Natural selection under conventional and organic cropping systems affect root architecture in spring barley

Root morphological traits

The wild-type parent ISR42-8 produced longer root length (RL) than the modern cultivar parent Golf and tested lines (Table S2, Fig. 1A.h,A.f [h = hydroponic; f = field]). The tested lines of the two evolving barley populations displayed significant variations under hydroponic conditions. Barley lines evolved under OCS had on average 3484 mm longer roots compared to CCS under hydroponic treatment (Fig. 1A.h, Table 2). Complementary results under field conditions show as well higher RL for the OCS lines, even though the variance was significantly less pronounced (Fig. 1A.f). In addition, a less evident variance was observed in the field within both groups compared to the hydroponic (Fig. 1A.d). Across both experimental setups, the observed range of RL was higher in the OCS lines [Standard deviation (SD)OCS = 883, SDCCS = 597] (Table 2).

Figure 1

Significantly variant root morphological phenotypes. Boxplots illustrate the overall distribution of observed data points for the parents Golf and ISR 42-8 as well as for the conventional (CCS) and organic (OCS) lines. Density plots highlight the overall distribution of organic and conventional adapted lines. (A)—Root length (RL)—the sum of all roots harvested in millimeters (mm), illustrated for all four groups. (A.h)—root length measured in the hydroponic experiment; (A.f)—field experiment; A.d—distribution histogram for root length in both field and hydroponic experiment for CCS and OCS adapted lines. (B)the ratio of root length to volume (L/V). Data available for hydroponics (B.h), field (B.f), and distribution of the ratio of root length to volume illustrated in B.d. (C.f)—Root mass density (RMD) from the field; (D.f)—Root angle (RA) from the field, distribution of the root angle illustrated in (D.d); (E.f)—root tip per plant count from the field, corresponding histogram visualized in (E.d). (F.f)—root fork per plant count from the field.

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Table 2 Comparison of organic and conventional population root phenotypes under field and hydroponic evaluation.
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The root length to volume (L/V) is an important indicator of the soil volume that can be explored by the roots. Under hydroponics conditions, variations were found for L/V between the parental genotypes as well as between the OCS and CCS populations (Tables 2 & S3). The organic lines were characterized by a significantly higher L/V, indicating a much more distinct exploration of the soil by these lines (Fig. 1B.h,B.f). In comparison to field, highest diversity in L/V was found under hydroponic experiments within both OCS and CCS populations (Fig. 1B.d).

The root mass density (RMD) is the ratio of root volume for a given root mass and is a key indicator of root thickness. Although significant variations existed between ISR42-8 and Golf under hydroponics conditions, such significant variations were not found between the OCS and CCS groups (P = 0.09) (Fig. 1C.f and Tables 2 and S3).

The root angle (RA) measurements were only performed under field conditions since plants grown under hydroponics conditions were placed in uniform growing vessels and the direction of root growth is restricted by tubes. Significant variation was observed for the RA between the two parental lines, which was also reflected in the CCS and OCS lines (Fig. 1D.f). ISR42-8 was characterized by an 11.5° average narrower RA than Golf (Table S3). The RA was 4.1° bigger in the OCS compared to the CCS population (P = 0.005) (Table 2). However, a higher diversity in RA was observed in the OCS compared to the CCS lines (Fig. 1D.d, Table 2).

In addition to the RA, the number of root tips and forks was measured under field conditions only. Both tips and forks indicate a similar pattern, where the OCS lines produced on average more for both PForks = 0.014, PTips = 0.0041 (Fig. 1E.f,F.f). After applying a P-adjustment, the number of forks count was no longer significantly different between OCS and CCS (PForks = 0.07, Table 2). Complementary, ISR 42-8 was observed to produce more tips and forks than Golf, which remained highly significant even after probability adjustment (Fig. 1E.f,F.f, Table S3). The distribution and the standard deviation of observed phenotypes highlight once more the fact that the OCS lines tend to have a higher variation (Fig. 1E.d, Table 2). Similarly, a significant increasing trend was recorded in root surface area (RSA) and root average diameter (RAD) by ISR42-8 as compared to Golf under hydroponics (Table S3). Contrasting to the parental genotypes, no variation was observed between OCS and CCS lines for RSA (Table 2).

Root anatomical traits

Within the observed anatomical traits, four were considered due to their relevance and variation between the systems. In both hydroponic and field experiments, significant variations were observed for the late metaxylem number (LMXN) between the parental lines as well as OCS and CCS lines (Tables 2 & S3, Fig. 2A.h,A.f). An increased LMXN for ISR 42-8 compared to Golf was observed (Fig. 2A.h). Regarding the CCS and OCS lines, a heterogenic scenario was presented over both experimental setups. While the median LMXN under CCS was identical with ISR 42-8 in the seedling stages of plant development (Fig. 2A.h), it was much lower in flowering stages under field conditions (Fig. 1A.f). Additionally, the LMXN was significantly higher in the CCS lines in the seedling stage compared to OCS lines, vice-versa LMXN was observed at the flowering time point (Fig. 2A.d).

Figure 2

Significantly variant root anatomical traits. Boxplots illustrate the overall distribution of observed data points for the parents Golf and ISR 42-8 as well as for the conventional (CCS) and organic (OCS) lines. (A) –Late metaxylem number (LMN)—the sum of all roots harvested and expressed by plant−1, illustrated for all four groups. A.h—Late metaxylem number measured in the hydroponic experiment; A.f—field experiment; A.d—distribution histogram for late metaxylem number in both field and hydroponic experiment for CCS and OCS adapted lines. (B)Aerenchyma area (AA). Data available for hydroponics (B.h), field (B.f), and distribution of the aerenchyma area illustrated in (B.d). (C.f)—Total cortical area (TCA) from the field; (D.f)—Root cross-section area (RA) from the field, distribution of the total cortical area and root cross-section area illustrated in C.d and D.d, respectively.

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The intercellular space, represented by the aerenchyma area (AA), was observed to be significantly more pronounced in the tested CCS compared to OCS lines in both environments (Fig. 2B.h,B.f). Furthermore, the OCS population did not show significant differences to both parents under hydroponics conditions, however, when grown under field conditions, it was noted that Golf had a significantly higher AA mean value as compared to the OCS population (Table S3). As illustrated by the values, the AA expended from early to late stages by a magnitude of 10-folds (Fig. 2B.d). In general, although the two parents did not indicate phenotypic variations, OCS and CCS lines showed significant variations (Table S4).

A 0.12 mm2 decreased average total cortical area (TCA) was recorded in the OCS compared to the CCS population under field conditions (P = 0.003, Fig. 2C.f), although substantial variations for TCA was observed within OCS and CCS populations (Fig. 2C.d). The root cross-section area (RXA) is a two-dimensional axis of the root which is an important indicator of root thickness. In the hydroponic examination of the seedling stage, significant variations existed between the CCS and ISR42-8 as well as OCS population (Tables 2 and S3). The complementary study under field conditions observed a noticeable variation for OCS from both parental genotypes and the CCS (Table S3). About 0.13 mm2 increased average value for RXA was identified for CCS (Fig. 2D.f), while consistent significant variations were also observed between the populations in the under field experiment, where 0.13 mm2 increased average value for RXA was identified for CCS (Fig. 2D.f). Analog (Fig. 2D.d). Analogue to the AA, the RXA indicates a lower root extension in the OCS compared to the CCS population. For the stele area (SA), significant variations were only observed at the flowering stage, where ISR42-8 generally had the highest SA and varies significantly between Golf and its progeny lines (Tables 2 and S3).

Shoot-related traits

Beyond the root-related phenotypic observations, above-ground characteristics were also recorded to assess the root-borne shoot dynamics (Figs. 3 and S2). Among the OCS and CCS populations and the parents, ISR42-8 had the longest duration of emergence. While CCS-adapted lines took on average 5.8 days of emergence (DE), OCS-adapted lines emerged 1.8 days later (7.6 days) (Fig. S2). No variation was observed for the tiller number (TN) throughout all tested groups, while ISR 42-8 tends to produce much more leaf number (LN), accompanied by a lower plant height (PH) and higher shoot dry weight (SDW) (Table S4, Fig. S2). The OCS and CCS plants significantly differed in PH as well as SDW (Fig. 3B,C). The LN was marginally above the probability threshold of 0.05 (p = 0.058, Fig. 3A), with a clear tendency of increased variability in phenotypic variation (Fig. 3D). Similar trend was recorded for the SDW (Fig. 3F).

Figure 3

Above-ground plant characteristics. Boxplots illustrate the overall distribution of observed data points for the parents Golf and ISR 42-8 as well as for the conventional (CCS) and organic (OCS) lines under the hydroponic experiment. (A)—Leaf number (LN) expressed by; (B)—Plant height (PH) and C-Shoot dry weight (SDW). The data distribution of the leaf number, plant height and shoot dry weight is illustrated in (D,E,F), respectively.

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Interconnection of root-shoot traits

We performed inter-trait correlation analysis to unravel association among root traits and in between root and shoot phenotypes (Fig. 4). Pearson correlation coefficient revealed significant correlations among root-shoot traits. LN, PH and SDW had strong positive associations with all root architectural traits under hydroponic conditions (P < 0.001, r =  > 0.30) in both CCS and OCS, while DE has negative association with all shoot traits (r = −0.17 to −0.48) (Fig. 4A.h,B.h). A consistent negative relationship was observed for L/V with shoot traits such as LN, PH and SDW and root morphological traits such as RL, RSA and RAD in both CCS and OSC populations (Fig. 4A.h,B.h). A strong negative association existed between RL and all shoot morphological, root architectural and anatomical traits in both populations, except for L/V where a weak negative (r = −0.09) association was displayed only in the OCS. Likewise, all above-ground traits and all root architectural traits exhibited significant positive associations with all root anatomical features in both groups with an exception for the AA (Fig. 4A.h,B.h). Moreover, correlation analysis revealed strong positive relationships in both groups of SDW and root dry weight (RDW) to all above-ground traits, below-ground traits including, RL, SA, and RAD, as well as in all root anatomical traits (Fig. 4A.h,B.h). This means that the growth of tissue and organ is proportional to the increase in total dry biomass. More importantly, we observed a significant positive correlation among most of the root morphological, architectural, and anatomical traits in both OCS and CCS adapted populations, with few exceptions such as L/V (Fig. 4A.h,B.h).

Figure 4

Correlation matrix for shoot morphological (only in hydroponic conditions; A.h and B.h), root architectural and anatomical traits in two groups of barley populations and their parental lines grown across two growing conditions. (A)—conventional and (B)—organic cropping systems. (A.h)—conventional under hydroponic, (B.h)—organic under hydroponic, (A.f)—conventional under field, and B.f—organic under field conditions. The color scale represents Spearman’s ranked correlation coefficient. A larger circle size indicates a smaller p-value; blank cells represent that correlation was non-significant at P < 0.05. Most strongly associated traits are ordered and grouped in black boxes based on hierarchical clustering. See Table 1 for trait description.

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Similar to hydroponics, correlation analysis for both groups under field conditions showed highly significant positive and negative correlations (Fig. 4A.f,B.f). While a positive correlation was observed under hydroponics, RDW only showed to be a positive significant relationship with other root morphological traits and a weak positive relationship with anatomical traits in response to both CCS and OCS. Interestingly, correlation analysis revealed the important association of RA to other root architectural and anatomical features, and showed a strong negative association between RL (r =  > −0.90) and RDW (r =  > −0.80) (Fig. 4A.f,B.f) for CCS and OCS populations respectively, which means that narrower the angle of the nodal roots, the longer was the root system. The two root branching traits, the number of tips and number of roots forks which were known to be associated and dependent on the RL have a strong positive correlation reflected by r = 0.81 and 0.90 in CCS and r = 0.74 and 0.84 in OCS developed lines, respectively, while they have a significant negative correlation with RA (r = −0.72 to −0.77) in both barley groups. In addition, RA had also strong negative relationship to RDW contributing architectural traits including, RMD (r = −0.81 to −0.84) and L/V (r = −0.34 to −0.44). However, no positive associations were observed for RA and all root anatomical traits in both OCS and CCS populations (Fig. 4A.f,B.f).

Allometry analysis

The correlation analysis identified interconnection among root and shoot-related traits. Therefore, we checked if these correlations can be explained by allometric relations (Tables 3 and 4).

Table 3 Summary of allometric analysis of root-shoot system traits under hydroponic condition.
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Table 4 Summary of allometric analysis of root-shoot system traits under field condition.
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In the hydroponic environment, we observed a total of ten allometric relations, from which six were annotated to the PH. The PH was allometrically related to the SDW, the RSA, the RV, the RDW, the SRL, and the RMD (Table 3). Besides, the SDW was allometrically associated with the RSD. Furthermore, the TCA was related to the RXA. Finally, an allometry relationship was detected between the LMXN and AA (Table 3).

In the field experimental setup, we detected in total ten allometric relations (P < 0.05). The most significant allometry was observed for the RL and the RA (Table 4). Besides, the RA was allometrically related to the RDW, the SRL, the RMD, and the number of root forks. Furthermore, the RAD growth was significantly related to the growth of RSA. Finally, we also observed an allometric relationship between TCA and the SA (Table 4).

Root system divergence exists in barley populations. Based on the root phenotypic traits, PCA was conducted to highlight variations between parental lines as well as OCS and CCS populations (Fig. 5). Lines that evolved in the OCS environment tend to have a higher variance compared to CCS adapted lines in both experimental setups (Hydroponics and field, Fig. 5A,B). The majority of variance is explained by the first component (PC1) (81.71% hydroponics, 97.46% field), while the second component (PC2) explains 18.28% and 2.52% for hydroponics and field experiments, respectively (Fig. 5). The principal components (PCs) of both environments highlight a similar range of values and extension of points.

Figure 5

The principal component output of the root morphology assessment in a hydroponic (A) and a field-based environment (B). The first two components are plotted, with the first PC on the x-axis and the second PC on the y-axis. The colors differentiate the conventional (magenta) and organic (purple) systems, and the wild donor (ISR 42-8, turquoise) from the cultivar (Golf, green). Each point represents an individual genotype. In (A), 150 genotypes were tested from the organic and conventional systems, while 100 genotypes each were tested in (B). Dashed ellipses are plotted based on the points if the sample size was sufficient. Both experiments illustrate higher genetic variance in the organic system as compared to the conventional system.

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Comparison of root phenotypes with the genomic background

To support the validity of the observed phenotypic variations between the OCS and CCS groups, we inspected the allele frequency patterns of root developmental-related QTL, as described by35. For that, we utilized the genetic and adaptation data of the two populations in the earlier generations, as described by26. Eighteen loci, clustered in three phenotype classes: root dry weight, root length, and root volume were selected and compared between the organic and conventional populations in the starting (BC2F3) and four later generations (12, 16, 22 and 23) for their ISR42-8 allele frequency levels. We observed significantly higher ISR42-8 allele frequencies for root length and root volume in the organic population (proot length = 0.015; proot volume < 0.001, Bonferroni corrected T-test, Fig. 6A) that showed evidence of increase, even when considering all generations (F3 = 0.09; O23 = 0.12). The opposite is true for the conventional system (F3 = 0.09; C23 = 0.06). Contrasting, both organic and conventional populations show a higher ISR42-8 allele frequency for the root dry weight-related alleles (Fig. 6B, F3 = 0.09, O23 = 0.18, C23 = 0.13). When investigating all 18 loci separately, it becomes apparent that for QTL locus Qrdw.S42IL.1H, affecting the root dry weight, the ISR42-8 allele frequency increased up to 50% in both environments (Fig. 6C).

Figure 6

The allele frequency pattern dissection of 18 selected QTL loci related to root architecture traits for both organic and conventional populations in 5 generations (F3, F12, F16, F22, F23). Genomic data derived from Schneider et al. (2021). (A) Boxplot of the ISR42-8 allele frequencies for the trait categories root dry weight, root length, and root volume. Stars indicate the significance level of adjusted p values (Bonferroni adjustment) between the environments. The data of all generations are included in this significance test. (B) Line plot showing the development of the ISR42-8 allele frequency for the same three categories, separated by the environments across the generations. (C) similar to B, but showing the evolution of the ISR42-8 allele frequency across the generations for each QTL locus, separately. Table—indicates the median allele frequency across all 18 QTL loci generation and environmentally wise.

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