Origin of current intermediate wheatgrass germplasm being developed for Kernza grain production.

Intermediate wheatgrass (IWG, Thinopyrum intermedium [Host] Barkworth & D. R. Dewey) has been developed as a perennial grain crop to provide ecosystem services, environmental benefits, and human food. Grain and products derived from IWG varieties improved for food production have been marketed under the registered trademark, Kernza. In the 1980s, a joint breeding effort between the Rodale Institute (RI) and the Big Flats Plant Material Center used IWG plant introductions (PI) from the National Plant Germplasm System (NPGS) and recurrent phenotypic selection to improve populations of IWG with the goal of developing a perennial grain. Initial selections were provided to The Land Institute where they were subsequently improved for grain production, yet the identity of the founder material of improved, food-grade IWG has not been publicly documented. Recently recovered original documents have been used to reconstruct the early breeding program to identify the most likely 20 PIs that form the founders of modern food-grade IWG. Molecular data using genotyping-by-sequencing in current elite breeding material, remnant seed from the initial RI selections, and preserved sample material have provided supporting evidence for the historical records. The genetic origin for food-grade IWG is focused between the Black Sea and Caspian Sea in the Stavropol region of Russia, with smaller contributions likely from collections as distant as Kazakhstan in the east to Turkey in the west. This work connects the flow of germplasm and utility of NPGS PIs to present day IWG grain cultivars being developed in multiple breeding programs around the world.

Discussion: Prioritize perennial grain development for sustainable food production and environmental benefits.

Perennial grains have potential to contribute to ecological intensification of food production by enabling the direct harvest of human-edible crops without requiring annual cycles of disturbance and replanting. Studies of prototype perennial grains and other herbaceous perennials point to the ability of agroecosystems including these crops to protect water quality, enhance wildlife habitat, build soil quality, and sequester soil carbon. However, genetic improvement of perennial grain candidates has been hindered by limited investment due to uncertainty about whether the approach is viable. As efforts to develop perennial grain crops have expanded in past decades, critiques of the approach have arisen. With a recent report of perennial rice producing yields equivalent to those of annual rice over eight consecutive harvests, many theoretical concerns have been alleviated. Some valid questions remain over the timeline for new crop development, but we argue these may be mitigated by implementation of recent technological advances in crop breeding and genetics such as low-cost genotyping, genomic selection, and genome editing. With aggressive research investment in the development of new perennial grain crops, they can be developed and deployed to provide atmospheric greenhouse gas reductions.

Climate change challenges, plant science solutions.

Climate change is a defining challenge of the 21st century, and this decade is a critical time for action to mitigate the worst effects on human populations and ecosystems. Plant science can play an important role in developing crops with enhanced resilience to harsh conditions (e.g. heat, drought, salt stress, flooding, disease outbreaks) and engineering efficient carbon-capturing and carbon-sequestering plants. Here, we present examples of research being conducted in these areas and discuss challenges and open questions as a call to action for the plant science community.

A high-throughput skim-sequencing approach for genotyping, dosage estimation and identifying translocations.

The development of next-generation sequencing (NGS) enabled a shift from array-based genotyping to directly sequencing genomic libraries for high-throughput genotyping. Even though whole-genome sequencing was initially too costly for routine analysis in large populations such as breeding or genetic studies, continued advancements in genome sequencing and bioinformatics have provided the opportunity to capitalize on whole-genome information. As new sequencing platforms can routinely provide high-quality sequencing data for sufficient genome coverage to genotype various breeding populations, a limitation comes in the time and cost of library construction when multiplexing a large number of samples. Here we describe a high-throughput whole-genome skim-sequencing (skim-seq) approach that can be utilized for a broad range of genotyping and genomic characterization. Using optimized low-volume Illumina Nextera chemistry, we developed a skim-seq method and combined up to 960 samples in one multiplex library using dual index barcoding. With the dual-index barcoding, the number of samples for multiplexing can be adjusted depending on the amount of data required, and could be extended to 3,072 samples or more. Panels of doubled haploid wheat lines (Triticum aestivum, CDC Stanley x CDC Landmark), wheat-barley (T. aestivum x Hordeum vulgare) and wheat-wheatgrass (Triticum durum x Thinopyrum intermedium) introgression lines as well as known monosomic wheat stocks were genotyped using the skim-seq approach. Bioinformatics pipelines were developed for various applications where sequencing coverage ranged from 1 × down to 0.01 × per sample. Using reference genomes, we detected chromosome dosage, identified aneuploidy, and karyotyped introgression lines from the skim-seq data. Leveraging the recent advancements in genome sequencing, skim-seq provides an effective and low-cost tool for routine genotyping and genetic analysis, which can track and identify introgressions and genomic regions of interest in genetics research and applied breeding programs.

Perennials as Future Grain Crops: Opportunities and Challenges.

Perennial grain crops could make a valuable addition to sustainable agriculture, potentially even as an alternative to their annual counterparts. The ability of perennials to grow year after year significantly reduces the number of agricultural inputs required, in terms of both planting and weed control, while reduced tillage improves soil health and on-farm biodiversity. Presently, perennial grain crops are not grown at large scale, mainly due to their early stages of domestication and current low yields. Narrowing the yield gap between perennial and annual grain crops will depend on characterizing differences in their life cycles, resource allocation, and reproductive strategies and understanding the trade-offs between annualism, perennialism, and yield. The genetic and biochemical pathways controlling plant growth, physiology, and senescence should be analyzed in perennial crop plants. This information could then be used to facilitate tailored genetic improvement of selected perennial grain crops to improve agronomic traits and enhance yield, while maintaining the benefits associated with perennialism.

Genetic architecture and QTL selection response for Kernza perennial grain domestication traits.

KEY MESSAGE: Analysis of multi-year breeding program data revealed that the genetic architecture of an intermediate wheatgrass population was highly polygenic for both domestication and agronomic traits, supporting the use of genomic selection for new crop domestication. Perennial grains have the potential to provide food for humans and decrease the negative impacts of annual agriculture. Intermediate wheatgrass (IWG, Thinopyrum intermedium, Kernza®) is a promising perennial grain candidate that The Land Institute has been breeding since 2003. We evaluated four consecutive breeding cycles of IWG from 2016 to 2020 with each cycle containing approximately 1100 unique genets. Using genotyping-by-sequencing markers, quantitative trait loci (QTL) were mapped for 34 different traits using genome-wide association analysis. Combining data across cycles and years, we found 93 marker-trait associations for 16 different traits, with each association explaining 0.8-5.2% of the observed phenotypic variance. Across the four cycles, only three QTL showed an FST differentiation > 0.15 with two corresponding to a decrease in floret shattering. Additionally, one marker associated with brittle rachis was 216 bp from an ortholog of the btr2 gene. Power analysis and quantitative genetic theory were used to estimate the effective number of QTL, which ranged from a minimum of 33 up to 558 QTL for individual traits. This study suggests that key agronomic and domestication traits are under polygenic control and that molecular methods like genomic selection are needed to accelerate domestication and improvement of this new crop.

Accelerated Domestication of New Crops: Yield is Key.

Sustainable agriculture in the future will depend on crops that are tolerant to biotic and abiotic stresses, require minimal input of water and nutrients and can be cultivated with a minimal carbon footprint. Wild plants that fulfill these requirements abound in nature but are typically low yielding. Thus, replacing current high-yielding crops with less productive but resilient species will require the intractable trade-off of increasing land area under cultivation to produce the same yield. Cultivating more land reduces natural resources, reduces biodiversity and increases our carbon footprint. Sustainable intensification can be achieved by increasing the yield of underutilized or wild plant species that are already resilient, but achieving this goal by conventional breeding programs may be a long-term prospect. De novo domestication of orphan or crop wild relatives using mutagenesis is an alternative and fast approach to achieve resilient crops with high yields. With new precise molecular techniques, it should be possible to reach economically sustainable yields in a much shorter period of time than ever before in the history of agriculture.

Re-imagining crop domestication in the era of high throughput phenomics.

De novo domestication is an exciting option for increasing species diversity and ecosystem service functionality of agricultural landscapes. Genomic selection (GS), the application of genomic markers to predict phenotypic traits in a breeding population, offers the possibility of rapid genetic improvement, making GS especially attractive for modifying traits of long-lived species. However, for some wild species just entering the domestication pipeline, especially those with large and complex genomes, a lack of funding and/or prior genome characterization, GS is often out of reach. High throughput phenomics has the potential to augment traditional pedigree selection, reduce costs and amplify impacts of genomic selection, and even create new predictive selection approaches independent of sequencing or pedigrees.

QTL for seed shattering and threshability in intermediate wheatgrass align closely with well-studied orthologs from wheat, barley, and rice.

Perennial grain crops have the potential to improve agricultural sustainability but few existing species produce sufficient grain yield to be economically viable. The outcrossing, allohexaploid, and perennial forage species intermediate wheatgrass (IWG) [Thinopyrum intermedium (Host) Barkworth & D. R. Dewey] has shown promise in undergoing direct domestication as a perennial grain crop using phenotypic and genomic selection. However, decades of selection will be required to achieve yields on par with annual small-grain crops. Marker-aided selection could accelerate progress if important genomic regions associated with domestication were identified. Here we use the IWG nested association mapping (NAM) population, with 1,168 F1 progeny across 10 families to dissect the genetic control of brittle rachis, floret shattering, and threshability. We used a genome-wide association study (GWAS) with 8,003 single nucleotide polymorphism (SNP) markers and linkage mapping-both within-family and combined across families-with a robust phenotypic dataset collected from four unique year-by-location combinations. A total of 29 quantitative trait loci (QTL) using GWAS and 20 using the combined linkage analysis were detected, and most large-effect QTL were in common across the two analysis methods. We reveal that the genetic control of these traits in IWG is complex, with significant QTL across multiple chromosomes, sometimes within and across homoeologous groups and effects that vary depending on the family. In some cases, these QTL align within 216 bp to 31 Mbp of BLAST hits for known domestication genes in related species and may serve as precise targets of selection and directions for further study to advance the domestication of IWG.

Nested association mapping reveals the genetic architecture of spike emergence and anthesis timing in intermediate wheatgrass.

Intermediate wheatgrass (Thinopyrum intermedium) is an outcrossing, cool season grass species currently undergoing direct domestication as a perennial grain crop. Though many traits are selection targets, understanding the genetic architecture of those important for local adaptation may accelerate the domestication process. Nested association mapping (NAM) has proven useful in dissecting the genetic control of agronomic traits many crop species, but its utility in primarily outcrossing, perennial species has yet to be demonstrated. Here, we introduce an intermediate wheatgrass NAM population developed by crossing ten phenotypically divergent donor parents to an adapted common parent in a reciprocal manner, yielding 1,168 F1 progeny from 10 families. Using genotyping by sequencing, we identified 8,003 SNP markers and developed a population-specific consensus genetic map with 3,144 markers across 21 linkage groups. Using both genomewide association mapping and linkage mapping combined across and within families, we characterized the genetic control of flowering time. In the analysis of two measures of maturity across four separate environments, we detected as many as 75 significant QTL, many of which correspond to the same regions in both analysis methods across 11 chromosomes. The results demonstrate a complex genetic control that is variable across years, locations, traits, and within families. The methods were effective at detecting previously identified QTL, as well as new QTL that align closely to the well-characterized flowering time orthologs from barley, including Ppd-H1 and Constans. Our results demonstrate the utility of the NAM population for understanding the genetic control of flowering time and its potential for application to other traits of interest.

Development of whole-genome prediction models to increase the rate of genetic gain in intermediate wheatgrass (Thinopyrum intermedium) breeding.

The development of perennial grain crops is driven by the vision of simultaneous food production and enhanced ecosystem services. Typically, perennial crops like intermediate wheatgrass (IWG)[Thinopyrum intermedium (Host) Barkworth & D.R Dewey] have low seed yield and other detrimental traits. Next-generation sequencing has made genomic selection (GS) a tractable and viable breeding method. To investigate how an IWG breeding program may use GS, we evaluated 3,658 genets over 2 yr for 46 traits to build a training population. Six statistical models were used to evaluate the non-replicated data, and a model using autoregressive order 1 (AR1) spatial correction for rows and columns combined with the genomic relationship matrix provided the highest estimates of heritability. Genomic selection models were built from 18,357 single nucleotide polymorphism markers via genotyping-by-sequencing, and a 20-fold cross-validation showed high predictive ability for all traits (r > .80). Predictive abilities improved with increased training population size and marker numbers, even with larger amounts of missing data per marker. On the basis of these results, we propose a GS breeding method that is capable of completing one cycle per year compared with a minimum of 2 yr per cycle with phenotypic selection. We estimate that this breeding approach can increase the rate of genetic gain up to 2.6× above phenotypic selection for spike yield in IWG, allowing GS to enable rapid domestication and improvement of this crop. These breeding methods should be transferable to other species with similar long breeding cycles or limited capacity for replicated observations.

Genomic prediction enables rapid selection of high-performing genets in an intermediate wheatgrass breeding program.

In an era of constrained and depleted natural resources, perennial grains could provide sustainable food production along with beneficial ecosystem services like reduced erosion and increased atmospheric carbon capture. Intermediate wheatgrass (IWG) [Thinopyrum intermedium (Host) Barkworth & D. R. Dewey subsp. intermedium] has been undergoing continuous breeding for domestication to develop a perennial grain crop since the 1980s. As a perennial, IWG has required 2-5 yr per selection generation, but starting in 2017, genomic selection (GS) was initiated in the breeding program at The Land Institute, Salina, KS (TLI), enabling one complete cycle per year. For each cycle, ∼4,000 seedlings were profiled using genotyping-by-sequencing (GBS) and genomic estimated breeding values (GEBVs) were calculated. Selection based on GEBVs identified ∼100 individuals to advance as parents each generation, while validation populations of 1,000-1,200 genets for GS model training were also selected using the genomic relationship matrix to represent genetic diversity in each cycle. The selected parents were randomly intermated in a greenhouse crossing block to form the subsequent cycle, while the validation populations were transplanted to irrigated and nonirrigated field sites for phenotypic evaluations in the following years. For priority breeding traits of seed mass, free threshing, and nonshattering, correlations between predicted values and observed data were >.5. The realized selection differential ranged from 11-23% for selected traits, and the expected genetic gains for these traits, including spike yield, ranged from 6 to 14% per year. Genomic selection is a powerful tool to speed the domestication and development of IWG and other perennial crops with extended breeding timelines.

Sequenced-based paternity analysis to improve breeding and identify self-incompatibility loci in intermediate wheatgrass (Thinopyrum intermedium).

KEY MESSAGE: Paternity assignment and genome-wide association analyses for fertility were applied to a Thinopyrum intermedium breeding program. A lack of progeny between combinations of parents was associated with loci near self-incompatibility genes. In outcrossing species such as intermediate wheatgrass (IWG, Thinopyrum intermedium), polycrossing is often used to generate novel recombinants through each cycle of selection, but it cannot track pollen-parent pedigrees and it is unknown how self-incompatibility (SI) genes may limit the number of unique crosses obtained. This study investigated the potential of using next-generation sequencing to assign paternity and identify putative SI loci in IWG. Using a reference population of 380 individuals made from controlled crosses of 64 parents, paternity was assigned with 92% agreement using Cervus software. Using this approach, 80% of 4158 progeny (n = 3342) from a polycross of 89 parents were assigned paternity. Of the 89 pollen parents, 82 (92%) were represented with 1633 unique full-sib families representing 42% of all potential crosses. The number of progeny per successful pollen parent ranged from 1 to 123, with number of inflorescences per pollen parent significantly correlated to the number of progeny (r = 0.54, p < 0.001). Shannon's diversity index, assessing the total number and representation of families, was 7.33 compared to a theoretical maximum of 8.98. To test our hypothesis on the impact of SI genes, a genome-wide association study of the number of progeny observed from the 89 parents identified genetic effects related to non-random mating, including marker loci located near putative SI genes. Paternity testing of polycross progeny can impact future breeding gains by being incorporated in breeding programs to optimize polycross methodology, maintain genetic diversity, and reveal genetic architecture of mating patterns.

Carbon and water relations in perennial Kernza (Thinopyrum intermedium): An overview.

Perennial crops have been proposed as a more sustainable alternative to annual crops, because they have extended growing seasons, continuous ground cover, reduced nutrient leakage, and sequester more carbon in the soils than annual crops. One example is intermediate wheatgrass (Thinopyrum intermedium), a perennial crop that has been used as a cool-season forage throughout the USA and Canada and also across its native range in Eurasia. Since the 1980's, intermediate wheatgrass has been under domestication to improve seed fertility and grain yield. Commercial products are being sold under the trade name Kernza, owned by The Land Institute, located in Salina, Kansas, USA. This review provides a comprehensive framework about the physical and biological aspects involving the water and carbon cycles in Kernza plants. The main aspects we highlight here are based on previous findings regarding Kernza: i) the ability of maintaining a relatively high water-use efficiency throughout the whole growing season, which is beneficial to mitigate water stress, representing an important physiological mean to acclimate under severe, unfavorable weather conditions, and ii) its higher evapotranspiration (ET) and net carbon uptake rates, particularly when compared to annual counterparts. Only a thorough multifaceted assessment of the repercussion for carbon and water fluxes of a shift from annual crops to Kernza will allow assessing the perspectives of such novel perennial crop to support food security and a number of ecosystem services, particularly under future climates.

New Food Crop Domestication in the Age of Gene Editing: Genetic, Agronomic and Cultural Change Remain Co-evolutionarily Entangled.

The classic domestication scenario for grains and fruits has been portrayed as the lucky fixation of major-effect "domestication genes." Characterization of these genes plus recent improvements in generating novel alleles (e.g., by gene editing) have created great interest in de novo domestication of new crops from wild species. While new gene editing technologies may accelerate some genetic aspects of domestication, we caution that de novo domestication should be understood as an iterative process rather than a singular event. Changes in human social preferences and relationships and ongoing agronomic innovation, along with broad genetic changes, may be foundational. Allele frequency changes at many loci controlling quantitative traits not normally included in the domestication syndrome may be required to achieve sufficient yield, quality, defense, and broad adaptation. The environments, practices and tools developed and maintained by farmers and researchers over generations contribute to crop yield and success, yet those may not be appropriate for new crops without a history of agronomy. New crops must compete with crops that benefit from long-standing participation in human cultural evolution; adoption of new crops may require accelerating the evolution of new crops' culinary and cultural significance, the emergence of markets and trade, and the formation and support of agricultural and scholarly institutions. We provide a practical framework that highlights and integrates these genetic, agronomic, and cultural drivers of change to conceptualize de novo domestication for communities of new crop domesticators, growers and consumers. Major gene-focused domestication may be valuable in creating allele variants that are critical to domestication but will not alone result in widespread and ongoing cultivation of new crops. Gene editing does not bypass or diminish the need for classical breeding, ethnobotanical and horticultural knowledge, local agronomy and crop protection research and extension, farmer participation, and social and cultural research and outreach. To realize the ecological and social benefits that a new era of de novo domestication could offer, we call on funding agencies, proposal reviewers and authors, and research communities to value and support these disciplines and approaches as essential to the success of the breakthroughs that are expected from gene editing techniques.

Roadmap for Accelerated Domestication of an Emerging Perennial Grain Crop.

Shifting the life cycle of grain crops from annual to perennial would usher in a new era of agriculture that is more environmentally friendly, resilient to climate change, and capable of soil carbon sequestration. Despite decades of work, transforming the annual grain crop wheat (Triticum aestivum) into a perennial has yet to be realized. Direct domestication of wild perennial grass relatives of wheat, such as Thinopyrum intermedium, is an alternative approach. Here we highlight protein coding sequences in the recently released T. intermedium genome sequence that may be orthologous to domestication genes identified in annual grain crops. Their presence suggests a roadmap for the accelerated domestication of this plant using new breeding technologies.

Enhancing Crop Domestication Through Genomic Selection, a Case Study of Intermediate Wheatgrass.

Perennial grains could simultaneously provide food for humans and a host of ecosystem services, including reduced erosion, minimized nitrate leaching, and increased carbon capture. Yet most of the world's food and feed is supplied by annual grains. Efforts to domesticate intermediate wheatgrass (Thinopyrumn intermedium, IWG) as a perennial grain crop have been ongoing since the 1980's. Currently, there are several breeding programs within North America and Europe working toward developing IWG into a viable crop. As new breeding efforts are established to provide a widely adapted crop, questions of how genomic and phenotypic data can be used among sites and breeding programs have emerged. Utilizing five cycles of breeding data that span 8 years and two breeding programs, University of Minnesota, St. Paul, MN, and The Land Institute, Salina, KS, we developed genomic selection (GS) models to predict IWG traits. Seven traits were evaluated with free-threshing seed, seed mass, and non-shattering being considered domestication traits while agronomic traits included spike yield, spikelets per inflorescence, plant height, and spike length. We used 6,199 genets - unique, heterozygous, individual plants - that had been profiled with genotyping-by-sequencing, resulting in 23,495 SNP markers to develop GS models. Within cycles, the predictive ability of GS was high, ranging from 0.11 to 0.97. Across-cycle predictions were generally much lower, ranging from -0.22 to 0.76. The prediction ability for domestication traits was higher than agronomic traits, with non-shattering and free threshing prediction abilities ranging from 0.27 to 0.75 whereas spike yield had prediction abilities ranging from -0.22 to 0.26. These results suggest that progress to reduce shattering and increase the percent free-threshing grain can be made irrespective of the location and breeding program. While site-specific programs may be required for agronomic traits, synergies can be achieved in rapidly improving key domestication traits for IWG. As other species are targeted for domestication, these results will aid in rapidly domesticating new crops.

Genome mapping of quantitative trait loci (QTL) controlling domestication traits of intermediate wheatgrass (Thinopyrum intermedium).

Allohexaploid (2n = 6x = 42) intermediate wheatgrass (Thinopyrum intermedium), abbreviated IWG, is an outcrossing perennial grass belonging to the tertiary gene pool of wheat. Perenniality would be valuable option for grain production, but attempts to introgress this complex trait from wheat-Thinopyrum hybrids have not been commercially successful. Efforts to breed IWG itself as a dual-purpose forage and grain crop have demonstrated useful progress and applications, but grain yields are significantly less than wheat. Therefore, genetic and physical maps have been developed to accelerate domestication of IWG. Herein, these maps were used to identify quantitative trait loci (QTLs) and candidate genes associated with IWG grain production traits in a family of 266 full-sib progenies derived from two heterozygous parents, M26 and M35. Transgressive segregation was observed for 17 traits related to seed size, shattering, threshing, inflorescence capacity, fertility, stem size, and flowering time. A total of 111 QTLs were detected in 36 different regions using 3826 genotype-by-sequence markers in 21 linkage groups. The most prominent QTL had a LOD score of 15 with synergistic effects of 29% and 22% over the family means for seed retention and percentage of naked seeds, respectively. Many QTLs aligned with one or more IWG gene models corresponding to 42 possible domestication orthogenes including the wheat Q and RHT genes. A cluster of seed-size and fertility QTLs showed possible alignment to a putative Z self-incompatibility gene, which could have detrimental grain-yield effects when genetic variability is low. These findings elucidate pathways and possible hurdles in the domestication of IWG.

Managing for Multifunctionality in Perennial Grain Crops.

Plant breeders are increasing yields and improving agronomic traits in several perennial grain crops, the first of which is now being incorporated into commercial food products. Integration strategies and management guidelines are needed to optimize production of these new crops, which differ substantially from both annual grain crops and perennial forages. To offset relatively low grain yields, perennial grain cropping systems should be multifunctional. Growing perennial grains for several years to regenerate soil health before rotating to annual crops and growing perennial grains on sloped land and ecologically sensitive areas to reduce soil erosion and nutrient losses are two strategies that can provide ecosystem services and support multifunctionality. Several perennial cereals can be used to produce both grain and forage, and these dual-purpose crops can be intercropped with legumes for additional benefits. Highly diverse perennial grain polycultures can further enhance ecosystem services, but increased management complexity might limit their adoption.