Towards the Development of Perennial Barley for Cold Temperate Climates—Evaluation of Wild Barley Relatives as Genetic Resources

: Perennial cereal crops could limit the negative impacts of agriculture on the environment and climate change. In cold temperate climates, perennial plants must be adapted to seasonal changes and abiotic stresses, such as frost, to be able to regrow for several years. Wild crop relatives that are perennials and already adapted to cold temperate climates may provide genetic resources for breeding new perennial cereal grain crops. Barley ( Hordeum vulgare ) is one of the most important cereals in northern agricultural areas, and its related perennial species may be good candidates for the development of perennial cereals. We evaluated a diverse set of 17 wild perennial Hordeum species represented by 67 accessions in ﬁeld conditions with a cold winter climate and long days during summer in Central Sweden (latitude 60 ◦ N). Six species ( H. brevisubulatum , H. bulbosum , H. fuegianum , H. jubatum , H. lechleri and H. secalinum ) showed regrowth and formation of spikes for four seasons. The most distant perennial relative of barley, H. stenostachys , showed weak regrowth. H. bulbosum , the closest perennial barley relative, had a large number of accessions with wide geographic origins that showed good regrowth. Together with its storage bulbs and its cross-compatibility with barley, this makes H. bulbosum an important genetic resource for the development of perennial Hordeum grains using either the domestication or the wide-hybridization strategy.


Introduction
Perennial grasses play major roles in temperate ecosystems and ley cultivation. With their deep and extensive root systems and earlier and extended growth period during the vegetation season they can provide several benefits over annual grasses with regard to their impact on the environment and climate. The perennial growth habit is associated with reduced soil erosion, more efficient use of mineral nutrients, higher soil quality and carbon sequestration [1][2][3][4]. Cropping systems that include perennial cereals may, therefore, offer one of several solutions to mitigate ongoing climate change and improve food security [5,6]. To meet demands on food security and resilience against climate change, a diversity of perennial cereal crops need to be developed.
Perennial cereal crops that are relatives of sorghum [7], rice [8], and the wheat-relative intermediate wheatgrass (Thinopyrum intermedium) [9] are under rapid development for sub-tropic and mild temperate regions. For cold temperate climates, the development of perennial cereals is at an earlier stage, and intermediate wheatgrass and rye-relatives [10] are ongoing candidates. In cold climates, perennial plants should be adapted to the seasonal changes and stressful conditions caused by

Pre-Cultivation in Climate Chamber and Greenhouse, and Trait Evaluation in Field
Fifty accessions of the 17 perennial Hordeum species were planted as two-year-old adult plants in the field (Table 1). These plants had large root systems at planting that ensured good establishment and growth in the first season, which allowed evaluation of survival and regrowth ability in the following years. To also evaluate regrowth of plants planted at earlier development stages, young plants at booting stage of six of the 17 perennial species were planted in the field. In addition, three of these six perennial species were seeded directly in the field. The two-year-old plants, the young plants and the seed for all species were planted in the field in May 2013. Prior to the planting, both the two-year old adult plants and the young plants were pre-cultivated in a climate chamber.
Two-year-old adult plants: In early spring 2011, seedlings derived from the 17 perennial species were cultivated in pots with a low nutrient commercial soil potting mix (S-jord, Hasselfors Garden, Örebro, Sweden) in a climate chamber (4.6 square meter, 2.4 m high; model BDW50, Conviron, Winnipeg, MB, Canada) at the Phytotron Plant Cultivation Facility, Uppsala BioCenter, SLU Uppsala, Sweden. After germination, the plants were cultivated for eight weeks in the climate chamber with a day/night cycle of 22/18 • C and 16/8 h light/dark period with a photosynthetic active radiation of 300 µmol m −2 s −1 . The plants were then given a vernalization treatment for six weeks at 4 • C under an 8 h photoperiod at 100 µmol m −2 s −1 , to induce flowering. After vernalization, the plants were maintained in the climate chamber for two years. Before planting in the field, the plants were acclimatized for two weeks in a greenhouse at 22 to 25 • C with a 16/8 h light/dark period with supplementary light from metal halogen lamps. Morphologies of spikes, root systems, and bulbs were observed. For comparison, five accessions from three annual Hordeum species were also vernalized as seedlings and cultivated in climate chamber as above until they reached flowering stage, followed by two weeks in the greenhouse until planted in the field.
Young plants: Seedlings derived from six of the 17 perennial species and of three annual Hordeum species were also pre-cultivated in the climate chamber in early spring 2013 as described above. Three of these six perennial species were also given the vernalization treatment as above before planting in the field.
The plants and seed were planted in clay soil in a farmer's field north of Uppsala, Central Sweden (60 • 00 N, 17 • 42 E). The field was located at an organic farm, and a low level of animal manure fertilizer was added. Manual weeding was carried out, and no pesticides or herbicides were applied. Climate data for the cultivation period during 2013-2016 is presented in Figure S1. Two-year-old plants a growth habit either annual (A) or perennial (P) [14], b see Table S1 for details about the accessions, c at least one plant per accession showed regrowth, d accessions not possible to identify are indicated by -. Regrowth was assessed based on the presence of new leaves originating from old plant parts in May and August 2014 (second growing season), in July 2015 (third growing season) and in August 2016 (fourth growing season). Presence of leaves was scored from 0 (no leaves) to 5 (50 or more leaves). Presence versus absence of spikes was recorded in August 2014 and in July 2015.

Data Analysis
To analyze and display the distribution of the perennial Hordeum species based on regrowth (leaf and spike) data from 2014 and 2015, we applied multivariate Principal Coordinate Analysis (PCoA) to a dissimilarity matrix constructed from the dataset treated as qualitative variables. To test the distribution of the two species with larger numbers of accessions (H. bulbosum and H. stenostachys) along the three first PCoA components, and residuals for each of the two species were checked for deviations from normality. As the residuals for all three components did not show normal distribution for both species, we applied the non-parametric Kruskal-Wallis test. The same test was also employed for the analysis of differences in individual traits. The statistical analyses were carried out using the software XLSTAT 2018.3 (Addinsoft, New York, NY, USA).

Diversity in Regrowth Ability
The two-year-old plants of the perennial species and the adult plants of the annual species established well in the field 2013 ( Figure 1). Regrowth was assessed based on the presence of new leaves originating from old plant parts in May and August 2014 (second growing season), in July 2015 (third growing season) and in August 2016 (fourth growing season). Presence of leaves was scored from 0 (no leaves) to 5 (50 or more leaves). Presence versus absence of spikes was recorded in August 2014 and in July 2015.

Data Analysis
To analyze and display the distribution of the perennial Hordeum species based on regrowth (leaf and spike) data from 2014 and 2015, we applied multivariate Principal Coordinate Analysis (PCoA) to a dissimilarity matrix constructed from the dataset treated as qualitative variables. To test the distribution of the two species with larger numbers of accessions (H. bulbosum and H. stenostachys) along the three first PCoA components, and residuals for each of the two species were checked for deviations from normality. As the residuals for all three components did not show normal distribution for both species, we applied the non-parametric Kruskal-Wallis test. The same test was also employed for the analysis of differences in individual traits. The statistical analyses were carried out using the software XLSTAT 2018.3 (Addinsoft, New York, NY, USA).

Diversity in Regrowth Ability
The two-year-old plants of the perennial species and the adult plants of the annual species established well in the field 2013 ( Figure 1).  Accessions of all perennial species, except H. muticum (47 out of 50 accessions), showed regrowth in the spring of the second growing season (2014) following the first winter (Table 1). In total 36 accessions of 13 perennial species also survived the second winter and produced new leaves and tillers during the third season (2015). Seven species showed relatively strong regrowth in both 2014 and 2015, while six species showed weak regrowth ( Table 2). Table 2. Regrowth ability in perennial wild Hordeum species planted in 2013 as two-year-old plants, as young plants at booting stage or as seed in a farmer's field in Uppsala, Sweden (latitude 60 • N), located in a cold temperate climate. Mean and standard error of presence of leaves after regrowth scored from 0 (no leaves) to 5 (50 or more leaves) and presence of spikes scored from 0 (no spikes) to 1 (presence of spikes) of plants of each species. See Table 1 for additional information.

Species
Presence Two-year-old plants The two species with large numbers of studied accessions, H. bulbosum and H. stenostachys, showed significant differences in regrowth ability (p < 0.0001, Kruskal-Wallis test, Figure 2a (Table 1). Thus, the same plants of these accessions had survived four consecutive years in the field. None of the species described as annuals survived the first winter in the field.
Spikes were formed by accessions planted as two-year-old plants in 13 of the 16 species living in 2014, and in nine of the 10 species remaining in 2015 (Table 3). Forty-two and 19 accessions formed spikes in 2014 and 2015, respectively. Almost all species with a strong regrowth in 2015 also formed spikes ( Table 2). The accessions of H. bulbosum showed a significantly higher ability to form spikes than the accessions of H. stenostachys in both 2014 and 2015 (p < 0.0001, Kruskal-Wallis test, Figure 2d,e).          Table S1 for details about the accessions, b at least one plant per accession showed presence of spikes.
PCoA based on the recorded leaf regrowth and spike formation in 2014 and 2015 for all the perennial Hordeum species planted as two-year-old plants, showed that most H. bulbosum accessions were different from the accessions of H. stenostachys (Figure 4). The distribution of the two species along the first PCoA component was significantly different (p < 0.0001, Kruskal-Wallis test). The distributions of several of the other species present as one to two accessions, for example, H. bogdanii, H. brevisubulatum, H. fuegianum, and H. secalinum, overlapped with the distribution of H. bulbosum and were not significantly different from the distribution of H. bulbosum (p > 0.05, Kruskal-Wallis test). However, compared to H. stenostachys they showed a significantly different distribution (p < 0.05, Kruskal-Wallis test).
Vernalized young plants of the three studied perennial species and non-vernalized young plants of two of three species showed regrowth the following season (Table 1). However, H. bulbosum present as eight accessions and H. brevisubulatum with one accession, were the only species with relatively strong regrowth ( Table 2) Table 3).
The proportion (approx. 90%) of perennial accessions that showed regrowth in 2014 was similar for plants planted in the field as two-year-old adult plants and as young plants (Table 1). However, in 2015, the proportion of the accessions that showed regrowth was higher for plants planted as two-year-old plants (77% versus 46%). The ability to form spikes was higher in the two-year-old plants both in 2014 and in 2015 (Table 3).
along the first PCoA component was significantly different (p < 0.0001, Kruskal-Wallis test). The distributions of several of the other species present as one to two accessions, for example, H. bogdanii, H. brevisubulatum, H. fuegianum, and H. secalinum, overlapped with the distribution of H. bulbosum and were not significantly different from the distribution of H. bulbosum (p > 0.05, Kruskal-Wallis test). However, compared to H. stenostachys they showed a significantly different distribution (p < 0.05, Kruskal-Wallis test).

Diversity in Spike Morphology and Storage Bulbs
Substantial variation in spike morphology, for example, spike and awn length, was observed among (Figure 5a) and within the perennial Hordeum species, as exemplified by H. bulbosum (Figure 5b). H. bulbosum spikes were brittle at maturity; the spikelets gradually shattered from the top of the spike (Figure 3c,d).
Even though the Hordeum species like all grasses are hermaphrodites, they show different rates of outcrossing, varying from mainly inbreeding as in H. chilense and H. jubatum, to obligate outcrossing with self-incompatibility systems as in H. bulbosum and H. brevisubulatum [14,23]. Since obligate outcrossing species require pollination by compatible genotypes for seed production, in contrast to inbreeding species, which reproduce through self-fertilization, seed production was difficult to compare between species and was therefore not scored in the field trial. However, several of the H. bulbosum accessions produced seed during three seasons (2014 to and 2016, Figure 3d), showing that cross-pollination occurred between the large number of H. bulbosum accessions planted in the field.
Presence of bulbs at the base of tillers on H. bulbosum was confirmed and was not observed for the other perennial Hordeum species (Figure 5c-f). Diversity in bulb shape among H. bulbosum accessions was found as well as the number of bulbs per tiller (Figure 5e,f). Some H. bulbosum accessions formed only one bulb per tiller, while others also formed more than one bulb on each tiller.

Diversity in Spike Morphology and Storage Bulbs
Substantial variation in spike morphology, for example, spike and awn length, was observed among (Figure 5a) and within the perennial Hordeum species, as exemplified by H. bulbosum ( Figure  5b). H. bulbosum spikes were brittle at maturity; the spikelets gradually shattered from the top of the spike (Figure 3c,d).

Discussion
Breeding of perennial cereal grain crops for cultivation in perennial cropping systems is one strategy to improve the impact of agriculture on the environment and climate change. Perennial cereals could also contribute to increase the resilience of agriculture to climate change and to enhance food security. The importance of agriculture across geographic regions and climate zones demands a diversity of perennial cereals with a variety of adaptations. Barley and other Hordeum species are widespread and well adapted to a wide range of habitats and could provide candidates that can fill the gap of perennial cereals in cold temperate climates. With the goal to develop perennial barley, we have as a first step evaluated a diverse group of perennial Hordeum species. This evaluation was done in field conditions during several seasons to identify candidate Hordeum species that could survive cold winters and show vegetative and reproductive regrowth during repeated seasons. This approach made it possible to narrow down the possible candidates among the range of wild perennial barley relatives and to identify accessions with favorable traits as genetic resources for further breeding.
The 17 studied perennial Hordeum species showed large diversity in the ability to regrow over seasons, where some species had good regrowth and others did not survive the first or second winter. Interestingly, accessions of the species H. brevisubulatum, H. bulbosum, H. fuegianum, H. jubatum, H. lechleri and H. secalinum survived and regrew for four seasons under the long day temperate cold climate, even though they originated from geographic areas with a climate and photoperiod at least partially different compared to at the evaluation site. All of these species except H. lechleri also formed spikes after regrowth. For H. brevisubulatum and H. bulbosum winter survival and regrowth was found in plants when planted as two-year old plants as well as young plants and seed. In PCoA based on multiple traits associated with regrowth, H. brevisubulatum, H. fuegianum, and H. secalinum showed overlapping distribution with the many accessions of H. bulbosum providing additional support for the similar performance. Interestingly, H. stenostachys, a species with many available accessions, and its close relatives H. cordobense, H. erectifolium and H. muticum clearly differed from their very distant relative, H. bulbosum, in their performance due to their weak regrowth ability. In addition, the four closely related Hordeum species all have a South American origin, while H. bulbosum originates from Europe and Asia [14].
A factor contributing to the good winter survival and regrowth of H. brevisubulatum could be its strong ability to form rhizomes [14,22]. Also, H. brevisubulatum and H. secalinum have been shown to have a high tolerance to salt stress [24,25], which has similarities to frost stress with regard to dehydration, and they induce similar plant responses such as the production of osmotically active compounds [26]. Tolerance to salt was also found to be higher in tetraploid H. bulbosum compared to diploid forms [27]. The traits of H. brevisubulatum and H. secalinum and their ability for regrowth, suggest that these species, as well as the other species with similar performances to H. bulbosum, deserve further evaluation as domestication candidates or as potential donors in wide-hybrid crosses with barley.
Most H. bulbosum accessions in this study were tetraploids and they displayed good regrowth over several consecutive years. Also, the few diploid accessions showed regrowth. Both the tetraploid and diploid accessions produce bulbs at the base of the tillers for storage of carbohydrates, which may play an important role to support regrowth until the plant begins active photosynthesis.
Whereas most Hordeum species have a narrow distribution, H. bulbosum grows in a wide range of habitats such as dry grasslands, shrubland, dry hillsides, wet meadows and coastal sites and can also be found at abandoned fields and roadsides [14]. The diploid form occurs along the Mediterranean coast from Spain eastwards to Greece and along the North African coast to Egypt, whereas the tetraploid form is found in the regions south of the Black Sea eastwards to Afghanistan and southern Tadzjikistan. H. bulbosum with its wide geographic distribution, different ploidy forms, and adaptability to various habitats can provide potential genetic resources for perennial barley development. In fact, most of the 29 studied accessions of H. bulbosum showed good vegetative regrowth and ability to form spikes for several seasons, and are interesting breeding material in the development of perennial barley for cold temperate climates.
Another advantage of H. bulbosum as a candidate species compared to other wild perennial Hordeum species is the close relationship to barley seen in the high levels of conserved synteny between the barley and H. bulbosum genomes [28][29][30]. All the technical resources that are available for barley breeding are therefore also accessible and highly valuable for breeding towards perennial barley. These resources significantly increase the possibilities to identify genes important for agronomic and quality traits, and to apply this knowledge in perennial cereal breeding. Also, characterization of genetic diversity in H. bulbosum breeding populations is greatly facilitated by its similarities to barley and the availability of the resources.
The close genetic relationship of H. bulbosum and barley has also made it possible to obtain fertile offspring in crosses between the two species, in contrast to crosses between barley and most other perennial Hordeum species [16]. Using the wide-hybridization strategy, offspring have been made into introgression lines (ILs) carrying different H. bulbosum segments introgressed into a barley genetic background through backcrossing or selfing [12,31]. These ILs were developed to improve pathogen resistance in barley [12]. However, based on the good regrowth ability found in a majority of the studied accessions of H. bulbosum in a field site in Uppsala, these ILs are also interesting genetic resources for the breeding of perennial barley for cold temperate climates. We have therefore evaluated a diverse set of ILs where different parts of chromosomes of H. bulbosum have been introgressed into barley cultivars in a companion paper in this issue (Westerbergh et al. 2018). Based on our findings and advantages discussed above, H. bulbosum is suggested to be a valuable genetic resource for the development of perennial barley using either the domestication strategy or the wide-hybridization strategy.

Conclusions
This pre-breeding study provides the first evaluation of candidate species towards the development of perennial barley. Among a large set of perennial Hordeum species, some candidates were identified, and H. bulbosum was considered as the most promising genetic resource for future work towards a barley-related perennial crop. The continuous regrowth over several seasons in a cold temperate climate, and its close relatedness to barley that makes crosses and introgressions of genes and traits possible, as well as the immediate access to the technical resources for barley breeding, all contribute to making H. bulbosum a strong candidate.
Supplementary Materials: The following are available online at http://www.mdpi.com/2071-1050/10/6/1969/s1. Table S1: Species and accessions included in the comparison of regrowth of wild Hordeum species in cold temperate climate. Figure S1: Monthly mean temperatures and precipitations at the field site in Uppsala, Sweden (latitude 60 • N).