How Could the Use of Crop Wild Relatives in Breeding Increase the Adaptation of Crops to Marginal Environments?

Abstract

Alongside the use of fertilizer and chemical control of weeds, pests, and diseases modern breeding has been very successful in generating cultivars that have increased agricultural production several fold in favorable environments. These typically homogeneous cultivars (either homozygous inbreds or hybrids derived from inbred parents) are bred under optimal field conditions and perform well when there is sufficient water and nutrients. However, such optimal conditions are rare globally; indeed, a large proportion of arable land could be considered marginal for agricultural production. Marginal agricultural land typically has poor fertility and/or shallow soil depth, is subject to soil erosion, and often occurs in semi-arid or saline environments. Moreover, these marginal environments are expected to expand with ongoing climate change and progressive degradation of soil and water resources globally. Crop wild relatives (CWRs), most often used in breeding as sources of biotic resistance, often also possess traits adapting them to marginal environments. Wild progenitors have been selected over the course of their evolutionary history to maintain their fitness under a diverse range of stresses. Conversely, modern breeding for broad adaptation has reduced genetic diversity and increased genetic vulnerability to biotic and abiotic challenges. There is potential to exploit genetic heterogeneity, as opposed to genetic uniformity, in breeding for the utilization of marginal lands. This review discusses the adaptive traits that could improve the performance of cultivars in marginal environments and breeding strategies to deploy them.

Full text

fpls-13-886162 November 30, 2022 Time: 12:6 # 1
REVIEW
published: 16 June 2022
doi: 10.3389/fpls.2022.886162
Edited by:
Diego Rubiales,
Institute for Sustainable Agriculture
(CSIC), Spain
Reviewed by:
Michael Benjamin Kantar,
University of Hawai‘i, United States
Harsh Raman,
New South Wales Department
of Primary Industries, Australia
*Correspondence:
Juan Pablo Renzi
renzipugni.juan@inta.gob.ar
Petr Smýkal
petr.smykal@upol.cz
Jan Brus
jan.brus@upol.cz
Specialty section:
This article was submitted to
Plant Breeding,
a section of the journal
Frontiers in Plant Science
Received: 28 February 2022
Accepted: 11 May 2022
Published: 16 June 2022
Citation:
Renzi JP, Coyne CJ, Berger J,
von Wettberg E, Nelson M, Ureta S,
Hernández F, Smýkal P and Brus J
(2022) How Could the Use of Crop
Wild Relatives in Breeding Increase
the Adaptation of Crops to Marginal
Environments?
Front. Plant Sci. 13:886162.
doi: 10.3389/fpls.2022.886162
How Could the Use of Crop Wild
Relatives in Breeding Increase the
Adaptation of Crops to Marginal
Environments?
Juan Pablo Renzi1,2*, Clarice J. Coyne3, Jens Berger4, Eric von Wettberg5,6,
Matthew Nelson4,7 , Soledad Ureta2, Fernando Hernández2, Petr Smýkal8*and
Jan Brus9*
1Instituto Nacional de Tecnología Agropecuaria, Hilario Ascasubi, Argentina, 2CERZOS, Departamento de Agronomía,
Universidad Nacional del Sur (CONICET), Bahía Blanca, Argentina, 3USDA Agricultural Research Service, Pullman, WA,
United States, 4Agriculture and Food, Commonwealth Scientific and Industrial Research Organisation, Wembley, WA,
Australia, 5Department of Plant and Soil Science, Gund Institute for Environment, University of Vermont, Burlington, VT,
United States, 6Department of Applied Mathematics, Peter the Great St. Petersburg Polytechnic University, Saint
Petersburg, Russia, 7The UWA Institute of Agriculture, University of Western Australia, Crawley, WA, Australia, 8Department
of Botany, Faculty of Science, Palacký University, Olomouc, Czechia, 9Department of Geoinformatics, Faculty of Sciences,
Palacký University, Olomouc, Czechia
Alongside the use of fertilizer and chemical control of weeds, pests, and diseases
modern breeding has been very successful in generating cultivars that have
increased agricultural production several fold in favorable environments. These typically
homogeneous cultivars (either homozygous inbreds or hybrids derived from inbred
parents) are bred under optimal field conditions and perform well when there is sufficient
water and nutrients. However, such optimal conditions are rare globally; indeed, a
large proportion of arable land could be considered marginal for agricultural production.
Marginal agricultural land typically has poor fertility and/or shallow soil depth, is subject
to soil erosion, and often occurs in semi-arid or saline environments. Moreover, these
marginal environments are expected to expand with ongoing climate change and
progressive degradation of soil and water resources globally. Crop wild relatives (CWRs),
most often used in breeding as sources of biotic resistance, often also possess traits
adapting them to marginal environments. Wild progenitors have been selected over
the course of their evolutionary history to maintain their fitness under a diverse range
of stresses. Conversely, modern breeding for broad adaptation has reduced genetic
diversity and increased genetic vulnerability to biotic and abiotic challenges. There is
potential to exploit genetic heterogeneity, as opposed to genetic uniformity, in breeding
for the utilization of marginal lands. This review discusses the adaptive traits that could
improve the performance of cultivars in marginal environments and breeding strategies
to deploy them.
Keywords: abiotic stress, adaptation, breeding, crop wild relatives, legumes, marginal environment
INTRODUCTION
When coupled with the use of irrigation, fertilizers and pesticides, modern breeding has increased
agricultural production several fold in favorable environments (Evenson and Gollin, 2003).
However, such high-input agriculture also has a high environmental impact. High performing
Frontiers in Plant Science | www.frontiersin.org 1June 2022 | Volume 13 | Article 886162

fpls-13-886162 November 30, 2022 Time: 12:6 # 2
Renzi et al. CWR Breeding for Marginal Lands
cultivars are designed under good field conditions and perform
well when there is sufficient water and nutrients, e.g., fertile soil.
However, such conditions are often not available because there
is a large proportion of arable land in marginal environments
globally (Sebby, 2010;Pingali, 2012). Marginal land exists
worldwide (e.g., Kang et al., 2013), and typically, it is of
poor soil fertility. In many areas these soils are prone to
erosion, with shallow soil layers, and may be prone to
drought, salinity, or other abiotic challenges. Moreover, these
environments are expected to expand with ongoing climate
change and increasing degradation of soil and water resources
(El-Beltagy and Madkour, 2012).
Throughout evolutionary history, plants have had to
adapt to a range of environmental conditions including
adverse habitats. As a consequence, plant species including
crop wild relatives typically exhibit a high level of genetic
and phenotypic variation within their distribution range
(Anderson et al., 2011;Russell et al., 2014;Smýkal et al., 2017;
Hellwig et al., 2021). Understanding the genetic, biological
and ecological bases of local adaptation is relevant to
manage the impact of climate change on crop and animal
production. In widely distributed plant species inhabiting
different environments, ecotypic differentiation has been
detected for populations along altitudinal, latitudinal or
environmental gradients (e.g., Polechová and Barton, 2015;
Berger et al., 2017). Understanding the extent to which such
distinct ecotypes are formed will help not only to reveal and
recognize their evolutionary adaptive patterns, but also will help
breeders to use their diversity to maximize productivity under
target environments.
It is widely accepted that past domestication processes have
resulted in a narrower genetic base of cultivated germplasm
that is prone to pests and diseases (Tanksley and McCouch,
1997;McCouch et al., 2020;Khoury et al., 2022). Unlike their
domesticated crops, crop wild relatives (CWRs) continued to
evolve to adapt to their environments and thus provide a
key resource to counteract the effects of climate change on
the world’s food supply. To fully understand and utilize this
process, studies in the geographical centers of origin are needed
combining ecology, physiology and genetics. This approach
was pioneered by N.I. Vavilov in the 1920s–1930s starting
with surveys, collecting missions and subsequent evaluation
of germplasm collections in diverse environmental settings
(Vavilov, 1957). Today, with the available tools of genomics,
geospatial analysis and modeling combined with improved
phenotyping methods, it has become all the more important
to analyze wild accessions and use them as sources of stress
tolerance for breeding purposes (Warschefsky et al., 2014;
Coyne et al., 2020). Wild species have been used mainly
for the introgression of disease and insect resistance into
crops (e.g., Hajjar and Hodgkin, 2007); drought, cold, heat
and salinity tolerance have also been addressed in some
staple crops (Prohens et al., 2017). Biotic and abiotic stress
resistances have been explored in wild relatives of many
crops including chickpea, barley and maize (Brozynska et al.,
2016;Fustier et al., 2017;von Wettberg et al., 2018;Liu
et al., 2020). A complementary approach is the de novo
domestication of new crops (Smýkal et al., 2018;Fernie and Yan,
2019).
Environmental fluctuations such as soil salinity, cold and
drought stress represent a major constraint on agricultural
productivity. Water availability and temperature extremes are
the major factors controlling the distribution of vegetation
over the earth’s surface. Crop yields are more dependent
on an adequate supply of water than on any other single
factor; environmental stress represents the primary cause of
crop losses. With current climate change projections, extremely
hot weather will become more frequent and rainfall will be
more erratic in many regions of the world (Foyer et al.,
2016). As many elite cultivars tend to be drought sensitive,
ensuring food security will require development of more
drought resistant varieties. Dissection of adaptation mechanisms
to harsh conditions naturally occurring in wild relatives of
domesticated crops may provide a solid foundation for crop
improvement under the changing climate. Drought and heat
stresses impact every plant developmental stage, starting from
seed germination, through vegetative growth. However, the
most adverse effects (from a crop production perspective)
are on crop establishment (germination and young seedling
phase) and reproduction, which are very sensitive to suboptimal
conditions. Small seedlings and pollen grains happen to
be particularly sensitive to lack of water and excess heat
(Dürr et al., 2015;Yu et al., 2019;Chaturvedi et al., 2021;
Chen et al., 2021).
MARGINAL ENVIRONMENTS AND THE
IMPACT OF CLIMATE CHANGE
The term "marginal" land originated in the field of agricultural
economics during the 19th century (Kang et al., 2013) by
Ricardo (2005) using the categorization from his land rent
theory which was the basis of marginal productivity theory.
Marginal lands are subject to socio-economic and biophysical
constraints, and lower productivity. Nevertheless, it is difficult
to define "productivity" within this concept because this
varies depending on land use. For example, land that is too
marginal for crop production might be suitable for grazing,
and "fragile" land can be susceptible to soil degradation, but
it may still be suitable for sustainable forestry. Many dry-
land countries have not assessed the extent or characteristics
of these lands, nor their sustainability as biofuels or food
crops production. These lands are often difficult to exploit
economically and sustainably for agriculture, and can lead
to abandonment (Campbell et al., 2008). Land can also be
degraded due to an intensive and unsustainable use (Campbell
et al., 2008;Dauber et al., 2010). Marginal lands are areas
with low rainfall, high temperatures, low quality soils, steep
terrain, shallow soil depth (less than 50 cm), poor fertility,
coarse texture, stony, heavy cracked clays, salt-affected lands,
waterlogged, marshy land, barren and rocky soils, and other
problems. If a land is considered marginal it means that it
does not have enough capacity to produce food, or other
agricultural activities.
Frontiers in Plant Science | www.frontiersin.org 2June 2022 | Volume 13 | Article 886162

fpls-13-886162 November 30, 2022 Time: 12:6 # 3
Renzi et al. CWR Breeding for Marginal Lands
FIGURE 1 | Map shows global land system archetypes classification by Václavík et al. (2013). Within the global scale, marginal lands in the developed world cover
about 9% of terrestrial ecosystems.
Nearly one billion people live in poverty today, a high
percentage living in these marginal lands in mostly developing
countries (Ahmadzai et al., 2021). These areas with low
soil fertility and limited agricultural value are also highly
vulnerable to climate change (Castro and Castro, 2019). The
research concluded that in total 29% of the agricultural area
is marginal in European Union. The most common reason
is the rooting limitations; 12% of Europe’s agricultural area
required intervention to improve soil structure and depth
to allow farming. This is followed by adverse climate and
excessive soil moisture occurring in respectively 11 and
8% of the agricultural land (Elbersen et al., 2018). Within
the global scale marginal lands in developed world covers
about 9% of terrestrial ecosystems (Václavík et al., 2013).
These lands occur predominantly in Western United States,
Australia, Argentina, but also in North and South Africa
(Figure 1). A more precise estimation of area covered by
marginal lands is limited by uncertainties and problems
of land cover classification (Brus et al., 2018). Marginal
lands are most vulnerable to climate events like drought
and floods, thus, most likely to be affected by climate
change. Climate change has made it difficult to maintain
established farming practices in recent decades. Within
the US the prevalence of local yield deficits confirms that
individual farmers looking to expand their operations are
generally confined to cultivating increasingly marginal land,
though widespread variation exists from location to location
(Lark et al., 2020).
The effects of climate change are manifested as erratic
precipitation and weather changes, which have led to rapid
fluctuations and unpredictable temperatures. Adaptation to this
requires new approaches to agriculture. For example, research
on agro-climatic conditions has shown that farmers in dryland
areas are more aware of climate change impacts than those
in wetland areas. Similar findings have also been reported
for Ecuador (Skarbø and VanderMolen, 2014). It is unclear,
however, if agricultural producers can keep up with climate
change at the incredible pace it is predicted to occur in
coming years (Jones et al., 2012). Adaptation to climate change
is difficult and impacts are based on various assumptions.
In addition to estimating future impacts, researchers need to
make subjective value judgments. This is especially true of
social and economic development in marginal regions. As
a result, the effects of climate change on these areas are
difficult to forecast. Although the impacts of climate change
are not always measurable, they are still a huge concern
for governments and local communities alike. Some areas of
the world are particularly vulnerable, and these risks can be
mitigated by developing new approaches and technologies in
crop production. One such approach is the implementation of
Frontiers in Plant Science | www.frontiersin.org 3June 2022 | Volume 13 | Article 886162

fpls-13-886162 November 30, 2022 Time: 12:6 # 4
Renzi et al. CWR Breeding for Marginal Lands
local and regional adaptation plans. However, it is crucial to
consider the broader context of marginal environments when
developing policies and adaptation strategies for agricultural
producers. This research is particularly important in Latin
America, where smallholder farmers are heavily impacted by
climate change. Moreover, rural and subsistence farming are
often location-specific systems, which are highly vulnerable
to the stressors of the climate. As a result, the research
needs to take this into account. Agro-ecosystems are critical
for food production and sustainability, but they are also
prone to pests. Consequently, many current crop varieties will
have to be replaced as a result of climate change. Failure
to address these challenges will have a profound impact on
the global economy and on social well-being. If we fail
to protect and restore natural ecosystems, it will become
impossible to grow crops in these marginal areas in the
future. One of the possible way how to cope with this
situation is implementation of Climate-Smart Agriculture (CSA)
(Lipper et al., 2014).
CONTEXT: HOW AND WHY DO CROPS
AND THEIR WILD RELATIVES DIFFER?
Crop wild relatives typically harbor far greater genetic
diversity than modern elite crops as a result of primary and
secondary domestication bottlenecks; the founder effect(s)
and subsequent fixation of domestication syndrome and
post-domestication traits including cultural selection for
local preference (Tanksley and McCouch, 1997;Purugganan
and Fuller, 2009). Moreover, the evolutionary trajectory
and resultant selection pressures encountered by wild
progenitors and elite cultivars are very different. While
we can now deploy an impressive array of technical and
analytical approaches for handling complexity in the search
for adaptive genes (Cortes and Lopez-Hernandez, 2021),
here we argue that understanding the divergent selection
pressures encountered by wild progenitor and elite cultivar
adds invaluable context for the use of CWR in improving
crop adaptation.
Wild progenitors are subject to natural selection in response
to environmental stressors, herbivory, pathogen attack and
inter-plant competition from the same or other species
occupying similar ecological niches in a variable climate.
While viable seed production is the ultimate measure of
‘fitness,’ reproductive investment in CWR is usually lower
than in their domesticated counterparts (Berger et al., 2017;
Berger J. et al., 2020;Garibaldi et al., 2021). This implies
that competition for resources such as light, nutrients or
water is selecting for greater vegetative investment (roots,
shoots, leaves) in CWR relative to the domesticated crop,
akin to the competitors in Grime’s triangle (Grime, 1974). In
some examples the genomic consequences of this divergent
selection for reproductive versus vegetative investment have
been identified, such as the selection for increased apical
dominance (e.g., reduced branching) in the domestication of
maize (Clark et al., 2004). Seed dormancy, a key domestication
syndrome trait, can both exacerbate and ameliorate seasonal
variability in CWR. In many Mediterranean CWR seed physical
dormancy has been selected as bet-hedging against false breaks
(heavy rainfall that precedes the growing season start and
which would expose the recently germinated seedling to
extreme heat and drought) and other disturbances such as
catastrophic herbivory (Piano et al., 1996;Norman et al.,
2002;Smýkal et al., 2014;Berger et al., 2017). Typically,
there is wide ranging dormancy within populations (Berger
et al., 2017;Hradilová et al., 2019;Renzi et al., 2019),
that in combination with variable seed placement will drive
staggered germination across the CWR population over the
growing season, exposing individuals to different micro-habitats
in space and time.
Conversely, crop plants are subject to a combination of
natural and artificial selection giving rise to the domestication
syndrome (large seed size, loss of dormancy, and seed dehiscence
etc.) and subsequent selection for local adaptation and food
preferences (Fuller and Allaby, 2009;Purugganan and Fuller,
2009;Smýkal et al., 2018). Compared to CWR, modern crops
lead a strictly regulated life in a tightly defined growing
season. Non-dormant seeds are accurately positioned with
respect to soil depth and neighbor distance in a prepared
seedbed during an optimal window, matching phenology to
target environment to minimize frost, drought, and other stress.
Ripening is both uniform and timely because of the use of
determinate cultivars (e.g., cereals), selection for earliness (e.g.,
chickpea, lupin, canola), or the application of desiccants (e.g.,
canola, chickpea). Herbivory and diseases are minimized by
management interventions. Importantly, crops are selected on
the basis of communal, rather than individual yield, whereas
the opposite is the case for CWR. As a result, modern crops
tend to be weak-moderate competitors (Donald, 1981;Weiner
et al., 2017) with a greater reproductive investment than their
wild progenitors (Berger et al., 2017;Berger J. et al., 2020;
Garibaldi et al., 2021).
Finally, domestication and subsequent global dispersal further
widens the evolutionary dichotomy between crop and CWR
(Purugganan and Fuller, 2009). Chickpea represents an extreme
exemplar, having been domesticated from a very narrowly
distributed southeastern Anatolian wild progenitor with a
Mediterranean winter annual lifecycle into a global crop grown
on all continents except Antarctica. It has been hypothesized
to have returned to the Mediterranean as a spring-sown
late season crop after a 2000 years archeological gap (Abbo
et al., 2003). The much more recently domesticated narrow-
leafed lupin (L. angustifolius) and yellow lupin (L. luteus)
represent the opposite extreme. Here the wild progenitors
range widely around the Mediterranean basin (particularly
L. angustifolius), being domesticated in the 18th century as
spring-sown crops for limited sandy acid soil regions in
central and eastern Europe (Hondelmann, 1984), and then
returning to Mediterranean climates in the southern hemisphere
in the last 50 years (Gladstones, 1994). The combination of
divergent genetic diversity and evolutionary trajectory/selection
pressures leads to different adaptive traits in crops and
their wild relatives.
Frontiers in Plant Science | www.frontiersin.org 4June 2022 | Volume 13 | Article 886162

fpls-13-886162 November 30, 2022 Time: 12:6 # 5
Renzi et al. CWR Breeding for Marginal Lands
The regulation of phenology in Mediterranean crops and
wild relatives provides clear examples of divergent evolutionary
paths. For example, vernalization plays an important role
in regulating phenology in both wild Cicer and Lupinus,
presumably as a consequence of the cool-cold winters and
rapidly warming spring temperatures experienced in their
Mediterranean distribution range. Wild narrow-leafed and
yellow lupins are widespread species in which flowering time
and associated traits including water use, biomass production
and reproductive investment are strongly linked to rainfall
at origin (Berger and Ludwig, 2014;Berger et al., 2017;
Berger J. D. et al., 2020). Despite the importance of phenology in
ecotypic adaptation of wild lupins, there is very little variation
in vernalization response (Taylor et al., 2019). Similarly, most
wild Cicer species also appear to be consistently vernalization
responsive (Berger et al., 2005), suggesting that other regulatory
mechanisms such as photoperiod or temperature responses or
earliness per se are responsible for the phenological differences
observed across wild germplasm collections. Indeed, wild
Cicer species appear to be extremely photoperiod responsive
(Sharma and Upadhyaya, 2019) and investigations are ongoing
to reveal interactions between all three phenology regulators
(vernalization, photoperiod, temperature) in order to understand
the regulation of flowering time and what CWRs might
bring to the crop.
Conversely, downregulation of the vernalization response
has played a key role in the domestication of chickpea
and both lupin species as these crops moved away from
their cool-cold winter Mediterranean origins. Selection for
earliness and high early vigor were key breeding criteria
in both lupin species in their domestication as spring-sown
crops because timely maturity in the cooling late central-
eastern European summer was essential to reliable seed
production (Hondelmann, 1984). When the crops moved to
relatively warm-winter Mediterranean climates in Australia,
where vernalization is a mostly unreliable regulator of phenology,
it was necessary to identify unresponsive mutants to start the
industry effectively (Gladstones and Hill, 1969). Now, both
Australian and European Lupin breeding is dominated by
the vernalization unresponsive FT alleles (Taylor et al., 2019,
2021). In chickpea vernalization was presumably downregulated
in the Bronze Age introduction of the crop to South Asia
and its hypothesized return as a Mediterranean spring-sown
crop (Abbo et al., 2002, 2003;Redden and Berger, 2007).
Unlike lupin, the much older chickpea crop has formed
distinct ecotypes with different flowering regulatory mechanisms
appropriate to each region (Berger et al., 2011). Thus, chickpea
landraces become increasingly temperature responsive from
the Mediterranean through north, central and southern India,
temperature response being strongly correlated to collection site
temperature during the vegetative phase (Berger et al., 2011).
Among Eastern Mediterranean germplasm photoperiod and
temperature response were negatively correlated, a relationship
that is likely to have been essential in the colonization of warmer
climates in South Asia where a strong photoperiod responsive
is maladaptive, particularly in Southern India where chickpea
flowers under declining day length.
Divergence in adaptive strategies between crops and wild
relatives provides opportunities to modify crop adaptation to
new and marginal environments, while understanding which
traits are selected for what regions adds useful context to the
problem. For example, genes from chickpea wild relatives would
be invaluable in the creation of a winter cultivar because these
appear to have been lost in evolution of the crop. Indeed,
chickpea wild relatives are more winter hardy than domesticated
chickpea (Singh et al., 1990, 1994) and appear to have greater
reproductive chilling tolerance as well (Berger et al., 2012b).
Efforts are currently underway to improve cold tolerance in
chickpea using wild relatives. In narrow-leafed lupin the strong
selection for earliness has created a bottleneck which is limiting
yield potential in higher rainfall environments where longer
season cultivars will be better adapted (Berger et al., 2012a;
Chen et al., 2017). As a result, considerable effort is being
invested in the search for other regulatory levers with which to
delay flowering in lupin without re-introducing an obligatory
vernalization requirement (Taylor et al., 2019, 2021). Wild
germplasm is playing an important role in this. These examples
stand out because phenology has played such a central role
in the divergent evolution of crop and wild relative in both
chickpea and lupin.
When the differences between crop and wild relative are
more obscure, it becomes more difficult to generalize about the
adaptive potential that may be exploited from CWR. Drought
stress often occurs in marginal environments as described
earlier. Among annual plants the principal adaptation strategy
is stress avoidance through appropriate phenology, with some
very limited capacity for drought postponement through water
acquisition (e.g., deep roots) and conservative water use (e.g.,
stomatal closure), and very short-term tolerance through osmotic
adjustment (Ludlow and Muchow, 1990;Berger et al., 2016).
Given the widespread selection for earliness in domesticated
crops compared to their wild relatives, it is difficult to argue
that CWR will improve the capacity for drought avoidance in
our crops. However, CWR may be a useful source of drought
postponement or tolerance traits, given that these are likely to be
under selection in wild populations where individuals compete
amongst themselves and often also have long, indeterminate
lifecycles which include periods of transient drought stress
(Berger et al., 2016). Lysimetry has demonstrated that the wild
Cicer species, C. echinospermum and C. reticulatum extract more
water both under terminal drought and well-watered conditions
than does domestic chickpea (Berger J. et al., 2020). At this
stage the underlying mechanism is unknown. If the greater water
use extraction of wild Cicer is explained by greater vegetative
investment and associated lower water-use efficiency, it will be
maladaptive in the chickpea crop; if it represents a capacity to
tolerate lower water potential, however, it may be useful. Wild
L. luteus has a greater capacity to maintain leaf water content
at low water potential than the domesticated crop, probably
through osmotic adjustment (Berger and Ludwig, 2014;Berger
J. D. et al., 2020). Surprisingly this capacity was only found
in high, rather than low rainfall ecotypes and was suggested
as a bet-hedging strategy to postpone self-induced transient
drought caused by very high water-use of high rainfall ecotypes.
Frontiers in Plant Science | www.frontiersin.org 5June 2022 | Volume 13 | Article 886162

fpls-13-886162 November 30, 2022 Time: 12:6 # 6
Renzi et al. CWR Breeding for Marginal Lands
Currently, there are not enough ecophysiological studies of
annual adaptive strategies over contrasting climates to generalize
about the likelihood of finding these sorts of adaptive traits
among CWR. There has been no evidence for similar drought
stress tolerance on low and high rainfall ecotypes of wild
L. angustifolius (Berger J. D. et al., 2020), Conversely Yousfi
et al. (2010) detected greater osmotic adjustment capacity in
low compared to high rainfall ecotypes of Medicago truncatula
and M. laciniata. We hope that future germplasm screening
efforts used in crop improvement will address this shortcoming
by studying material from contrasting, well characterized
collection sites.
DESIRABLE CROP WILD RELATIVES
PHENOTYPIC TRAITS TO IMPROVE
CROP ADAPTATION TO MARGINAL
ENVIRONMENTS
Selection attributes according to the crop objective are related
to classical domestication traits, such as reduced seed and
fruit dispersal, changes in plant structure, plant phenology,
seed dormancy, palatability, or acquisition of modified fruit
size and shape in vegetable crops (Alonso-Blanco et al., 2009;
Sakuma et al., 2011;Meyer et al., 2012;Zohary et al., 2012;
Abbo et al., 2014;Fuller and Allaby, 2018;Ogutcen et al.,
2018;Smýkal et al., 2018;Iqbal et al., 2020). Much progress
has been made in the most widely grown and intensively
selected crops (rice, wheat, soybean, sugarcane, tomato and
potato), but improvement using CWR is more recently (Hajjar
and Hodgkin, 2007;Shelef et al., 2017;Zhang et al., 2018;
Zsögön et al., 2018;Fernie and Yan, 2019). CWRs provide
unique reservoirs of genetic diversity for crop improvement
(Cowling et al., 2009;Redden, 2013;Matesanz et al., 2020;Bohra
et al., 2021). They have typically been used in breeding to
introgress a small number of major genes (for insect/disease-
resistant varieties), in contrast to adaptation that is controlled by
many small-effect quantitative trait loci and complex interactions
which are more difficult to harness (Hajjar and Hodgkin, 2007;
Zhang et al., 2018;Zsögön et al., 2018). The introgression of
adaptive diversity from CWR to domesticated crops provides
breeders with new tools for crop improvement (Smýkal et al.,
2018). As environmental changes associated with domestication
should favor resource-acquisition strategies compared with the
resource-conservation strategies of wild relatives (Redden, 2013;
von Wettberg et al., 2014;Vilela and González-Paleo, 2015),
breeders are faced with the challenge of a trade-off between
productivity and adaptation. The development of a variety with
high adaptation (survival) under a marginal environment, but
very low productivity would not be desirable (Richards, 2006;
Berger et al., 2016).
Cultivars with tolerance to drought, heat, salinity, soils with
extreme pH (alkaline or acidic) and low fertility, as well as
various biotic factors are known to be required in marginal
environments (Redden, 2013;Quezada-Martinez et al., 2021).
Irrespective of the pre-breeding technique it is important to
understand the phenotypic traits that confer adaptation for
identification in any given marginal environment and for
agronomic management (Wilke and Snapp, 2008). Appropriate
phenotypic traits (morphological, anatomical, and phenological)
that are easily distinguishable and inexpensive to measure in
the field, suitable for pre-breeding programs in developing
countries, where marginal environments predominate, could be
considered. However, the breeding efficiency might be low (Araus
and Kefauver, 2018). On the contrary, adaptation to salinity
through the evaluation of osmoprotectants, reactive oxygen
scavengers (ROS), stress proteins and ion/proton transporters,
or the resistance to diseases-pests not strongly associated with
specific phenotypic traits, requires more complex screening
and modern techniques (Araújo et al., 2015). Since many
CWR can help stabilize and increase productivity in marginal
agroecosystems, the phenotypic traits that provide these and
other types of services of candidates for domestication should
be described. Table 1 presents some phenotypic traits that are
related to the plant responses to marginal environmental factors.
These traits can guide breeders who are seeking new germplasm
for domestication.
Note that some traits are not directly related to yield,
or even appear to be indirectly associated, but will favor
adaptation in a marginal environment in the long term (Duc
et al., 2015;Vilela and González-Paleo, 2015). For example,
it is known that the use of high reproductive biomass (high
harvest index) as a key selection criterion leads to increased
wheat yield (Reynolds et al., 2007). However, in marginal
environments in the northern Patagonian region of Argentina,
it may be preferable to select high plant height and late-
cycle wheat to cope with transient stress while maintaining
the yield ceiling (∼1–1.5 t ha−1). Tall crops could favor
competition with weeds, and the late-season genotype could
prolong the duration of root growth, allowing the utilization
of water and nutrients deeper in the soil profile (Richards,
2006). In addition, higher post-harvest residues with firmer
stubble could increase soil cover, favoring soil moisture retention
and avoid erosion processes that frequently occur in bare
soils (Silenzi et al., 2012). Often remnant vegetative biomass
is used as forage or for fuel. This is one of the reasons why
traditional local varieties are still used instead of modern ones
in this marginal environment (Cantamutto et al., 2016). In the
case of forage legumes, the simple selection of a major trait
(biomass) with four adaptive traits was useful for improving
a semi-domesticated species such as hairy vetch (Vicia villosa)
(Figure 2). The adaptive traits were late phenology, aboveground
morphology (high plant branching), dense leaf pubescence and
high seed dormancy. The long period of vegetative production
was associated with the ability to modify the growth and
flowering phases according to the occurrence of rainfall events.
Successful adoption by farmers has occurred with the cultivar
Patagonia INTA, with a high biomass and regrowth capacity
after grazing, tolerance to transient drought, resistance to cold,
and adequate natural reseeding (Renzi, 2020). The lesson is that
adaptive traits have to be considered with the target environment
and end user in mind; seed yield may not always be the
dominant criterion.
Frontiers in Plant Science | www.frontiersin.org 6June 2022 | Volume 13 | Article 886162

fpls-13-886162 November 30, 2022 Time: 12:6 # 7
Renzi et al. CWR Breeding for Marginal Lands
TABLE 1 | Linking phenotypic traits and adaptation strategies that could be used as pre-breeding criteria in a population improvement program in grain
and pasture crops.
Specific adaptation strategy Phenotypic trait of interest Target environment References
Drought escape. High seed size.
Rapid germination and growth rates
(high seedling vigor).
Thomson and Siddique, 1997;Berger et al., 2002,
2016;Richards, 2006;Reynolds et al., 2007;Sadras
and Richards, 2014;Araújo et al., 2015;Duc et al.,
2015;Gardarin et al., 2016
The life cycle is completed before a
severe water deficit develops.
Early flowering and physiological
maturity phenology.
Determinate growth habit.
High specific leaf area (SLA).
Stay-green.
Dry matter remobilization and high
reproductive investment (harvest index).
Lengthened seed-filling period.
Terminal drought (or
end-of-season drought)
Drought avoidance/postponement. Phenology adjustment (ability to modify
growth and flowering phases according
to occurrence of rainfall events).
Root:shoot ratio increase.
Transient stress (or
droughts occurring at
any time of the growing
season)
Berger et al., 2002, 2016;Richards, 2006;Reynolds
et al., 2007;Wilke and Snapp, 2008;Fernández et al.,
2012
When maximizing water uptake and/or
reduced soil water depletion.
Leaf area reduction.
Leaf thickness increase.
SLA*reduction.
Leaf senescence delay.
Leaf movement (paraheliotropic, rolling).
Leaf (canopy) temperature reduction.
Drought and temperature stress
tolerance.
Specific adaptation to water deficit
through organs or processes.
Dense leaf pubescence.
Smaller and thicker leaf.
High thick leaf cuticle.
High leaf wax.
Low SLA*.
Deep root.
High root:shoot ratio.
Green photosynthetic organs other than
leaf (stems on shrubs, beard in cereals).
Nyctinastic leaf movements.
Transient and terminal
drought
Sandquist and Ehleringer, 1997;Passioura, 2006;
Richards, 2006;Reynolds et al., 2007;De Andrés et al.,
2008;Wilke and Snapp, 2008;Araújo et al., 2015;Vilela
and González-Paleo, 2015;De la Rosa et al., 2020
Leaves highly reflective or orientated at
steep angles.
Weed competitiveness Low seed dormancy
Rapid growth rates.
High plant height.
High specific leaf area.
Dense leaf canopies.
Spread root systems.
High tillers/stems per plant.
High dry matter.
Mild, transient stress Hoad et al., 2006;Duc et al., 2015;Gardarin et al.,
2016
Winter survival Survival testing (at <0◦C)
Median lethal temperature (LT50 )
Regrowth ability after freezing
Pubescence
Freezing winter Badaruddin and Meyer, 2001;Brandsæter et al., 2002;
Wilke and Snapp, 2008;Araújo et al., 2015;Wiering
et al., 2018
Prevents germination in unfavorable
conditions
Seed dormancy
High seedling growth rate
Initial drought Loi et al., 2005;Norman et al., 2005;Gardarin et al.,
2016
Ability to recover from transient damage
(grazing, predation and biotic stress)
Hypogeal emergence.
Phenology plasticity (to extend the
growing season).
Secondary stem/shoot production.
Indeterminate growth habit.
High allocation to roots or storage
organs. Low HI or RE (reproductive
effort).
Deep-rooting.
Grassland – Pasture
crop
Wilke and Snapp, 2008;Annicchiarico et al., 2015;
Araújo et al., 2015;Duc et al., 2015;Renzi, 2020
*Specific leaf area (SLA) = leaf surface area (cm2)/leaf dry mass (g).
Frontiers in Plant Science | www.frontiersin.org 7June 2022 | Volume 13 | Article 886162

fpls-13-886162 November 30, 2022 Time: 12:6 # 8
Renzi et al. CWR Breeding for Marginal Lands
FIGURE 2 | Seed collection (A), plant breeding (B), multiplication (C), and agronomic use (D) of the improved cultivar of hairy vetch (Vicia villosa) cv. Patagonia INTA
as forage and cover crop.
A less common but promising approach is the domestication
of perennial grain crops. Traditional systems relying on annual
crops have substantial negative impacts on ecosystem functions
(i.e., nutrient cycling, water quality and carbon emissions,
salinity, soil erosion and degradation) (Tilman et al., 2011).
These problems can be reduced through the introduction of
perennial crops in some environments (González-Paleo and
Ravetta, 2015). This is illustrated by perennial Silphium oilseed
(Heliantheae), with ideotypes with desirable traits for improving
long-term yields and ecosystem services (Runck et al., 2014;
Van Tassel et al., 2017). Kernza, a large-grained variety of
intermediate wheatgrass (Thinopyrum intermedium) (DeHaan
et al., 2020), is similar. The introduction of greater diversity
in cropping systems through biotypes for mixed cultivation
(“ideomixes”), with high resource-use complementarity, is also
used as a novel approach by breeders (Litrico and Violle,
2015). For forage purposes, the domestication of shrub-tree
legumes for livestock production has the potential not only
to enhance productivity, but it could also provide multiple
environmental benefits, such as providing shade for cattle,
restoring degraded lands and mitigating greenhouse gas (GHG)
emissions (Muir et al., 2018). The concept of the traditional
ideotype in marginal environments needs to be discussed, and
the ecosystem services of these should be considered (Duc et al.,
2015). While introduction of perennial crops may not suit every
marginal environment (such as those that experience extended
hot and dry summer seasons), more research effort should be
directed toward exploring the potential fit of perennial crops to
different marginal environments.
Different drought scenarios (opportunity, extent, intensity
and frequency of drought) and heat-stress are the most common
and important stress factors in marginal lands (Chapman
et al., 2000;Reynolds et al., 2007;Berger et al., 2016;Sehgal
et al., 2018). This combination of changes in temperature
and rainfall patterns is driving the development of drought-
adapted crops. Table 1 details the different adaptive strategies
to drought and the associated phenotypic traits; however, the
integration of traits into an adaptive strategy that explains
how species or genotypes address drought stress should be
analyzed in a specific context that considers environmental
selection pressures.
The phenotypic traits between drought postponement and
tolerance may be overlapping in the same directions (co-
gradient variation). Plant adjustments under stress conditions
(plasticity) as opposed to tolerance as a constitutive trait of the
species independent of water and/or heat stress are considered.
Both the genetic differentiation in phenotypic traits and the
plasticity of those traits are complementary mechanisms
contributing to plant adaptation to environmental resource
fluctuation (Matesanz et al., 2020). Uniform-rich habitats
or consistently stressful environments (homogeneous and
predictable low-resource environments) promote genetic
differentiation rather than plasticity. On the contrary,
unpredictable environments can lead to increased phenotypic
Frontiers in Plant Science | www.frontiersin.org 8June 2022 | Volume 13 | Article 886162

fpls-13-886162 November 30, 2022 Time: 12:6 # 9
Renzi et al. CWR Breeding for Marginal Lands
plasticity (Vilela and González-Paleo, 2015;Espeland
et al., 2018). Thus, the plasticity could represent most of
the phenotype variation in some traits (phenology, leaf
morphology) when compared with the genetic contribution;
it has been suggested that it could be beneficial under climate
change scenarios (Nicotra et al., 2010). In order to detect
“plastic ideotypes” and exploit the plasticity in pre-breeding
programs, a wide range of environments in order to fit
models of phenotype versus environment should be included
(de Felipe and Alvarez Prado, 2021).
In this context, CWR show high genetic variation for
adaptation and may represent potential candidates for
introduction into marginal areas. In addition to adaptation
to different stress conditions, the ability to fix atmospheric
nitrogen (N) is important in legumes and should be considered
(Kebede, 2021). The percentage of nodulated plants and the
number of nodules per plant are associated with N fixation
in some species (Provorov and Tikhonovich, 2003). For
pre-breeding purposes, the qualitative plant vigor rating,
biomass, and the N measurements from non-destructive
samples collected during flowering or maturity can be used
(Muller et al., 2021). As the plant-associated microbes are
closely linked, abiotic stresses can affect either the host
plant, the Rhizobium, or the Rhizobium-legume symbioses,
decreasing the N-fixing activity (Zahran, 1999). Marginal
environments harbor and select diverse microbial communities,
and the isolation of effective rhizobia from wild legumes
for inoculating other legume crops could be an effective
strategy (Ayangbenro and Babalola, 2020). Screening for
tolerant N-fixing bacteria strains, as in other growth-
promoting (PGP) organisms, should be considered in the
selection of plant species/cultivar-specific microbial populations
(Coba de la Peña and Pueyo, 2011).
Stress tolerant wild plants, such as halophytes,
secretohalophytes, xerophytes and thermophytes, with a suite
of desired features (food, economical or ecological) represent
useful germplasm (Zhang et al., 2018), and the ecological niche
models can be used to predict the adaptation in a target marginal
environment (Phillips et al., 2006;Redden, 2013). Understanding
the adaptive strategy simplifies crop improvement by allowing
breeders to introgress new traits through traditional breeding
methods or modern selection techniques (Berger et al., 2016).
Likewise, knowledge of phenotypic traits in wild species would
estimate the probability of adaptation a priori, and the global
plant functional trait databases may be used as a screening tool
(Kattge et al., 2011;Saatkamp et al., 2019).
THE POTENTIAL USE OF
DEMOGRAPHIC MODELING FOR CROP
WILD RELATIVES WITH HIGH
NON-DOMESTICATION TRAITS, SUCH
AS LOW-COST MANAGEMENT
Domestication could result in the loss of potentially useful “wild
traits” that confer biotic and abiotic stress tolerance or specialized
resource-use strategies (Van Tassel et al., 2017;Nagel et al.,
2019). Pizza et al. (2021) showed that in Clarkia pulchella, after
the eighth generation of farm cultivation, the relative fitness
of the wild plants was significantly greater than the farmed
plants, especially under drought stress. Genetic diversity lost over
generations may be mitigated by growing out a large number
of unrelated individuals (Basey et al., 2015). On the other hand,
obtaining an ideal cultivar for a marginal environment can take
many years of breeding resulting in abandoned programs due
to low financial resources (Hajjar and Hodgkin, 2007;Muir
et al., 2014;Fernie and Yan, 2019). Demographic modeling,
especially in adapted pasture biotypes due to the predominance
of livestock systems in marginal environments, can be used to
advance their use in real production systems, until getting an
advanced cultivar.
Adapted biotypes, which are not necessarily domesticated,
can be very difficult to cultivate. Poor agronomic performance
introduces numerous practical issues for breeders, such as
difficulty in recovering a sufficient number of seeds and not
being able to use standard equipment (Nutt and Loi, 1999;
Nichols et al., 2012). For fodder production or restoration
purposes, these traits can be used for the dispersal of desirable
biotypes through demographic modeling. Dynamic population
models have proven to be very useful for understanding survival
and dispersal strategies of wild and semi-domesticated species.
This approach is widely documented for reducing survival in
weed species under different production systems (González-
Andujar and Fernández-Quintanilla, 1993, 2004;Forcella et al.,
2000;Colbach et al., 2005;Holst et al., 2007;Gardarin et al.,
2012), as well as promoting it in legume and grass pastures
(Taylor et al., 1991;Komatsuzaki, 2007;Thapa et al., 2011;
Renzi et al., 2017). These models, in their simplest form,
consist of five stages from seed production, seed dispersal
and input into soil seed banks, through germination and
seedling emergence to the reproductive phase (adult plants)
(Figure 3). The study of these stages and processes can be
the basis for identifying the causes of failure or success in
the establishment and performance of the selected biotypes
(Renzi et al., 2019).
The first requirement for this approach is to find the
biotype(s) adapted to the target marginal environments, with
acceptable forage yield and nutrition. Secondly, to reach a
successful commercial scale, it is important to have seed available
for farmers (Runck et al., 2020). To improve the harvestable
seed yield, less seed shedding after physiological maturity, is
a key objective for pre-breeding programs (Nichols et al.,
2012). When the number of hectares that can be planted
for cultivation I from a single hectare of seed production
(S) is lower than 20 (C/S <20), models that assist natural
reseeding could be a good option. This is the case of
Vicia villosa which is well-adapted to semi-arid temperate
environments, but incompletely domesticated due to extreme
indeterminate growth, non-uniform maturity, large seed losses
due to uneven pod dehiscence, and seed dormancy (Kissing
Kucek et al., 2020a,b;Renzi et al., 2020). The demographic
model was useful for expanding their adoption by farmers
in marginal lands in Argentina (Renzi et al., 2017, 2019).
Frontiers in Plant Science | www.frontiersin.org 9June 2022 | Volume 13 | Article 886162

fpls-13-886162 November 30, 2022 Time: 12:6 # 10
Renzi et al. CWR Breeding for Marginal Lands
Several options of legumes with self-regeneration potential
have been published (Carr et al., 2005;Loi et al., 2005, 2012;
Norman et al., 2005;Ovalle et al., 2005;Driouech et al.,
2008;Walsh et al., 2013). In a scenario of very low seed
production, with a C/S ratio <10, the natural seed dispersal
should be considered in addition to the field emergence model.
Under this premise, planting in patches or stripes (<<seed
doses per ha) in function of the dispersal ability together
with appropriate management will reduce the need for high
initial seed availability. This management could be carried
out in intercropping using biotypes with high resource-use
complementarity (Litrico and Violle, 2015;Mazzafera et al.,
2021). Thus, estimates of plant dispersal distances could be
useful not only for ecological studies, but also for agronomic
purposes. The dispersal traits of tall perennial pasture grasses
suggest the potential applicability of this modeling approach
(Thomson et al., 2011;Bullock et al., 2017). Nevertheless,
complementary studies of temporal and spatial demographic
dynamics are necessary.
PRE-BREEDING TECHNIQUES FOR
TRANSFERRING USEFUL TRAITS TO
THE CROP
Crop wild relatives are an untapped source of genetic diversity
and a critical resource to meet food security needs and the
challenges of new production systems, especially in response
to climate change (Zhang et al., 2017;Bohra et al., 2021).
However, three major factors discourage the use of CWRs in
plant breeding (Zamir, 2001): (1) poor agronomic performance
of CWRs, mostly due to the presence of undesirable traits such
as shattering and seed dormancy; (2) crossing barriers between
crops and their wild relatives, which limits the production
of fertile offspring; and (3) the transfer of undesirable traits
physically linked to the target trait, known as linkage drag
(Zamir, 2001;Hajjar and Hodgkin, 2007;Shelef et al., 2017;
Fernie and Yan, 2019). Therefore, to facilitate the use of CWRs in
breeding programs, it is necessary to focus on the development
of systematic strategies for the characterization and use of
CWRs. Pre-breeding here refers to all activities designed to
identify and transfer desirable traits or alleles from genetic
materials that cannot be used directly in plant breeding (Prohens
et al., 2017;Tefera, 2021). Pre-breeding produces intermediate
genetic materials that can be used for breeders to produce
elite cultivars. This section provides a review of distinct pre-
breeding strategies to facilitate the use of CWRs in breeding
programs, focused on the utilization of CWR for improving
the tolerance to stressful conditions associated to marginal
agricultural lands.
In the broad sense, strategies to incorporate CWR into
breeding programs can be classified as “choose first” or
“cross first” (Dempewolf et al., 2017). In “choose first” wild
accessions are chosen from a larger number of accessions,
based on phenotypic, genotypic, or eco-geographic merits.
Chosen accessions are expected to carry a desirable trait,
then they are used in a target crossing design. In “cross
FIGURE 3 | Conceptual model of the life cycle of an annual or perennial
pasture biotype. Boxes indicate state in the plant life cycle. Gray arrows
indicate the processes, and black arrows the agronomic traits of interest for
pre-breeding. Process variables: pre-d, predation pre-dispersal; post-d,
predation post-dispersal and seed rain losses; m, mortality of seeds in the
soil; e, germination and seedling emergence; s, seedlings survivorship; fs,
seed fecundity; and fv, vegetative reproduction.
first” one or more wild accessions are crossed with elite
cultivars, and then the progeny is evaluated, this approach is
useful when the trait of interest cannot be directly measured
in the wild donor.
Choose First Strategy: Target
Hybridization
Target hybridization is the classic example of the “choose first”
strategy. Generally, a single wild accession carrying a single trait
of interest (e.g., herbicide resistance, disease resistance) is used as
donor and one elite cultivar, with good agronomic performance
but lacking the trait of interest, is used as recurrent parent
(Breseghello and Coelho, 2013). Crossing and backcrossing with
positive selection for the target trait are used to incorporate
the target trait and to recover most of the recurrent parent
genome. This approach is routinely used in breeding programs
to improve tolerance to pests and diseases (Hajjar and Hodgkin,
2007;Larkan et al., 2013;Seiler et al., 2017;Bohra et al.,
2021). Using this approach, molecular markers can be used
to map the region underlying the trait of interest, and to
accelerate the recovery of the elite genome through assisted
backcrossing (Breseghello and Coelho, 2013). The first step of
target hybridization is to identify the suitable wild accession
carrying the trait of interest (Zamir, 2001;Bahrami et al.,
2021). As mentioned above, wild accessions can be selected
based on phenotypic, genotypic, or eco-geographic merits. For
Frontiers in Plant Science | www.frontiersin.org 10 June 2022 | Volume 13 | Article 886162

fpls-13-886162 November 30, 2022 Time: 12:6 # 11
Renzi et al. CWR Breeding for Marginal Lands
phenotypic selection, several wild accessions are directly screened
for the trait of interest along with elite cultivars; accessions
outperforming elite cultivars for the target trait are selected for
the next stage (Bahrami et al., 2021). Phenotypic screening has
been used to identify wild accessions with increased tolerance
to several stressful conditions associated to marginal agricultural
lands, such as salinity in wild tomato (Pailles et al., 2020),
freezing in wild sunflower (Hernández et al., 2020), drought
in wheat and barley (Nevo and Chen, 2010), heat in wild rice
(Scafaro et al., 2016;Bheemanahalli et al., 2017), wild sunflower
(Hernández et al., 2018, 2020), and wild barley (Bahrami et al.,
2021). Genotypic information can also be used to identify
valuable wild accessions. For example, Buitrago-Bitar et al. (2021)
identified wild accessions of tepary bean carrying novel alleles
for previously identified drought-responsive genes. With the
increasing amount of reported candidate genes for abiotic stress
tolerance, genes-specific hybridization probes can be designed
to target multiple candidate genes at once in many samples
before next generation sequencing (Weitemier et al., 2014), thus
accelerating the discovery of novel alleles of relevant genes.
Lastly, eco-geographic tools can be used to identify possible
sources of abiotic stress tolerance (e.g., Khazaei et al., 2013;
Kantar et al., 2015;Stenberg and Ortiz, 2021). Eco-geographic
approaches gather environmental variables from collection sites
such as temperature, precipitation, and soil characteristics. This
approach assumes that wild accessions from sites prone to a
given abiotic stress (e.g., drought, cold or heat) likely carry
adaptive alleles to cope with these environmental challenges
(Kantar et al., 2015;Anderson et al., 2016). Phenotypic,
genotypic, and eco-geographic approaches can be combined.
Landscape genomics is a powerful approach that combines
eco-geographic and genomic data to identify loci underlying
environmental adaptation (Joost et al., 2007). This approach does
not require phenotypic experiments, thus allowing the evaluation
in silico of hundreds to thousands of accessions. Several climatic
databased, including WorldClim (Fick and Hijmans, 2017),
Envirem (Title and Bemmels, 2018), and SoilTemp (Lembrechts
et al., 2021), along with genomic information can be used
to link geographic coordinates of any accession with local
environmental conditions and genomic data, increasing the
value of ex situ collections. Recent examples focused on wild
relatives of soybean (Anderson et al., 2016), wheat (Brunazzi
et al., 2018), narrow-leafed lupin (Mousavi-Derazmahalleh et al.,
2018), and chickpea (von Wettberg et al., 2018). Once the
suitable accession is identified, a crossing and backcrossing
design with an elite cultivar is initiated. As this approach
uses only one wild accession as donor, genome-wide increases
of genetic diversity are not expected. The advantage of this
approach is the efficient transfer of traits regulated by a few
genes, such as disease resistance (Seiler et al., 2017), and it
is well-known by breeders. However, it is time-consuming
and its success in traits with complex genetic architecture
is not guaranteed.
Cross First Strategy: Wide Hybridization
The cross first strategy consists of developing populations of
lines with introgressions from CWR into the genetic background
of crops, aiming to generate elite materials carrying genome
fragments from CWR (Tanksley and McCouch, 1997;Dempewolf
et al., 2017;Prohens et al., 2017). Such populations can be
used to identify genomic regions underlying adaptive traits,
but they can also be directly incorporated into breeding
programs (von Wettberg et al., 2018;El Haddad et al.,
2021). There are many ways for developing populations with
introgression from CWR; Chromosome Segment Substitution
Lines (CSSL), Nested Association Mapping (NAM) populations,
and Multiparent Advanced Generation Intercross (MAGIC)
populations. These populations are often developed in an
unfocused manner regarding target traits, i.e., the aim is
to generate populations with as high genetic diversity from
CWR as possible, thus populations are useful to map most
agronomic traits in many environments. However, multi parent
populations can be developed in a focused manner, selecting
parents with particular features, such as tolerance to biotic or
abiotic stress (reviewed in Prohens et al., 2017;Scott et al.,
2020).
Chromosome Segment Substitution
Lines
Chromosome Segment Substitution Lines were designed and
used for mapping QTL in many crops (Balakrishnan et al.,
2019). CSSL form a population of lines with chromosomal
segments from one wild accession into an elite background
(Balakrishnan et al., 2019). Ideally, each CSSL harbors a
single segment of the donor and the whole donor genome
is distributed segment-wise in the entire population of CSSL.
In pre-breeding, CSSL has been used in several crops, such
as rice, wheat, barley, maize, and soybean to identify and
transfer alleles from wild species, including alleles associated
to drought, heat, and freezing stress (reviewed in Balakrishnan
et al., 2019). By definition, with this approach a single wild
accession is used as donor and a single elite cultivar as
recurrent parent, although CSSL populations using the same
recurrent parent and distinct wild donors can be combined
(Singh et al., 2020).
Multiparent Populations
In the last decades, multiparent populations of crop species were
developed and largely used to explore the genetic architecture of
agronomic traits (Scott et al., 2020). Nested Association Mapping
(NAM) and Multiparent Advanced Generation Intercross
(MAGIC) populations are the most popular designs. They
were designed to improve the resolution of QTL analysis with
biparental populations and to reduce the confounding effects
of population structure of association mapping (Gage et al.,
2020). While NAM populations can be seen as a collection of
biparental populations with a common parent (e.g., Yu et al.,
2008;Hu et al., 2018), MAGIC populations have more complex
crossing designs, commonly involving 4, 8, or 16 parents (Zamir,
2001;Gage et al., 2020;Scott et al., 2020). Such differences
in the crossing designs determine that lines from NAM and
MAGIC populations differ in their genetic composition, even
when the same parents are used. In the NAM populations,
Frontiers in Plant Science | www.frontiersin.org 11 June 2022 | Volume 13 | Article 886162

fpls-13-886162 November 30, 2022 Time: 12:6 # 12
Renzi et al. CWR Breeding for Marginal Lands
lines are grouped into families, with as many families as donor
parents, thus the genomes of NAM lines are a mosaic of only two
parents, and haplotype diversity is constrained by the common
parent (Zamir, 2001;Gage et al., 2020). On the contrary, the
genomes of MAGIC lines are a mosaic of all founders, and
haplotype diversity grows with the number of founders, although
it is constrained by the number of lines in the population
(Zamir, 2001;Scott et al., 2020). More than 50 multiparent
populations were developed for several crop species (reviewed
in Scott et al., 2020), however, only four of them included CWR
as founders, three for barley and one for maize (Scott et al.,
2020). In multiparent populations, the selection of founders
is the most critical step, as it determines the pool of genetic
variation segregating in the population. Choosing founders as
genetically diverse as possible maximizes the population’s utility
for mapping agronomic traits in multiple environments (Gage
et al., 2020;Scott et al., 2020). However, for pre-breeding, a
more conservative selection of founders will produce populations
with higher overall agronomic performance, and therefore more
attractive for breeders. In the case of NAM populations, after
crossing the elite cultivar with CWR, one or more cycles of
backcrosses can be implemented before selfing to improve the
overall agronomic performance of the population (Chen et al.,
2019). In the case of MAGIC populations, a trade-off between the
overall agronomic performance and the novel genetic variation
is expected, i.e., the larger the proportion of elite cultivars as
founders the larger the overall agronomic performance and the
lower the novel genetic variation, and vice versa.
Genomic Selection and Multiparent
Populations
The complex genetics controlling most abiotic stress tolerances
presents a challenge for crop breeders to incorporate many CWR
alleles into their breeding programs. Genomic selection (GS) is an
efficient approach that was pioneered in animal breeding, where
complex, polygenic traits are the norm (Goddard and Hayes,
2007). Genomic selection has been refined for plant breeding
and adopted as a core methodology by many breeding programs
(Crossa et al., 2017). This approach could be used to accumulate
increasing numbers of alleles associated with stress tolerance and
shift the breeding populations from stress-sensitive to stress-
tolerant over many generations.
Genomic selection is increasingly common for a range
of crops, e.g., barley (Gonzalez et al., 2021), pea (Al Bari
et al., 2021), chickpea (Varshney et al., 2021), wheat (Muleta
et al., 2017), and sorghum (Yu et al., 2016). However, GS
to select within CWR collections has been proposed, but
examples have yet to be published (Bohra et al., 2021).
With the genotyping of larger CWR collections (e.g., Song
et al., 2015;Sansaloni et al., 2020), it is expected this
approach may be used in the future as it has been proven
effective in plant breeding programs (Merrick and Carter,
2021). The importance of GS may increase for CWR pre-
breeding programs as it allows for efficient introgression of
beneficial alleles for polygenic traits with reduced linkage
drag (Wang et al., 2017). Genetic selection has the advantage
of selecting superior progeny based on genotypes alone
using phenotyped training populations (Meuwissen et al.,
2001). The first demonstration of GS in pre-breeding was
in exotic maize populations successfully selecting for yield
(Combs and Bernardo, 2013). Genomic selection in CWR
pre-breeding was also validated recently in soybean (Beche
et al., 2021). Very good prediction accuracies for several
quantitative traits were reported using a NAM breeding
population with three wild Plant Inventory parents (Beche
et al., 2021). An excellent example for pre-breeding for
marginal lands using focused identification of germplasm
strategy (FIGS) is found in studies with barley, wheat
and faba bean landraces (Endresen et al., 2011;Khazaei
et al., 2013). FIGS is another machine learning application
that takes multivariate data sets of a priori knowledge
(phenotypes, environmental) to identify the potential most
useful accessions (Bari et al., 2012). Stenberg and Ortiz (2021)
suggest improving the FIGS model of adaptive processes
(natural selection) by adding non-adaptive processes
(gene flow and genetic drift) to hit the most intensive
evolutionary hotspots to capture elusive traits followed by
genomic selection.
In summary, the “choose first” strategy is more suitable
for incorporating traits regulated by one or a few major loci
(e.g., herbicide or disease resistance alleles) while the “cross
first” is more suitable for polygenic traits, which is expected
for most traits associated to adaptation to marginal agricultural
lands. Multiple parent populations, especially MAGIC, are well-
suited for the cross first strategy. Such populations are useful
to discover agronomically relevant loci, to improve genetic
diversity of pre-bred populations, and to directly incorporate
them to breeding schemes. However, we identified a lack of
multiparent populations (MPPs) that include CWR as founders,
meaning that current MPPs are exploiting only a small fraction
of species’ genetic diversity. Therefore, we call for a systematic
development of MAGIC populations, which should include
CWR, elite cultivars, and landraces as founders. The outlined
advantages of MAGIC populations for both basic science and
plant breeding should facilitate the creation of public-private
pre-breeding partnership programs aiming to develop cultivars
well-adapted to marginal agricultural lands. Although universal
MAGIC populations can be used to discover agronomically
relevant loci by multiple research groups, we encourage (when
possible) the development of MAGIC populations with local
founders to exploit local adaptation.
DE NOVO DOMESTICATION AND
GENOME EDITING
Since domestication began approximately 12,000 years ago, some
significant events determined a deep change in the evolution of
crops and their wild relatives. Fernie and Yan (2019) divided the
history of crop improvement into four breeding generations, the
first generation with breeding based on phenotype selection; the
second generation with hybrid breeding and green revolution
with dwarf plants and higher yields; a third generation called
Frontiers in Plant Science | www.frontiersin.org 12 June 2022 | Volume 13 | Article 886162

fpls-13-886162 November 30, 2022 Time: 12:6 # 13
Renzi et al. CWR Breeding for Marginal Lands
or “biotechnology-based breeding” with the arrival of genetically
modified crops. Currently, the fourth generation of breeding
is running, with genome editing and precision breeding. In
fact, the fourth-generation breeding is determined by new plant
breeding techniques like cis-genesis (Jacobsen and Schouten,
2007) and genome editing (Zsögön et al., 2017), mainly
CRISPR/Cas (Sukegawa et al., 2021). Cis-genesis consists of the
genetic transformation of a crop with CWR’s genes, without
the introduction of reporters or selectable markers from other
organisms, avoiding linkage drag. This is particularly interesting
in the case of the secondary and tertiary genepool species (Harlan
and de Wet, 1971) with strong hybridization barriers.
Genome editing techniques involve zinc-finger nucleases
(ZFNs), transcription activator-like effector nucleases (TALENs),
and clustered regularly interspaced short palindrome repeats
associated protein 9 (Cas9), CRISPR/Cas systems (Manghwar
et al., 2019;Sedeek et al., 2019;Van Tassel et al., 2020;Molla
et al., 2021). ZFNs and TALENs are protein-based and require
protein engineering for every user-defined sequence. Instead,
CRISPR/Cas is a RNA-guided system that induces double-
stranded DNA breaks by the action of Cas9 nuclease at a genome
corresponding location (Belhaj et al., 2013;Altpeter et al., 2016;
Rodríguez-Leal et al., 2017). Among genome editing techniques
in plants, CRISPR/Cas9 has become the most popular (Altpeter
et al., 2016;Pacher and Puchta, 2017;Das et al., 2019). The
CRISPR/Cas9 system can generate stable and heritable mutations
in genes that cannot be distinguished from a natural mutation
at the same locus, without affecting the existing valuable traits
(Belhaj et al., 2013;Arora and Narula, 2017). Since the main
domestication genes in most of the major crops have been studied
(see reviews by Kantar et al., 2017;Purugganan, 2019), the
new gene editing tools would allow neo-domestication avoiding
the linkage drag. De novo domestication or neo-domestication
is defined as the targeted introduction of domestication genes
into non-domesticated plants, this represents an important
opportunity for fitting cultivated species to the climatic niche
where they live (Fernie and Yan, 2019). In addition, genome
editing allows to incorporate new adaptation attributes present
in wild materials into cultivated ones, bringing resilience under
the unfavorable conditions of climate change (Van Tassel et al.,
2020). This is possible since the whole genome sequences for
many crops and their wild species are now available (Bohra et al.,
2021;Ramsay et al., 2021). However, it should be emphasized that
this approach has yet to be fully tested and is unlikely to transfer
all the beneficial traits accumulated in crops over centuries of
generations, such as complex traits like grain yield, which is
controlled by many genes.
Genome editing (GE) can be used for several purposes like to
activate or suspend the function of any gene. Ghoshal et al. (2021)
showed that with epigenome engineering it is possible to achieve
heritable methylation, gene silencing, and delayed flowering time
phenotypes. Hence, stable heritable epigenetic modifications in
flowering time would allow researchers and breeders to overcome
the crossing barriers of the secondary and tertiary genepool,
facilitating crossings of CWRs with related crops (Cable et al.,
2021). Genome editing can also be used to knock out genes.
For example, increasing grain number and larger grain size in
rice was possible by knocking out three mutations on negative
regulators of yield (Mishra et al., 2018). In addition, knocking out
the self-incompatibility gene S-Rnase allowed re-domestication
of potato into an inbred-line based diploid crop for genetic
improvement (Ye et al., 2018). This demonstrated the utility
of this approach in four different Solanum tuberosum clones,
opening possibilities in both future diploid breeding and basic
research in self-incompatible crops.
Another application for GE is to generate novel alleles of
any gene. For breeding drought tolerance in maize, novel allelic
variation for ARGOS8 locus was generated by changing the DNA
sequence at the native ARGOS8 (Shi et al., 2017). Genome editing
could also add genes that do not exist in an original genome.
There is also work on CRISPR/Cas9-mediated HDR (homology-
directed repair) for editing genes that confer herbicide resistance,
and to transfer an allele from a wild rice variant into a cultivated
variant to increase yield potential. In addition, GE makes it
possible to delete any sequence including large chromosomal
fragments or even the entire chromosome (Xiao et al., 2013).
This could be an advantage for the secondary and tertiary
genepool species (Harlan and de Wet, 1971). Introgression
of beneficial alleles from CWR into the cultivated gene pool
has been a challenge when reproductive barriers are present.
For instance, the genus Helianthus, native to North America,
comprises 52 species including cultivated sunflower, H. annuus L.
which has emerged as a model for genetic studies of adaptation,
hybridization, and speciation. In particular, H. annuus and
H. petiolaris are chromosomally divergent species, differing by a
minimum of seven translocations and three inversions (Rieseberg
et al., 1995). Notably, the first source of cytoplasmic