed could influence our observations. In our analysis, we have assumed that field records and that sampling were completely accurate, with similar assumptions for genetic profiling. If errors occurred, after the passage of time it would be almost impossible to identify or correct. Along with potential field errors, the IWG accessions evaluated by RI mainly came from the NPGS system after having been collected in the 1970s and earlier. Until the 1990s there was no isolation protocol in accession regenerations (Johnson et al. 1996) and IWG pollen dispersal has been documented to mainly occur within 10 m (Bajgain et al. 2022) indicating that there could have been the possibility of admixture among the NPGS accessions before they were received for phenotypic evaluation by RI in the 1980s and later genomic profiling. However, for many of the PIs used in Polycross-1, records from NPGS indicate that there had been only one seed increase since collection and distribution to RI therefore RI received NPGS PI accessions as close to the original collection as possible.

Notably, both historic records and genomic data often indicate many NPGS accessions come from a series of adjacent collections. For example, the PI 4400xx series formed a large portion of the selected 20 accessions for Polycross-1. As many of the accessions were collected during the same expeditions and most likely in chronological or spatial order, it is not surprising that these accessions would often be similar to each other. Work by Crain et al. (2023) showed a strong correlation between geographic distance and genetic distance within the IWG NPGS collections, suggesting that accessions collected near each other are more likely to share alleles. Even with potential germplasm, field, and laboratory errors, the data clearly indicate a small subset of IWG NPGS accessions that are the most likely primary founders of improved IWG germplasm that is currently used for Kernza grain production.

Conclusion

This work identifies direct linkages between NPGS accessions and improved IWG germplasm that is currently grown for Kernza grain production, showcasing how plant germplasm collections and repositories can be utilized for breeding and the development of new crops. By identifying the most likely genetic origins of food-grade IWG, plant breeders can continue to utilize the NPGS accessions for additional genetic diversity for enhanced crop production as well as better understand the domestication effort behind this grain. Confirming the small number of genets (14–20) within the RI breeding program that have been used to improve IWG helps indicate likely effective population size within this new crop and could guide future genetic research. Finally, identifying the primary geographic origin between the Black and Caspian Seas provides areas for future germplasm collections, in-situ conservation initiatives, and identifying accessions that can be used to broaden the current germplasm pool for improved IWG.