Roadmap for Accelerated Domestication of an Emerging Perennial Grain Crop

Current grain crops are annuals that must be sown every year, giving their root systems little time to develop during the growing season.A perennial grain crop with a long-lived extensive root system would improve soil quality, store carbon belowground, and utilize water and minerals more efficiently.Domestication genes of the annual grass wheat are highly conserved in the perennial intermediate wheatgrass (Thinopyrum intermedium), providing an opportunity for accelerated domestication of a perennial grain using a mutagenesis approach. 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. 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. Crop production is facing unprecedented challenges. In 2050, the human population will likely exceed nine billion [1.Food and Agriculture Organization of the United Nations The Future of Food and Agriculture: Trends and Challenges. FAO, 2017Google Scholar], increasing the demand for staple crops and livestock by 60% [2.Springmann M. et al.Options for keeping the food system within environmental limits.Nature. 2018; 562: 519-525Crossref PubMed Scopus (1143) Google Scholar]. Climate change is expected to drastically constrain plant productivity, necessitating the development of cultivars with increased tolerance to abiotic stresses such as heat, drought, soil salinization, and flooding. 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Annual crops must be sown every year, which disturbs the soil and exposes it to erosion through tillage or clearing of vegetation with herbicides. Furthermore, in the beginning of the growth season, the shallow root systems are inefficient at taking up water and nutrients, which is a major cause of ground and surface water pollution by nitrate leaching. A perennial grain crop that does not need to be sown every year would develop a long-lived deep root system (Figure 1A ) that sequesters carbon and takes up nutrients and water efficiently [7.Crews T.E. et al.Is the future of agriculture perennial? Imperatives and opportunities to reinvent agriculture by shifting from annual monocultures to perennial polycultures.Glob. Sustain. 2018; 1: 1-18Crossref Scopus (87) Google Scholar,8.Culman S.W. et al.Soil and water quality rapidly responds to the perennial grain Kernza wheatgrass.Agron. J. 2013; 105: 735-744Crossref Scopus (145) Google Scholar]. 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In 1988, a Eurasian forage grass called intermediate wheatgrass (Thinopyrum intermedium), which is a close perennial relative of wheat, was selected as a promising perennial grain candidate at the Rodale Institute [12.Wagoner P. Perennial grain: new use for intermediate wheatgrass.J. Soil Water Conserv. 1990; 45: 81-82Google Scholar]. The intention was to create a productive grain crop through phenotypic selection within the species. Two cycles of breeding for improved grain production were completed in New York, USA, between 1990 and 2000 [13.DeHaan L. et al.Development and evolution of an intermediate wheatgrass domestication program.Sustainability. 2018; 10: 1-19Crossref PubMed Scopus (65) Google Scholar]. The Land Institute's domestication program for intermediate wheatgrass began in 2003 and continues to the present time [13.DeHaan L. et al.Development and evolution of an intermediate wheatgrass domestication program.Sustainability. 2018; 10: 1-19Crossref PubMed Scopus (65) Google Scholar]. Eight generations of selecting and intermating the best plants based on their yield, seed size, shatter resistance, and other traits have been performed, resulting in improved populations of T. intermedium that are currently being evaluated and further selected at The Land Institute and by collaborators in diverse environments. However, these breeding approaches have not yielded T. intermedium varieties that would be profitable for farmers to produce at large scale. On-farm yields of T. intermedium varieties are currently less than 20% of that of wheat and seed mass is about 25% that of wheat seeds. The breeding program is currently focused on selecting for several traits, including yield, shatter resistance, free threshing ability, seed size, and grain quality. Although progress is steady, the urgent need to sustainably boost food production necessitates the development of methods to dramatically accelerate the pace of domestication. A high-quality genome sequence of T. intermedium (available at https://phytozome-next.jgi.doe.gov/info/Tintermedium_v2_1), which promises to facilitate future breeding approaches, was released in early 2019 by the US Department of Energy Joint Genome Institute. Like wheat, it has a large and complex allohexaploid genome (2n = 6x = 42) with three genomes in one and containing approximately 11 912 million nucleotide base pairs. Given the near-obligate outcrossing nature of T. intermedium, allelic diversity is very high in every population studied so far. Therefore, no population will be genetically identical to the reference genome. Because the genome is hexaploid, six alleles of a given gene may be present in a single individual. In a larger population, many allelic variants are expected at every locus. This inherently large diversity makes it extremely challenging to identify mutants by phenotypic evaluation. Novel variation induced by mutagenesis and already present genetic variation can be identified by target induced local lesions in genomes (TILLING) and ecotype TILLING (EcoTILLING) approaches, respectively [14.Barkley N. Wang M. Application of TILLING and EcoTILLING as reverse genetic: approaches to elucidate the function of genes in plants and animals.Curr. Genomics. 2008; 9: 212-226Crossref PubMed Scopus (74) Google Scholar]. If a recessive mutation is required to produce a desired phenotype, each of the six alleles might need to be mutated. Therefore, searches for new promising recessive alleles may be futile if they are based on the phenotype of mutant plants. An alternative, and perhaps the only feasible way to search for useful variation, is to locate variation directly at the DNA level in the genome. In the case of recessive mutations, one would need to identify mutations in all three genomes and then breed to obtain homozygosity at all three loci. This would have to be accomplished through marker-assisted breeding and selection. There is substantial divergence between the three genomes of T. intermedium and in many cases large deletions in gene orthologs suggest that many of the alleles are already nonfunctional. Therefore, it may be possible to identify instances where mutations are only needed in one of the three genomes in order to produce a desired phenotype. However, to design a working strategy, one would need to sequence every allelic form present in the individual to be mutated. T. intermedium is primarily outcrossing (Figure 1B,C), which means that any seed pool will be heterozygous and heterogeneous. Distinguishing new mutations from existing mutations in such material is challenging. However, self-pollination for one or two generations is possible. Selfing could likely be used to obtain homozygosity at particular loci if these were tracked with markers, but it has not yet been possible to breed to full homozygosity. The sequenced individual used for the reference genome was a spontaneous and extremely rare haploid. In our studies, T. intermedium has had a low tolerance to the mutagen ethyl methanesulfonate (EMS), with 100% mortality resulting from the typical dosage of 0.8% (v/v) used in wheat [15.Chen L. et al.Development and characterization of a new TILLING population of common bread wheat (Triticum aestivum L.).PLoS One. 2012; 7e41570Crossref PubMed Scopus (77) Google Scholar]. Furthermore, a transformation protocol for T. intermedium has not yet been developed, posing a major constraint to future genome editing approaches. Domestication, the reiterative selection of plants with a desired trait, has resulted in genetic changes that distinguish domesticated taxa from their wild ancestors in traits such as shatter resistance, ease of threshing, and seed dormancy [16.Gross B.L. Olsen K.M. Genetic perspectives on crop domestication.Trends Plant Sci. 2010; 15: 529-537Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar]. Molecular genetic studies have revealed several domestication genes in plants (mainly encoding transcription factors), loci underlying crop diversity (mainly encoding enzymes and structural proteins), and mutations that result in cis-regulatory changes [17.Olsen K.M. Wendel J.F. A bountiful harvest: genomic insights into crop domestication phenotypes.Annu. Rev. 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Two major hypotheses have been proposed to explain the molecular mechanisms of domestication: the genetic tinkering hypothesis (i.e., slow gain of new functions) posits that domestication proceeds via the modification of genes and physical features that are already present rather than the addition of fundamentally new characteristics or the loss of existing ones [20.Denison R.F. et al.Darwinian agriculture: when can humans find solutions beyond the reach of natural selection?.Q. Rev. Biol. 2003; 78: 145-168Crossref PubMed Scopus (143) Google Scholar,21.Doebley J. Unfallen grains: how ancient farmers turned weeds into crops.Science. 2006; 312: 1318-1319Crossref PubMed Scopus (64) Google Scholar]. The 'tinkering' aspect of this process may resemble that of the evolutionary adaptation found in nature [20.Denison R.F. et al.Darwinian agriculture: when can humans find solutions beyond the reach of natural selection?.Q. Rev. Biol. 2003; 78: 145-168Crossref PubMed Scopus (143) Google Scholar]. In support of this model, few genes that contributed to the domestication of diploid and ancient polyploid species are null alleles; gene mutations often cause altered protein function and/or gene expression rather than eliminate protein function. For example, de novo domestication of wild relatives of tomato (Solanum lycopersicum) was possible by altering protein expression through CRISPR-Cas9-assisted modification of cis-regulatory regions or upstream open reading frames [22.Li T. et al.Domestication of wild tomato is accelerated by genome editing.Nat. Biotechnol. 2018; 36: 1160-1163Crossref Scopus (281) Google Scholar]. The second hypothesis is the genetic disassembling hypothesis (i.e., rapid loss of function), which posits that a major portion of phenotypes associated with domestication, even those associated with gain of function and/or unaffected protein function, result from loss-of-function ('crippling') mutations (reviewed in [23.Østerberg J.T. et al.Accelerating the domestication of new crops: feasibility and approaches.Trends Plant Sci. 2017; 22: 373-384Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar]). Loss-of-function is not restricted to proteins but can also be loss or weakening of cis-regulatory mechanisms that help the plant survive in nature. Whereas these mutations are generally recessive, they can also be dominant (e.g., if they result from the loss of repressor sites in promoter regions). Furthermore, in polyploid species, null mutations of one homologous gene copy may have only subtle dosage effects and so appear as 'tinkering' mutations. Thus, domestication by 'crippling' resulted in the loss of many properties essential for plant survival in the wild and is consistent with the observation that domesticated plants are generally completely dependent on humans and can no longer compete in nature. Loss-of-function mutations that occur in coding regions often have profound pleiotropic effects, which weaken the plant overall. One solution that has been proposed for new domestications is to prioritize mutations in cis-regulatory mutations, which have more subtle effects and can produce the desired phenotype without weakening the organism in other ways [24.Dehaan L.R. Van Tassel D.L. Useful insights from evolutionary biology for developing perennial grain crops.Am. J. Bot. 2014; 101: 1801-1819Crossref PubMed Scopus (35) Google Scholar]. It has been hypothesized that domestication is caused by changes in only a few domestication genes and that these events can be mimicked by mutagenesis of homologous genes in wild plants [23.Østerberg J.T. et al.Accelerating the domestication of new crops: feasibility and approaches.Trends Plant Sci. 2017; 22: 373-384Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar,25.Shapter F.M. et al.High-throughput sequencing and mutagenesis to accelerate the domestication of Microlaena stipoides as a new food crop.PLoS One. 2013; 8e82641Crossref PubMed Scopus (40) Google Scholar,26.Zsögön A. et al.Genome editing as a tool to achieve the crop ideotype and de novo domestication of wild relatives: case study in tomato.Plant Sci. 2017; 256: 120-130Crossref PubMed Scopus (82) Google Scholar]. The phenotypic changes involved in domestication often have parallel genetic underpinnings, where mutations in homologous loci underlie the same phenotype across species [19.Purugganan M.D. Evolutionary insights into the nature of plant domestication.Curr. Biol. 2019; 29: R705-R714Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar]. Recent work has demonstrated de novo domestication of wild relatives of tomato (S. lycopersicum) by introducing mutations in the form of small insertions or deletions (indels) in as few as five genes, providing strong support for the genetic disassembling hypothesis [22.Li T. et al.Domestication of wild tomato is accelerated by genome editing.Nat. Biotechnol. 2018; 36: 1160-1163Crossref Scopus (281) Google Scholar,27.Zsögön A. et al.De novo domestication of wild tomato using genome editing.Nat. Biotechnol. 2018; 36: 1211-1216Crossref Scopus (353) Google Scholar]. The general applicability of these findings remains to be tested in a range of plant species. However, if this approach would allow for accelerated domestication of wild plants, it could have enormous potential for agriculture [28.Eshed Y. Lippman Z.B. Revolutions in agriculture chart a course for targeted breeding of old and new crops.Science. 2019; 366: 705Crossref Scopus (121) Google Scholar,29.Bailey-Serres J. et al.Genetic strategies for improving crop yields.Nature. 2019; 575: 109-118Crossref PubMed Scopus (428) Google Scholar]. Recently, the orphan crop groundcherry (Physalis pruinosa) was partially domesticated by mutating orthologous domestication genes of tomato [30.Lemmon Z.H. et al.Rapid improvement of domestication traits in an orphan crop by genome editing.Nat. Plants. 2018; 4: 766-770Crossref PubMed Scopus (236) Google Scholar]. The genome of T. intermedium is closely related to that of wheat [31.Avni R. et al.Wild emmer genome architecture and diversity elucidate wheat evolution and domestication.Science. 2017; 357: 93-97Crossref PubMed Scopus (473) Google Scholar, 32.The International Wheat Genome Sequencing Consortium (IWGSC) Shifting the limits in wheat research and breeding using a fully annotated reference genome.Science. 2018; 361eaar7191Crossref PubMed Scopus (1646) Google Scholar, 33.Maccaferri M. et al.Durum wheat genome highlights past domestication signatures and future improvement targets.Nat. Genet. 2019; 51: 885-895Crossref PubMed Scopus (334) Google Scholar], which suggests that domestication genes in wheat are represented by orthologous genes in T. intermedium. Below, we will capitalize on this knowledge and expand on it to provide a roadmap for accelerated domestication of T. intermedium (Figure 2). Many domestication genes have been discovered in a variety of crop plants. Here we will focus on some that govern traits that were essential for the early domestication of grasses. During the process of crop domestication, human selection resulted in plants with larger seeds that were easier to harvest. Most wild grasses have abscission layers that cause the heads to break into smaller fragments that disperse their propagules. This seed shattering process is essential for survival of wild plant species, but would generate enormous losses in agriculture. Hence, one of the most important events in crop domestication has been the elimination of the natural mode of propagule dispersal. Two main natural modes of dispersal exist: (i) fruits separate (abscise) from the plant upon ripening, thus allowing seeds to come into contact with the soil; (ii) fruits remain attached to the plant, but open (dehisce) to release the seeds, which in turn abscise from the mother tissues. Abscission of fruits or seeds is dependent on the mechanical properties of the relevant tissues, and several domestication genes involved in seed shattering were shown to control lignification or the development of abscission layers [34.Di Vittori V. et al.Convergent evolution of the seed shattering trait.Genes (Basel). 2019; 10: 68Crossref Scopus (26) Google Scholar]. Wild type T. intermedium plants exhibit seed shattering and most seeds remain attached to a hull (Figure 1D,E). Phenotypic selection has produced individual plants that are nearly nonshattering and mostly free threshing. As the traits are highly polygenic and T. intermedium is an outcrossing polyploid, the challenge is to obtain a population that breeds true for the desired traits. This may be achieved in a matter of generations using marker-assisted/genomic selection, but could turn out to be a persistent problem. Seed shattering in barley (Hordeum vulgare) and other grasses is the result of detachment of the spikelet from the spike. The central part of the spike is the rachis, composed of a number of short nodes and internodes. Spikelets grow at the nodes. Wild barley spikes present thin primary and secondary cell walls at rachis nodes, which makes them brittle and prone to shedding the seeds at maturity [35.Pourkheirandish M. et al.Evolution of the grain dispersal system in Barley.Cell. 2015; 162: 527-539Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar], while domesticated barley varieties have thickened cell walls at rachis nodes. Although the exact mechanism is unknown, this phenotype is related to loss of function of either one of two genes, namely Btr1 or Btr2 [35.Pourkheirandish M. et al.Evolution of the grain dispersal system in Barley.Cell. 2015; 162: 527-539Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 36.Zhao Y. et al.Btr1-A induces grain shattering and affects spike morphology and yield-related traits in wheat.Plant Cell Physiol. 2019; 60: 1342-1353Crossref PubMed Scopus (16) Google Scholar, 37.Nave M. et al.Wheat domestication in light of haplotype analyses of the Brittle rachis 1 genes (BTR1-A and BTR1-B).Plant Sci. 2019; 285: 193-199Crossref PubMed Scopus (9) Google Scholar], and it was suggested that these genes were significant drivers in the evolution of the rachis-type disarticulation system of Triticeae grasses. In barley, closely related genes named Btr1-like and Btr2-like have also been identified, but they are not functional paralogs of Btr1 and Btr2 as they cannot complement btr1 and btr2 in cultivated barley [35.Pourkheirandish M. et al.Evolution of the grain dispersal system in Barley.Cell. 2015; 162: 527-539Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar]. Spikes of T. intermedium have multiple brittle break points. In some genotypes, the break points are above the node, while in others they are below, which suggests that avoiding shattering is a complex problem. The T. intermedium genome has several genes that resemble Btr1 and Btr1-like and also Btr2 and Btr-2-like (Table 1). Among the encoded proteins, nine show more than 50% amino acid sequence identity to Btr1 and nine more than 50% identity to Btr2. The hydrophobicity profile of the barley Btr1 protein is distinct from that of barley Btr1-like in that two transmembrane spanning segments appear to be present in Btr1 but absent from Btr1-like [35.Pourkheirandish M. et al.Evolution of the grain dispersal system in Barley.Cell. 2015; 162: 527-539Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar]. At least the six best hits among putative Btr1 proteins have a hydrophobicity profile that resembles that of barley Btr1. This would suggest that multiple homologs of Btr1 and possibly also Btr2 are present in the T. intermedium genome. Btr1 therefore appears to be a challenging target, but mutation of Btr2 may be sufficient to obtain a reliable non-brittle rachis.Table 1Candidate Domestication Genes That Align with Intermediate Wheatgrass QTLsTraitGeneGene nameSourceIWG HGaAbbreviations: HG, homoelogous group; IWG, intermediate wheatgrass.IWG V2.1 gene modelIdentity (%)bPercent identity of predicted amino acid sequences.RefsNon-brittle rachisBtr1Non-brittle rachis1Barley (3)3Thint.09G0046000Thint.07G0115700Thint.07G0115500OtherscSix physically close genes have a sequence identity of >50%: Thint.07G0115400, Thint.07G0116600, Thint.07G0116000, Thint.08G0083500, Thint.09G0044900, and Thint.09G0045800.87,2488,2786,73[35.Pourkheirandish M. et al.Evolution of the grain dispersal system in Barley.Cell. 2015; 162: 527-539Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar,82.Civáň P. Brown T.A. 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