Root Trait Variability in Co ﬀ ea canephora Genotypes and Its Relation to Plant Height and Crop Yield

: Co ﬀ ee breeding based on root traits is important to identify productive genotypes under adverse environmental conditions. This study assessed the diversity of root traits in Co ﬀ ea canephora and its correlation with plant height and crop yield. Undisturbed soil samples were collected down to 60 cm from 43 co ﬀ ee genotypes, in which one of them was propagated by seed and all others by stem cutting. The roots were washed, scanned, and processed to quantify root length density, root volume, root superﬁcial area, and root diameter. Additionally, plant height and crop yield were also assessed. Root length density ranged from 40 to 1411 mm cm − 3 , root volume from 6 to 443 mm 3 cm − 3 , root superﬁcial area from 61 to 1880 mm 2 cm − 3 , and root diameter from 0.6 to 1.1 mm. Roots were concentrated in the topsoil (0–20 cm) for most genotypes. In deeper depths (30–60 cm), root length density, root volume, and root superﬁcial area were higher in genotypes 14, 25, 31, and 32. Positive correlations were found between root traits and both plant height and crop yield. The results of this work may contribute to the overall cultivation of C. canephora , specially for crop breeding in adverse environmental conditions.

aspects, aiming not only to identify genotypes with higher yields per se, but also genotypes with higher adaptability and stability in conditions of abiotic stresses [14][15][16][17].
In perennial crops, such as C. canephora, the root systems are important not only for nutrient and water uptake, but also for ecosystem services such as carbon sequestration, improving soil structure and genetic conservation [18]. Regarding the mechanisms related to coffee adaptation in adverse environments, studies about root performance in coffee during its productive stage are still scarce [19]. Such scarcity is related to the long crop cycle, as coffee is a permanent crop, along with the lack of proper methods for assessing and monitoring coffee roots. According to Ryan et al. [20], such knowledge is unquestionably important, as it has broad and significant implications for the global crop productivity.
Assessments related to root traits have been reviewed for selecting coffee genotypes with better performance under drought conditions [21]. Deeper root systems were associated with drought tolerance in C. canephora genotypes due to the larger root dry mass [22]. One of the reviewed studies have assessed both root systems and crop yield, although root assessments were performed in plantlets and yield data were retrieved from previous on-farm data [23]. As root assessments might be cumbersome, they are not widely used in crop breeding programs. Thus, considering that root development and performance are largely influenced by the environment, on-farm studies of the root systems of coffee trees are essential to understand crop performance.
In this study we have characterized the diversity of root traits, plant height and crop yield of 43 C. canephora genotypes with a fourfold objective to: (i) assess root distribution (length density, volume, superficial area, and diameter) at the reproductive stage, (ii) analyze the diversity of root traits, (iii) identify potentially promising genotypes to cope with water stress conditions, and (iv) test the correlation between root traits and both crop yield and plant height.

Study Site, Soil Characterization and Coffee Genotypes Studied
The study was performed in the municipality of Nova Venécia, northern Espírito Santo State, Brazil (18 • 39 43 S, 40 • 25 52 W and 199 m above sea level). The region's mean annual temperature is 23 • C, with a Aw climate, tropical with hot and humid summer and dry winter, according to Köppen classification [24]. The studied soil was classified as Ferralsol [25], which corresponds to a Latossolo Amarelo in the Brazilian soil classification system [26]. Particle size distribution and soil chemical properties are shown in Table 1.
Coffee plantlets with about five pairs of leaves were transplanted in May 2014 with spacing of 3 m × 1 m, which corresponds to a crop density of 3333 coffee trees per hectare. Coffee pruning was performed in order to maintain from three to four orthotropic branches per tree (10,000-12,000 plagiotropic branches per hectare). Farming managements were performed according to the technical guidelines for the crop, aiming to achieve nutritional and phytosanitary needs. Liming and fertilization were performed according to the regional recommendations [27]. The annual rates for N, P 2 O 5 , and K 2 O were 500, 100, and 400 kg ha −1 , respectively. In relation to soil micronutrients, a total of 2 kg ha −1 Zn, 1 kg ha −1 B, 2 kg ha −1 Cu, and 10 kg ha −1 Mn were applied annually. The area was irrigated by drip irrigation.
A total of 43 coffee genotypes were arranged in randomized blocks with three replicates. Each genotype was considered as a treatment, wherein a group of seven coffee trees of the same genotype constituted an experimental unit, leading to a total of 21 coffee treatments of each genotype and a total number of 903 plants in the experiment. The design was arranged in the double factorial 6 × 43 (six soil depths and 43 coffee genotypes). From the 43 assessed genotypes, 42 were propagated from stem cutting and one genotype was propagated by seed (genotype ID 39), as shown in Table 2.   1  Verdim R  16  Pirata  31  Cheique  2  B01  17  Peneirão  32  P2  3  Bicudo  18  Z39  33  Emcapa 02  4  Alecrim  19  Z35  34  Emcapa 153  5  700  20  Z40  35  P1  6  CH1  21  Z29  36  LB1  7  Imbigudinho  22  Z38  37  122  8  AD1  23  Z18  38  Verdim D  9  Graudão HP  24  Z37  39  Sementes  10  Valcir P  25  Z21  40  Emcapa 143  11  Beira Rio 8  26  Z36  41  Ouro negro 1  12  Tardio V  27  Ouro Negro  42  Ouro negro 2  13  AP  28  18  43  Clementino  14  L80  29  Tardio C  15 Bamburral 30 A1 Most of the genotypes have not been tested in experimental studies before, as they were selected by regional coffee farmers. Within the C. canephora groups, the genotypes assessed are part of the conilon coffee group, which is genetically distinct of the robusta group [28,29]. Genotype 33 belongs to the Emcapa 8111 cultivar, and the genotypes 34 and 39 belong to the Emcapa 8131 cultivar [30], which are late-season cultivars. The genotypes IDs 1, 11, 15, 16, 30 and 43 belong to the Tributum cultivar [8,31], which is recommended for areas under 500 m of altitude in the Brazilian States of Espírito Santo, Southern Bahia, and East Minas Gerais. The genotypes IDs 30 and 35 belong to the Andina cultivar [9], which is recommended for Brazilian states with latitude lower than 22 • S, altitude lower than 900 m and minimum air temperature not lower than 8 • C for more than 10 days in a year.

Root Traits, Plant Height and Crop Yield Assessments
Undisturbed soil samples of about 27 cm 3 were taken with a tubular sampler from six different soil depths (0-10; 10-20; 20-30; 30-40; 40-50; 50-60 cm) in each treatment during February 2018. From each experimental unit, the soil samples were taken from the fourth coffee trees in each one of the three replicates of the experimental plot, which led to a total of 774 soil samples (43 genotypes, three replicates and six soil depths). Each sample was taken from a distance of 30 cm from the coffee stem in relation to the inter-row ( Figure 1A), which is a region known for concentrating coffee roots [32]. The reason for sampling the soil down to 60 cm in the soil profile is because this depth best reflects the coffee plant's water status, due to the relation between the vegetative vigor of coffee plants and soil moisture in the root zone [33]. The samples were placed in plastic bags, sealed, and stored under −10 • C until further assessments. The samples were thereafter washed ( Figure 1B) under running water in a 30 mesh (0.595 mm) sieve in order to separate the roots from the soil ( Figure 1C). The few roots with more than 3 mm were excluded from the data set as they were considered outliers. The roots were thereafter scanned with a Nikon 24.1 MP camera model D5200 (Tokyo, Japan), and the images were taken 50 cm above the roots. The resulting images were analyzed with the Safira software version 1.1 [34]. The following traits were assessed: root length density (mm cm −3 ), root volume (mm 3 cm −3 ), root superficial area (mm 2 cm −3 ), and root diameter (mm).
Coffee tree height was assessed with a measuring tape, from base to top. Crop yield was assessed as coffee production per unit of area. It is important to note that the coffee plantlets were transplanted in 2014 and assessments were performed in 2018, wherein the coffee tree height was related to the four-year-old plagiotropic branches. In relation to crop yield, as the coffee plantlets were transplanted in May 2014, the crop yield was assessed annually for three years (2015, 2016, and 2017), and the average crop yield within these three years was used to calculate the correlations with root traits. For both coffee tree height and crop yield, three plants in each experimental unit were assessed in the three replicates, totaling nine plants for each genotype.

Statistical Analyses
In order to check the analysis of variance assumptions, the data normality was tested by the Shapiro-Wilk test (p > 0.05) and the homogeneity of variances by the Bartlett's test (p > 0.05) for each soil depth. As the assumptions were not met, data transformation was performed according to the method of Box and Cox [35], using a lambda value (λ) of −1 and the transformed data (Y) equals to Y −1 = 1/Y 1 . After data transformation, the assumptions of normality and homogeneity were met. Thereafter, an analysis of variance for the root traits (variables) versus different coffee genotypes (treatments), considering the different soil depths as different environments, and a Scott-Knot test (p < 0.05) were performed in R version 3.6.1 [36].
In order to group the coffee genotypes according to the assessed traits (root length density, root volume, root superficial area and root diameter), Mahalanobis distance was calculated and used as a measure for dissimilarity. Cluster analysis was then performed by two methods, the Tocher optimization method [37] and the hierarchical unweighted pair group method using arithmetic averages (UPGMA) [38]. The relative contribution of each trait for the diversity within 43 genotypes was analyzed according to Singh [39]. Moreover, Pearson correlation between root traits and both plant height and crop yield were calculated. The analyses were performed in the software Genes [40].

Root Length Density, Volume, Superficial Area, and Root Diameter
Most roots sampled were either short or average-sized (Table 3), with an average mean value for all genotypes and soil depths of 321.13 mm cm −3 . The short roots were mostly whitish colored with less than 1 mm diameter. Significant differences were observed between the 43 genotypes of C. canephora and the six soil depths for root length density (Table 3), root volume (Table 4), root superficial area (Table 5), and root diameter ( Table 6). There was no interaction between coffee genotype and soil depth for root diameter (Table 7), suggesting different root distribution patterns within the assessed coffee genotypes.      Most coffee roots were concentrated in the 0-10 and 10-20 cm depths. This layer (0-20 cm) concentrated 61.56 % of total root length density (Table 3), 61.57% of total root volume (Table 4), and 61.10% of total root superficial area (Table 5). Results from deeper depths were more evenly distributed.
Three out of the four root assessments, root length density (Table 3), root volume (Table 4), and root superficial area (Table 5), grouped the studied genotypes in four distinct groups (as the lowercase letters ranged from a to d) based on data from all depths combined. However, considering the assessed soil depths, the 20-30 and 30-40 cm layers formed at least three distinct groups for all four root assessments (Tables 3-6).

Cluster Analyses
Coffee genotypes were grouped into four different groups (Figure 2), considering a cut-off point of 38% of dissimilarity in the dendogram, as recommended by Mojena [31]. In order to better understand the five different groups formed, the mean averages for root length density, root superficial area, and root volume were calculated for each group (Figure 3). Group I was composed of genotypes ID 1, 2, 5, 6, 7, 9, 10, 11, 12, 16, 17, 18, 20, 21, 22, 23, 26, 27, 28, 29, 31, 33, 34, 36, 37, 39, and 42. This group differed from the others with 39% of accuracy. Root length density ( Figure 3A), root volume ( Figure 3B) and root superficial area ( Figure 3C) were lower in this group than all others. Furthermore, it allocated genotypes ID 21 and 34, which showed the highest similarity between two genotypes. Within group I, the genotype ID 31 concentrated 20.35% of total root length density ( Figure 3B), 23.28% of root volume ( Figure 3D) and 22.11% of root superficial ( Figure 3F) area in the 50-60 cm soil depth.  Group II was composed of genotypes ID 3,4,8,13,15,19,24,30,35,38,40,41,and 43. Group III was composed only of genotype ID 32, which had the highest root volume ( Figure 3C), mainly concentrated in the topsoil ( Figure 3D). The root traits of group IV (comprising genotypes ID 14 and 25) were more evenly distributed within the six soil depths than all other groups (Figure 3). Group IV differed from all other groups, with a dissimilarity to all other genotypes over 99%. This group had the highest root length density ( Figure 3A,B) and root superficial area ( Figure 3E,F).
It is important to note the results for the seed-propagated genotype (genotype 39) in relation to all others, propagated by stem cutting. This genotype is within group I, the group with the highest number of different coffee genotypes (Figure 2). Intermediate values were found for the root traits in the seed-propagated genotype, in which other genotypes (propagated by stem cutting) had higher, similar, and lower values.
The Tocher clustering method formed ten distinct groups ( Table 8). The higher number of groups indicates a broad genetic diversity between coffee genotypes, as the method seeks to minimize intragroup distance and maximize intergroup distance. Most (25 out of 43) of the genotypes were located within groups I and II. Group III was composed of five coffee genotypes (ID 02, 27, 29, 33, and 42). Groups IV, V, VI, VII, VIII, and IX were composed of two genotypes each, and group X was composed of only one coffee genotype (31). Root superficial area, root length density, root volume and root diameter had a relative contribution for the diversity within genotypes of 43.1%, 33.9%, and 6.5%, respectively.

Root Traits, Plant Height and Crop Yield Correlations
There were significant positive correlations between root traits and plant height, as well as root traits and crop yield (Figure 4), with different levels of significance. However, such correlations were weak (r < 0.3) or moderate (0.3 < r < 0.6). Within root traits, the highest correlation was found between root length density and superficial area (0.99), followed by root superficial area and root volume (0.96).

Root Length Density, Volume, Superficial Area, and Root Diameter
Short or average-sized (Table 3) roots are considered absorbing roots [41]. In ideal conditions for root development, coffee genotypes grow efficiently with absorbing roots concentrated within the topsoil (down to 30 cm deep). However, under adverse conditions, such as severe drought, coffee genotypes with deeper roots may access more water and thus be better adapted to such conditions [42]. Studies from Ronchi et al. [43] and Isaac et al. [44] indicated that the root system is directly related to crop adaptation and crop yield.
The root distribution patterns, which were concentrated in the topsoil, are in accordance with the work of Partelli et al. [45], who found about 60% of coffee roots within soil depths of 0-20 and 25-50 cm. Furthermore, in the subsoil, the 20-30 and 30-40 cm layers formed at least three distinct groups for all four root assessments (Tables 3-6). Similarly, Defrenet et al. [46] assessed root biomass and necromass in a coffee agroforestry system and found that most roots were fine and located in the topsoil. Crop breeding for potentially promising coffee genotypes under water stress should thus be performed in soil depths of 40 cm or deeper.
It is also important to note that deeper soil depths generally imply higher soil resistance to root penetration due to higher soil compaction [47], although coffee roots may penetrate down to four meters in the soil profile [46]. Another possible factor related to higher resistance to root penetration in the soil subsurface is the higher aluminum content, which is responsible for soil acidity [48]. The above-mentioned relationships are in accordance with this study results, as the 20-40 cm layer presented higher Al content (Table 1) and lower root abundancy (Tables 3-6) than the topsoil.
According to Rao et al. [49], crop adaptation for infertile soils may be achieved from two options: either the growing medium may be changed or the plant genotype may be bred. Considering the current concerns about climate change and soil degradation, both options should be integrated [19].
According to Gould et al. [50], architectural root traits, as the ones studied in this work, positively impact soil structure, forming effective hydraulic pathways in the soil, and promoting a better environment for water and nutrients uptake.

Cluster Analyses
The cluster analysis suggests that group IV comprises promising genotypes to cope with water stress conditions, as their roots were more evenly distributed within soil depths, and it had consequently higher root abundance in deeper layers in comparison with groups I, II and III. The deeper root abundance in C. canephora genotypes are commonly related to drought tolerance due to the larger root dry mass [22]. Thus, genotypes from group IV (ID 14 and 25) may be a promising alternative for C. canephora breeding due to their root system distribution.
The intermediate root traits values for the seed-propagated genotype (39), suggest that the root system development is more related to the plant genetic than to the propagation method, as other genotypes (propagated by stem cutting) had higher, similar, and lower values. Partelli et al. [7] found similar results, with no difference in the root system distribution of C. canephora plants propagated by seeds in relation to plants propagated by stem cutting.
According to Hair Jr. et al. [51], cluster analyses may be divided in hierarchical and non-hierarchical methods. Ideally, studies should consider both approaches and thereafter ensure a more detailed result. Thus, apart from the UPGMA clustering (hierarchical method), this study also tested the Tocher optimization method (Table 8) by using the Mahalanobis distance as the genetic dissimilarity between genotypes. The Tocher method had been successfully used in C. canephora in previous studies [13,14,52] to identify promising genotypes with greater genetic variability.
Both clustering methods led to somehow similar results (Table 8). Genotypes ID 14, 25, and 32 were grouped separately in both methods, which strengthens the suggestion that they are promising alternatives for C. canephora breeding due to their distinct root system distribution. The UPGMA groups I and II were divided into subgroups by the Tocher method. The UPGMA group I, for example, were parted into five groups by the Tocher method, in which only genotypes 01 and 24 did not corresponded within both methods. Likewise, the UPGMA Group II were parted into three Tocher groups, whereas only the genotype 09 did not match the corresponding group. Similarities between the Tocher optimization method and the hierarchical unweighted pair group method using arithmetic averages (UPGMA) were also found by Giles et al. [31] and Dubberstein et al. [53]. In relation to the relative contribution of each root trait, as the root diameter has the lowest contribution (6.55%), it should not be considered as a main root trait for C. canephora breeding.

Root Traits, Plant Height and Crop Yield Correlations
The positive correlation between root traits and coffee yield suggests that the more abundant the root systems, higher coffee yield. As the correlations between root traits and plant height were weak or moderate, the root system of C. canephora genotypes should not be inferred merely based on plant height. Moreover, the correlation between plant height and coffee yield was weak. According to Carvalho et al. [54], plant height is more related to environmental aspects than to crop yield.

Main Limitations of the Study
There are a few limitations in this study which should be noted. Firstly, there was no water stress in the assessed coffee trees, as plants were irrigated by drip irrigation. The introduction of any stress may therefore cause differences in crop yield and root distribution. Secondly, neither soil porosity nor soil resistance to penetration have been assessed. It is well known that the soil structure and aggregation are closely related to root distribution and to the potential of water storage in the soil and the extraction by plant roots. Thirdly, there was only one genotype propagated by seeds, which makes it difficult to infer about the differences between genotypes propagated by stem cutting and by seeds. Future works should therefore fill these gaps in order to consolidate the knowledge about the relationship between root abundance and drought tolerance.

Conclusions
In this work, the diversity of root traits in 43 C. canephora genotypes and its correlation with plant height and crop yield were analyzed. Most roots were concentrated in the topsoil (0-20 cm) for all assessed genotypes. Considering the 30-60 cm depth, genotypes ID 14, 25, 31, and 32 had more roots than most others coffee genotypes, which suggest they are promising under adverse environmental conditions, such as drought.
Root traits significantly varied within the genotypes propagated by stem cutting, and the seed-propagated genotype (39) had intermediate values, indicating that root development is mostly related to the plant genetic background than to the propagation method.
There were positive correlations between the assessed root traits and both crop yield and plant height. The results of this work may contribute to the overall cultivation of C. canephora, especially for crop breeding towards abiotic stress tolerance.