Orphan crops: A genetic treasure trove for hunting stress tolerance genes

Abstract

Orphan crops, also known as minor crops, smart foods, and superfoods, have attracted great attention recently because of their unique ability to grow in resource-poor marginal lands, and under harsh environmental conditions without any intensive agricultural care. These crops possess inherent tolerance against different abiotic stresses such as drought, salinity, cold, and heat. Recent advancements in genomic resources and high-throughput phenotyping platforms have provided opportunities to explore the untapped potential of orphan crops to identify novel gene source(s) and mechanism(s) for developing abiotic stress-tolerant crops. Moreover, genomics-assisted investigations into the various physiological and molecular mechanism(s) could provide useful insights into stress tolerance mechanisms in these plants. Nevertheless, translating the hidden power of the tolerant gene pools from the orphan crops into major staple crops for enhancing their stress tolerance while maintaining yield is a challenging task. The contemporary tools of genomics can be used to unravel the secret of stress tolerance in orphan crops and employ these untapped genes for tailoring stress-tolerant crop varieties to ensure global food security in the era of climate change.

INTRODUCTION

The population of the world is expected to reach 9.7 billion by 2050 (United Nations, 2019). Ensuring food security to feed the growing population, particularly in the developing countries where the majority of the world population lives, is one of the major challenges (Byerlee & Fanzo, 2019). Global food security assumes even more importance in the face of climate change as the occurrence of weather extremes, such as heat, drought, and flood events has intensified over the years in terms of frequency as well as duration (Kadam et al., 2014). Just heat and drought events alone in the past four decades have caused a catastrophic impact on agricultural crops affecting millions of people and the world economy (Kadam et al., 2014; Perkins-Kirkpatrick & Lewis, 2020). Moreover, shrinking agricultural resources such as fertile land and freshwater are further aggravating the situation (Rossati, 2017). Thus, crop improvement programs are focused on developing climate-resilient crops, which can sustain yield under fluctuating environments and limited resources. This, however, needs redesigning of breeding programs with the integration of advanced molecular and high-throughput phenotyping tools. For example, the evolution of sequencing technologies coupled with the development of computational pipelines and exponential drop in sequencing costs have led to the availability of high-quality genome sequences for many crops. Some recent breakthroughs in agriculture have been achieved with the help of genomic tools like genomics-assisted breeding, transgenics, and genome editing (Anders et al., 2021; Varshney, Chen, et al., 2012, Varshney, Ribaut, et al., 2012; Zafar et al., 2020), and various elite cultivars have been developed with combinations of desired traits, such as high yield and stress tolerance (Pareek et al., 2010; Salgotra & Neal Stewart, 2020).

The green revolution witnessed an unprecedented success in food grain production by the introduction of high-yielding varieties, chemical fertilizers, pesticides, and agriculture mechanization (Briggs, 2009; Pingali, 2012). However, this culminated in the monoculture of a limited set of elite high-yielding genotypes of cereal crops, such as wheat, rice, and maize. Eventually, this practice has contributed toward the loss of beneficial alleles particularly for tolerance against biotic and abiotic stresses.

Recently, scientists have started exploring wild relatives of major crops to identify the novel gene(s) or mechanism(s), which can be utilized to enhance stress tolerance in elite cultivars (Rawat et al., 2021). Consequently, gene banks or seed vaults have become an essential resource for plant scientists to preserve and explore genetic resources. Besides wild relatives of major crops, a relatively neglected group of minor crops categorized as “orphan crops” have been identified as a useful genetic resource for important traits associated with stress tolerance and grain nutrients (Figure 1). However, the benefits of orphan crops for human health and nutrition, their potential to grow and produce optimum yields under marginal lands, and sustain under harsh climatic conditions were often ignored (Talabi et al., 2022). Conversely, adequate efforts and investment have not been made to introduce orphan crops into the mainstream food chain during the pre- and the post-green revolution era (Cullis & Kunert, 2017). Therefore, orphan crops have lagged behind major crops in terms of preference, production, and productivity. Interestingly, orphan crops drew attention recently as alternative nutrient-rich foods, and have been described as “super foods” or “smart foods” (Tadele, 2019). Additionally, orphan crops have also gained attention due to their stress tolerance features (Mabhaudhi et al., 2019). Unprecedented demand for orphan crops as food and genetic resource for agri-traits has resulted in the initiation of several global research initiatives, such as the African Orphan Crops Consortium (AOCC) (http://africanorphancrops.org/) and Crops for the Future (CFF) (http://www.cffresearch.org/), for further improvements and bringing orphan crops to the mainstream food chain (Tadele, 2019).

FIGURE 1. Evolution of crops and their domestication. The process of domestication in major crops led to substantial improvement in yield with a concomitant loss in stress tolerance. On the contrary, orphan crops continuously adapted to survive under harsh conditions, but compromised in yield.

Challenging global climatic conditions in the recent past and alarming condition of globally widespread malnutrition termed as “hidden hunger” has forced the scientific community to revisit orphan crops to explore their genetic potential for abiotic stress tolerance and superior nutritional properties (Cullis & Kunert, 2017; Kumar & Bhalothia, 2020; Mabhaudhi et al., 2019). On the contrary, the staple cereal crops, such as rice, and wheat, have been documented to lose trait(s)/gene(s) related to stress tolerance (Kumar et al., 2022) and nutritional property (Tripathi et al., 2022) during the process of domestication and yield-oriented breeding. Therefore, orphan crops could serve as an instant resource to identify superior traits contributing to stress tolerance and nutritional properties, which could be introgressed in elite varieties of staple crops. In the present review, we have highlighted the (i) importance of orphan crops as a genetic resource for the improvement of abiotic stress tolerance in other major staple crops, and (ii) recent advancements in the field of genomics and phenotyping platforms, which could speed up the crop improvement programs by utilizing orphan crops. The efforts made at several international consortiums focusing on harnessing the benefits of orphan crops for food security have also been critically evaluated.

ORPHAN CROPS AS GENETIC RESOURCE FOR BIOTIC AND ABIOTIC STRESS TOLERANCE

Orphan crops are documented to be nutritionally superior and highly stress tolerant, and hence, can be a key player for developing climate-resilient, high-yielding, and nutrient-rich staple crops (Figure 2). Despite their unique properties and stress tolerance potential, a number of indigenous crops have been tagged as orphan crops (Table 1) based on their limited area of cultivation, low market returns, and least investment in research and genetic improvement in the past (reviewed by Talabi et al., 2022). However, most of these orphan crops have recently received attention across the globe as a unique source of dietary nutrition and having the potential to sustain under suboptimal environment. During the past decade, efforts have been initiated to explore the genetic potential of orphan crops, which can help getting them into the mainstream of crop improvement programs (Tadele, 2019).

FIGURE 2. Orphan crops are a genetic treasure trove. Exploring these crops at the physiological and molecular level may pave the way toward improving stress tolerance in modern-day high-yielding crop plants.

TABLE 1 Representative orphan crops having natural tolerance to abiotic stress

Crop type	Common name	Scientific name	Stress tolerance	References
Cereals	African rice	Oryza glaberrima	Biotic and abiotic stress tolerance	Linares (2002)
	Barnyard millet	Echinochloa crus-galli	Abiotic stress tolerance	Upadhyaya et al. (2016)
	Finger millet	Eleusine coracana	Abiotic stress tolerance	Singh (2012), Wambi et al. (2021)
	Foxtail millet	Setaria italica	Abiotic stress tolerance	Taylor and Kruger (2016)
	Kodo millet *	Paspalum scrobiculatum	Abiotic stress tolerance	Upadhyaya et al. (2016)
	Little millet *	Panicum sumatrense	Abiotic stress tolerance	Upadhyaya et al. (2016), Das et al. (2020)
	Pearl millet	Pennisetum glaucum	Abiotic stress tolerance	Yadav et al. (2016)
	Proso-millet	Panicum miliaceum	Abiotic stress tolerance	Taylor and Kruger (2016)
	Tef	Eragrostis tef	Abiotic stress tolerance	Spaenij-Dekking et al. (2005)
	Red rice	Oryza longistaminata	Drought tolerance	Zhang et al. (2015)
	Quinoa	Chenopodium quinoa	Salinity tolerance	Jarvis et al. (2017), Zou et al. (2017)
	Grain amaranth	Amaranthus hypochondriacus	Salinity tolerance	Clouse et al. (2016)
	Wild rice	Oryza coarctata	Salinity tolerance	Mondal et al. (2017)
Leafy vegetable	Gynandra	Gynandropsis gynandra	Abiotic stress tolerance	Sogbohossou et al. (2018)
Legume	Bambara groundnut	Vigna subterranean	Drought tolerance	Mayes et al. (2019)
	Cowpea	Vigna unguiculata	Drought tolerance	Timko and Singh (2008)
	Grass pea *	Lathyrus sativus	Extreme drought tolerance	Girma and Korbu (2012)
	Horse gram	Macrotyloma uniflorum	Drought, salinity, heat, and heavy metal tolerance	Aditya et al. (2019)
Oil seed	Noug *	Guizotia abyssinica	Abiotic stress tolerance	Solomon and Zewdu (2009)
Root	Cassava	Manihot esculentum	Drought tolerance	Ceballos et al. (2004)
	Enset	Ensete ventricosum	Drought tolerance	Olango et al. (2014)
	Yam	Dioscorea spp	Drought tolerance	Raymundo et al. (2014)

Note: The genome sequence is available for all the crops, except those with asterisk (*) mark.

Since the orphan crops are scarcely cultivated both in terms of area and region, it is highly probable that the crops have not yet undergone the domestication process, and the allelic diversity in them and their wild relatives related to stress tolerance, nutritional value, and secondary metabolite content are still being inherited. Although breeding efforts are underway to enhance the yield potential of different orphan crops, they are less likely to be affected by yield-oriented breeding due to substantial awareness about their traits contributing to nutrition and stress resilience. Thus, the research focus is now being shifted toward genome sequencing, and resequencing of these orphan crops so as to understand, and explore the magnitude of genetic variation accommodated in these crops. Among the legume orphan crops, the pigeon pea (Cajanus cajan L.) genome was the first to be sequenced (Singh et al., 2012; Varshney, Ribaut, et al., 2012). It is the sixth most important legume crop in the world, which is a major source of protein, particularly for the population living in the semi-arid regions (Varshney, Chen, et al., 2012). Analysis of the genome of pigeon pea revealed a total of 48,680 genes, out of which 111 genes were found to be drought responsive, which was higher than the drought-responsive genes reported in Medicago truncatula (90), Lotus japonicus (58), and Glycine max (109) (Varshney, Ribaut, et al., 2012). Moreover, the homology search revealed 71 genes in pigeon pea encoding proteins with USP (Universal Stress Protein) domain, of which 49 genes were found to be drought responsive (Sinha et al., 2016). Specifically, C. cajan_29830 and C. cajan_33874 genes have been documented to contribute to the drought tolerance in pigeon pea. Apart from the abiotic stress, genes contributing resistance to Fusarium wilt and Sterility Mosaic Disease, which are among the most deadly diseases of pigeon pea were also identified (Singh et al., 2016).

Chickpea (Cicer arietinum L.), the second most widely grown legume worldwide with a high symbiotic N-fixing capacity, has been identified to harbor genes providing tolerance to drought, heat, and biotic stresses. The draft genome assembly of chickpea revealed the presence of 187 disease resistance gene homologs in chickpea (Varshney et al., 2013). Further, resequencing of 429 chickpea accessions collected from 45 countries brought to light that the nucleotide diversity in the wild relatives of chickpea was much higher than that in the landraces, indicating that genomic regions are influenced by the process of selection and domestication. During the domestication process, genomic diversity in chickpea has been reported to be reduced significantly from wild lines to landraces and then to the breeding lines. However, >200 candidate genes for biotic and abiotic stress tolerance and phenological traits have been identified from chickpea from the process of selection for crop breeding and post-domestication diversification (Varshney et al., 2019). Another orphan crop, rye (Secale cereale L.) is known to be inherently tolerant to biotic and abiotic stresses. Rye harbors several non-orthologous or functionally redundant nucleotide-binding site and leucine-rich repeat (NLR) motif genes, specifically linked to race-specific pathogen resistance, which could offer an excellent scope for mining novel alleles and QTLs for both biotic and abiotic stress tolerance (Rabanus-Wallace et al., 2021). Additionally, DRE-binding factors present in rye have been documented to result in the upregulation of 21 genes identified for low-temperature tolerance (Jung & Seo, 2019). Conversely, Indian barnyard millet (Echinochloa frumentacea) has been documented to be highly adaptive to drought due to robust photosynthetic machinery, chlorophyll binding, and ability to maintain photosynthesis even under drought stress (Jayakodi et al., 2019; Tadele, 2016). Similarly, another popular crop, pearl millet (Pennisetum glaucum (L.) R. Br.) is grown for food, fodder, and fuel, particularly in the arid and semi-arid regions of India, sub-Saharan Africa, and South Asia. Genetic analysis revealed that pearl millet contains a set of around 378 genes specifically for pathogen resistance. Moreover, the pearl millet genome highlights an expansion of the gene family involved in terpenoid backbone biosynthesis and monoterpenoid and diterpenoid biosynthesis, which eventually contributes to the remarkable heat and drought stress tolerance (Varshney et al., 2018).

Some other orphan crops are well recognized for their natural ability to grow under harsh environments such as finger millet is adapted to harsh climatic and soil conditions, particularly drought (Singh, 2012), Tef (Eragrostis tef) is tolerant to poorly drained soil (Assefa et al., 2011), cowpea is tolerant to drought and heat (Sanginga et al., 2000), grass pea (Lathyrus sativus) and cassava are extremely tolerant to drought (Ceballos et al., 2004; Girma & Korbu, 2012), horse gram (Macrotyloma uniflorum) is tolerant to several abiotic stresses such as drought, salinity, heat, and heavy metals (Aditya et al., 2019; Bhardwaj et al., 2013). Nevertheless, the genetic basis of natural tolerance existing in these orphan crops is not yet fully explored. Recently, Varshney, Ribaut et al. (2012) presented a detailed map of variation in 3171 cultivated and 195 wild accessions of chickpea as a resource for chickpea genomics research and breeding. Similarly, diverse germplasm collections in other orphan crops such as pigeon pea (13,632 accessions), pearl millet (10,764 accessions), finger millet (5940 accessions) (Upadhyaya et al., 2006, 2007, 2011), and similar germplasm collections in other orphan crops could be utilized for the traits and QTL identifications and introgression of potential traits into major staple crops as well as in crop improvement programs for orphan crops to develop nutrients rich, high-yielding varieties.

GENERATION OF GENOMIC RESOURCES FOR DIVERSE ORPHAN CROPS

Orphan crops were left behind in the genomics-based revolution due to the lack of complete genomic sequences as well as efficient and reliable phenotyping platforms for proper selection of germplasms (Kamenya et al., 2021; Zhao et al., 2019). Hence, during the past decades, these crops were reported to be suffered from a dearth of genomic and molecular-genetic resources (Varshney et al., 2009). However, concerted efforts by exploiting advanced genome sequencing and genotyping technologies, RNA sequencing, and transcript expression profiling techniques along with the use of high-throughput phenotyping platforms have improved the situation over recent years (Selvaraj et al., 2020; Sun et al., 2020). To date, genomes of 12 cereals (and their wild relatives) and 16 legumes have been sequenced (Ye & Fan, 2021). Eventually, large-scale genomic datasets have been developed, which can accelerate genomics-assisted breeding approaches. Breeding techniques that are mostly used for resource generation are marker-assisted selection (MAS), marker-assisted backcrossing (MABC), and marker-assisted recurrent selection (MARS) (Ibrahim Bio Yerima & Achigan-Dako, 2021). MARS is used frequently and extensively in cross-pollinated orphan crops including pearl millet, and cassava for pyramiding all superior alleles within a genetic background. Eventually, recombinant lines are subjected to phenotyping for precise selection of potential donors (http://www.generationcp.org/gcp-research/research-initiatives/cassava/cassava-mars-for-drought.html). In cassava, root yield is one of the traits of interest used for selection of superior genotypes. However, estimation of below-ground biomass throughout the growing season was not possible (Kengkanna et al., 2019). Selvaraj et al. (2020) generated a multispectral imagery along with analytic framework (CIAT Pheno-i) to measure canopy matrices and vegetative indices (both above and below ground) of cassava, which was found useful for germplasm and varietal selection. On the other hand, RNA-sequencing and transcriptome analysis techniques are also explored for the generation of genetic resources specifically for yield improvement, and stress tolerance in orphan crops (Nida et al., 2021). In this line, Sankar et al. (2021) exposed eight different inbred lines of pearl millet to heat stress during the seedling stage and analyzed the transcript expression profiling data of small Heat Shock Protein (sHSP) genes, viz, Pgcp70 and PgHSF. Authors reported substantially higher accumulation of sHSPs in heat-tolerant genotypes and suggested that transcript expression profiling could be utilized for the identification of unknown genes relevant to heat stress tolerance in pearl millet and other orphan (millet) crops. Similarly, transcriptome analysis of pearl millet resulted in the identification of 318 transcription factors (TFs) and 149 transcription regulators (TRs) differentially expressed under heat stress, while 315 TFs and 128 TRs differentially expressed under drought stress (Sun et al., 2020). Additionally, RNA sequencing data helped in identifying nearly 7000 and 6500 genes responsive to heat and drought stress, respectively. During the past two decades, various initiatives involving international collaborations have been incepted, which played a significant role in the creation and characterization of genomic and genetic resources for different orphan crops, which has boosted the breeding efforts for stress tolerance and nutrition-rich crop development programs. Significant achievements of some of these initiatives have been discussed in the following sections.

The generation challenge Program (GCP)

The Generation Challenge Program was a pioneering initiative started by the Consultative Group on International Agricultural Research (CGIAR) in 2003 as a 10-year program. Its objective was to utilize the genetic diversity of crops to breed for tolerance to drought and other harsh environmental conditions (Bruskiewich et al., 2006). This initiative focused on a few important orphan crops, such as cassava, cowpea, millets, yam, sorghum, groundnut, and chickpea, and provided breeders an access to resources for optimum utilization of available germplasms (Varshney et al., 2010). This initiative also helped to modernize breeding efforts by the creation of an Integrated breeding management platform (https://www.integratedbreeding.net/) (Varshney, Chen, et al., 2012). Eventually, it led to the characterization of genetic diversity in different orphan crops (Glaszmann et al., 2010), and deciphered the genetic basis of important agronomic traits contributing to yield and stress tolerance (Pandey et al., 2014). Further, elite alleles were also identified for introgression of potential traits through advanced breeding techniques to create crops with improved performance (Caniato et al., 2014; Carvalho et al., 2016). For example, the “stay-green” trait was introduced into sorghum through breeding for the development of tolerance against post-flowering drought stress (Kassahun et al., 2010). Similarly, several varieties of cassava and bean have been developed for enhanced tolerance to biotic and abiotic stresses under the GCP (http://www.generationcp.org/impact/variety-release-a-updates/variety-releases.html). In addition, GCP also led to gene discovery and identification of stress tolerance genes from landraces and crop wild relatives (CGIAR-IEA, 2014). Concerted efforts have enabled genomics-assisted breeding for the development of future crops. For example, a genomic region harboring QTL for drought tolerance was identified in chickpea using linkage mapping approach and introgressed in three chickpea varieties using marker-assisted backcrossing (Thudi et al., 2014). Similarly, a high-yielding, wilt-resistant chickpea variety Pusa Chickpea Manav was developed by introgressing QTL region for Fusarium wilt resistance from WR 315 line into chickpea variety 391 (AICRP on Chickpea Annual Report, 2019-20). Apart from using orphan crops as a genetic treasure for future crop generation, yield improvement of these crops was also taken into consideration for African countries, where these crops serve as staple food crops. For example, sorghum is cultivated as a staple crop in Africa, but its productivity is very low due to the deficiency of phosphorus (P) and toxicity of aluminum (Al). Hence, a project was led by EMBRAPA (Empresa Brasileira de Pesquisa Agropecuária), Brazil under GCP to identify and validate Pup1, QTL in sorghum (http://www.generationcp.org/phosphorous-efficiency/sorghum.html), which is responsible for P acquisition in rice under P-deficit condition. In another project led by ICRISAT, GCP targeted to establish a molecular breeding program for Alt_SB gene for Al tolerance and Pup1, QTL for P efficiency in sorghum (http://www.generationcp.org/aluminium-tolerance-a-phosphorous-efficiency/sorghum.html). These reports highlight the importance of such international collaborative efforts in bringing the orphan crops to the mainstream of research.

The African orphan crops consortium (AOCC)

The AOCC was initiated in 2011 with the support of the African Union. Its objective was to improve the nutrition, productivity, and climate resilience of local African crops by enhancing the research on these crops to overcome malnutrition and stunting among rural children of Africa. The AOCC aimed to sequence the genomes of 101 African orphan crops/trees (Hendre et al., 2019). Out of these, the reference genomes of several agriculturally, biologically, medicinally, and economically important orphan plants, namely, Bambara groundnut, dolichos bean, apple ring acacia, marula, moringa (Chang et al., 2018), African eggplant (Song et al., 2019), and two Artocarpus species, A. heterophyllus (jackfruit), and A. altilis (breadfruit; Sahu et al., 2019) have been sequenced, which helped in identification of genes involved in stress tolerance, disease resistance, and other economically important traits. Subsequently, independent studies followed the characterization of orphan crops by utilization of genetic resource developed through AOCC. For example, Gao et al. (2021) crossed two landraces of Bambara groundnut, S19-3 (from Namibia) and DodR (from Tanzania), and screened the F3 generation to identify QTLs associated with yield-related traits under well-watered and water-deficit conditions. The QTLs expressed under well-watered condition were associated with number of seeds per plant, number of pods per plant, and pod weight per plant, whereas QTL associated with internode length was expressed under water-stressed condition. Thus, QTLs expressed under optimum condition were primarily yield specific, and QTLs expressed under drought were crucial to sustain under water-deficit conditions. Besides genome sequencing, acquisition of transcriptome data of several important orphan crops under different environmental conditions was documented to be effective for the identification of unknown genes. For instance, transcriptome data of two Bambara groundnut landraces, DipC and TN, under water-deficit condition revealed some dehydration-responsive gene regulators, including WRKY40, PRR7, ATAUX2-11, CONSTANS-like 1, MYB60, and AGL-83. In addition, it was found that the landraces were in dehydration-ready mode (Khan et al., 2017). Thus, the identification and characterization of potential genes/QTLs by utilizing existing genomic resources have enormous potential to provide new insights in tolerance to stress environments.

Crops for the future (CFF)

Crops for the future is a research company supported by the Government of Malaysia and the University of Nottingham, intending to develop solutions to diversify future agriculture using orphan crops. Bambara groundnut was used as a representative crop to develop tools and resources that can be applied to other orphan crops as well. It is regarded as one of the potential crops that can ensure future food security, particularly under the changing climatic condition (Olanrewaju et al., 2022). Despite its unique features for stress tolerance and nutritional properties, two traits viz. ‘short-day photoperiod’ requirement for pod-filling and ‘hard-to-cook’ are major bottlenecks for its adoption worldwide (Kendabie et al., 2014; Mubaiwa et al., 2017). Researchers tried to select photoperiod neutral landraces of Bambara groundnut through consecutive studies under greenhouse and temperature-controlled environment under long photoperiodic condition. Eventually, TZA 1505 collected from Tanzania was identified as a promising landrace, which is neutral to the photoperiodic requirement during pod filling (Jørgensen et al., 2009). Similar efforts are being made for the improvement of other crops, such as foxtail and proso millets, amaranth, etc. to enhance yield (Gregory et al., 2019).

Apart from these three major programs, many other initiatives are playing an important role in the improvement of orphan crops. Many specialized resources have come up for specific crops, such as legumes (Cullis & Kunert, 2017).

GENOMICS-ASSISTED IMPROVEMENT FOR ABIOTIC STRESS TOLERANCE

Abiotic stresses, such as temperature extremes, drought, and salinity, are known to limit crop productivity. This limitation in productivity poses a huge challenge in efforts to eradicate world hunger and is expected to worsen due to current and future global climate change scenarios (Boyer, 1982). Hence, it is imperative to develop new crop varieties with enhanced abiotic stress tolerance. Crops have evolved to adapt to different environmental conditions using their inherent genetic ability. However, this adaptation has happened over thousands of years. The rapidly evolving climatic conditions due to global climate change require faster optimization of plant features and performance to meet the current demands. Classical breeding methods, which rely on combining diversity from different germplasms for the selection of desired traits are a time-consuming process and cannot meet the demands under rapidly evolving climatic conditions. Further, breeding and genetic engineering approaches to enhance abiotic stress tolerance have met with limited success due to several reasons such as (i) complexity of traits (Pareek et al., 2010), (ii) complexity of stress response mechanisms in plants (Suzuki et al., 2014), and (iii) lack of efficient selection techniques (Gilliham et al., 2017). Advances in modern biotechnology, coupled with the availability of complete genomes of many crop plants, could supplement and speed up breeding programs, and the use of speed breeding tools could fast-track the development of new improved varieties in shorter time (Chiurugwi et al., 2019). For example, the speed breeding protocol was standardized for chickpea that enabled rapid generation advancement to produce up to seven generations per year (Samineni et al., 2020). Similarly, in the case of grain amaranth, up to six generations per year could be achieved by optimizing crossing methods and growth conditions (Stetter et al., 2016). Nevertheless, the establishment of speed breeding facility requires controlled environmental conditions, which need to be optimized for each crop. Moreover, maintaining the required temperature, and light duration and quality throughout the crop growth season is costly, which may be a major limiting factor for resource-poor countries (Samantara et al., 2022).

Genomic insights into the orphan crops can help to establish a correlation between gene expression and their role in abiotic stress tolerance. Hence, it can help in the identification of novel gene(s) or mechanism(s), which can be translated to major crops to improve their productivity under stress conditions. These genomics-assisted interventions, courtesy of orphan crops, can help both in boosting crop productivity as well as improving stress tolerance of crops. Thus, genomics has the potential to usher in a new era of the green revolution that would be driven by discovery, delivery, and targeted manipulation of desired traits (Bilichak et al., 2020).

Genomic approaches to investigate orphan crops can be divided into two broad categories: structural genomics and functional genomics. These approaches generate massive amounts of data, which needs to be investigated from different viewpoints to gain useful insights for crop improvement. Genes associated with desired traits, such as yield and stress tolerance, can be identified through these genomic datasets. Further, investigations into their expression patterns under different abiotic stresses and their specific role in stress adaptation can help to design effective genetic engineering strategies for enhanced abiotic stress tolerance. Hence, genomics can help to analyze the crops at the molecular level, and design stress-resilient crops by targeting specific gene(s).

ADVANCEMENT OF GENOMICS TO HARNESS GENETIC POTENTIAL OF ORPHAN CROPS

Genomic advancements offer a bird's eye view of the combinatorial working of genes. Focus has shifted from single-gene studies to whole-genome analysis. This is particularly useful to tackle abiotic stresses, which are known for their complexity. A plethora of genomic tools and techniques are available, which have made it possible to perform an in-depth analysis of the various physiological mechanisms behind abiotic stress tolerance of orphan crops (Figure 3). The importance of genomics for crop improvement has been very well explained as a “5G concept” which involves genome sequencing, germplasm characterization, gene identification and characterization, genomics-assisted breeding, and gene editing (Varshney et al., 2020). Further, it is now possible to translate the knowledge from one crop to another using advanced gene-editing tools, such as CRISPR/Cas (Gaba et al., 2021; Zafar et al., 2020). Employing CRISPR/Cas-based gene editing approach may be challenging in many orphan crops due to the non-availability of efficient stable genetic transformation and regeneration protocol. Several studies have reported the CRISPR/Cas-based gene editing in many orphan crops such as citrus, cassava, yam, watermelon, banana, kiwifruit, melon, sorghum, cowpea, green foxtail, and chickpea for the varietal improvement (reviewed by Venezia & Krainer, 2021). Therefore, CRISPR/Cas-based gene/genome editing is a powerful tool to bring orphan crops to mainstream agriculture by editing undesired traits, which may restrict their acceptance and commercial cultivation.

FIGURE 3. Schematic diagram depicting the way forward to utilize orphan crops for ensuring global food security. The orphan crops have the potential for gene discovery and allele mining for stress tolerance. Using molecular tools, major crops, such as rice and wheat, can be made more resilient to climate change. Also, trait improvement in orphan crops per se can contribute directly to food security.

Comparative genomics

Comparative genomics uses synteny or sequence conservation between different species to identify homologous or orthologous regions. Hence, it can be used to translate studies done in one species to another species. For crops without genome sequences, it can help to clone species-specific alleles based on their sequence conservation. Similarly, it can help to extrapolate functions based on sequence similarity. However, the prediction of gene functions merely based on sequence homology is not entirely accurate, and hence, other omics techniques need to be used in conjunction to make accurate predictions. Further, the extrapolation of model species data for orphan crops has its potentials and limitations (Armstead et al., 2009). For instance, candidate genes in the unsequenced regions of ryegrass (Lolium perenne and multiflorum) were identified by linking these to the rice genome through in silico mapping of ryegrass marker DNA sequences (Armstead et al., 2009). However, the success of this approach also depends on other factors, such as the degree of synteny between the target species and the annotation of the model genomes. Structural studies between homologous genes from two different species can help to pinpoint regions responsible for functional dissimilarity. Homologous plant disease-resistance genes (R-genes) were mapped in both Arabidopsis thaliana and Brassica napus to identify candidate R-genes (Sillito et al., 2000). Similarly, efforts were made for the identification of abiotic stress-responsive genes in chickpea (Cicer arietinum) based on sequence similarity using known abiotic stress tolerance-related genes (Roorkiwal & Sharma, 2012). Cannarozzi et al. (2014) sequenced the genome E. tef for marker-assisted breeding and delineating the mechanisms conferring high nutritional and agronomic properties. Further, the authors suggested that genomic approach could be used to identify target traits and genes for genetic improvement of this orphan crop. Conversely, a de novo browser has been developed to analyze the next-generation sequencing data, which can help to expedite search and identification of candidate genes/transcripts and design specific and degenerate primers for gene expression analysis in desired orphan crop (Kamei et al., 2016).

Transcriptome analysis

Translating gene sequences into their function is a major challenge. The exponential increase in genomic datasets necessitates additional efforts to decipher these datasets to make functional sense. Transcriptomics contributes to predicting gene function by correlating expression with a particular biological condition. A global transcriptome study under particular conditions can help in the identification of key regulatory genes and pathways, which can be further analyzed individually to identify their functional role with accuracy. De novo transcriptome analysis of foxtail millet (Setaria italica) led to the identification of putative proteins, many of which were successfully annotated to reveal numerous potentially novel proteins (Jo et al., 2016). Transcriptome analysis of chickpea (Cicer arietinum) using the RNA-seq approach was performed to identify drought-responsive genes and gene-based molecular markers (Hiremath et al., 2011). Subsequently, a comparative transcriptome analysis of drought-tolerant (ICC 4958), drought-sensitive (ICC 1882), salinity-tolerant (JG 62), and salinity-sensitive (ICCV 2) chickpea genotypes revealed transcriptome dynamics and genes involved in drought and salinity stress response in chickpea (Garg et al., 2016). Similarly, a comparative transcriptome analysis of drought-tolerant (M-249) and drought-sensitive (M-191) horse gram genotypes identified drought-responsive genes and gene-based SSR markers (Bhardwaj et al., 2013). The first genomic resource for Capparis spinosa, generated by transcriptomic approach and de novo assembly was used to identify gene homologs associated with different abiotic stresses, metabolic pathways, lipid metabolism, and genes involved in stomatal development. Moreover, this transcriptome study was also used to uncover polymorphic SSR markers (Mercati et al., 2019).

Genotyping and marker-assisted breeding

The exponential drop in sequencing costs has enabled the resequencing of different accessions of the same species to identify genotypic variations. This has led to the identification of genome-wide molecular markers, including candidate genes, predictive markers, and functional markers, which can make genomics-assisted molecular breeding a possibility. Besides, it has been possible to create comprehensive genetic maps of marker segregation data on mapping populations. Genetic diversity is an indicator of gene pool richness and is the greatest resource for plant breeders for the selection of lines with the potential to enhance the quantity and quality of food (Dempewolf et al., 2017; Ye & Fan, 2021). Plant accessions stored in different gene banks around the world play a critical role in maintaining this genetic diversity. Different accessions of cowpea were evaluated by the Kenyan national gene bank for a study (Wamalwa et al., 2016). Variations were observed between the different accessions, and hence, they were clustered into two major groups, which can be exploited for the improvement of cowpea (Wamalwa et al., 2016). Recently, a Diversity Arrays Technology (DArT) is being used for genetic characterization and molecular marker development in orphan crops, and in combination with next-generation sequencing, this approach could ensure rapid SNP discovery in various orphan crops such as groundnut, finger millet, lupin, grass pea, etc. (Kamenya et al., 2021).

Molecular markers are a great resource for plant breeders to enhance the precision of the breeding process. Molecular markers obtained from different genomic and transcriptomic studies, such as simple sequence repeats (SSR) and single nucleotide polymorphism (SNP), help in trait dissection and enhance the efficiency of functional gene selection. Cowpea (Vigna unguiculata) is an important orphan crop, which is often called “poor man's meat” due to its high protein content. It is drought tolerant and hence, can contribute majorly to ensuring food security in the future, under limiting water resources. Many studies have been conducted over the years to access the genetic diversity of cowpea in different parts of the world. Different accessions of cowpea from different geographical regions of Sudan, Ethiopia, and Nigeria were evaluated using molecular markers, such as SSR (Ali et al., 2015; Desalegne et al., 2016; Igwe et al., 2017). Similarly, the genetic diversity of the common bean (Phaseolus vulgaris) was also evaluated using molecular markers (Dennis et al., 2014; Tshilenge-Lukanda et al., 2012). Wang et al. (2021) sequenced the white fonio (Digitaria exilis) genome with long-read SMRT-cell technology to reveal extensive genetic diversity and identification of several genes associated with traits such as shattering, plant height, and grain size, which can empower rapid domestication of fonio. These studies helped to gauge the genetic diversity within the local and regional populations, which can be utilized for the improvement of various orphan crops (Ye & Fan, 2021).

Genome-wide association studies (GWAS) can help to reveal the relationships between molecular markers and QTLs using linkage disequilibrium. The availability of comprehensive maps of genomic variability allows GWAS of complex traits and functional investigations of the evolutionary aspect of crops (Huang & Han, 2014). In barley (Hordeum vulgare), GWA mapping revealed high levels of linkage disequilibrium within and between chromosomes, and yet detected common alleles of high penetrance (Cockram et al., 2010). A combination of GWA mapping with a comparative analysis was utilized to resolve traits and identify the polymorphism level in unsequenced genomes (Cockram et al., 2010). A new method, XP-GWAS (extreme-phenotype GWAS), relies on genotyping pool of individuals from diversity panels having extreme phenotypes, and thus, does not require genotyping of a large number of individuals (Yang et al., 2015). GWAS has become a routine strategy to decode genotype–phenotype associations in crops and allows unparalleled opportunities to probe functional genomics (Liu & Yan, 2019). Thus, it is considered as one of the best tools to investigate complex traits caused by a single or a combination of stresses. Indeed, many novel candidate genes or QTLs responsible for abiotic stress tolerance have been identified using GWAS (Challa & Neelapu, 2018). A high-quality genomic resource was established by sequencing 183 cultivated and wild fonio (D. exilis), which revealed wide genetic diversity, and population structure was shaped up by climatic, geographic, and ethnolinguistic factors (Abrouk et al., 2020). Moreover, the authors explained that unlike the major cereal crops, selection of fonio was majorly confined to two genes associated with seed size and shattering, hence, other traits can be utilized for the improvement of this crop in hot and dry environments.

Genetic engineering and genome editing

Agricultural sciences have witnessed rapid development in improving diverse set of traits due to the advent of recombinant DNA technology. It is now possible to design and control the expression of almost any desirable trait or character in the plant system. Coupled with efficient transformation techniques, this technology has revolutionized the field of agriculture. Many genetically modified (GM) crops have been commercialized with improved pesticide and herbicide resistance, while crops with improved abiotic stress tolerance are in the pipeline (Singh et al., 2022). Rapid advances in genome-editing techniques, especially CRISPR/Cas, have made the manipulation of genomes very affordable. Targeted genome editing has emerged as an alternative to classical plant breeding and transgenics (GM). CRISPR/Cas9 was used to successfully mutate genes in groundcherry (Physalis pruinosa) that control plant architecture, flower reproduction, and fruit size, and hence, improved major productivity traits (Lemmon et al., 2018). Enhanced drought tolerance was introduced in corn and soybean using CRISPR/Cas, which was indistinguishable from the naturally occurring variation (Chilcoat et al., 2017). Moreover, several stress resistance genes well documented in major cereal crops such as rice, wheat, and maize, for which orthologs can be identified in orphan crops such as millets can be manipulated using genome editing (CRISPR/Cas9) to improve stress resilience and productivity of orphan crops in diverse environments (Numan et al., 2021). Further, these easy-to-use and robust gene-editing tools, coupled with the availability of annotated genome sequences of many crops, will enable the discovery of novel gene functions through reverse genetics (Char & Yang, 2020).

CONCLUSIONS

Abiotic stresses pose a significant challenge in ensuring global food security and are expected to worsen with the adverse climate conditions. Deciphering the complexities of abiotic stress tolerance mechanisms is a huge task and requires good model crops for investigation. Advancements in genomic technologies have made it possible to sequence and analyze a large number of orphan crops, which are known for their abiotic stress tolerance potential. Various global and national initiatives have generated large genomic datasets, which have provided useful insights into the mechanistic understanding of abiotic stress tolerance. Translation of the knowledge gained from orphan crops to some of the major crops, such as wheat, rice, and maize, is now possible due to advanced genomics-assisted breeding techniques, as well as gene-editing techniques like CRISPR/Cas. Moreover, the development of advanced high-throughput phenomics platforms has facilitated the characterization of large germplasm and mapping populations for association mapping (GWAS). Using a combination of these techniques, it is quite possible to develop new, improved varieties of both orphan and major crops, which would be superior in productivity as well as possess abiotic stress tolerance. Eventually, it will aid in ensuring global food security in this era of global climate change.

ACKNOWLEDGMENT

S.L.S.-P acknowledge ICGEB for core grant support.

FUNDING INFORMATION

No funding was received to support this research.

CONFLICT OF INTEREST

The authors have stated explicitly that there are no conflicts of interest in connection with this article.

DATA AVAILABILITY STATEMENT

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.