Rhizomes of Atractylodes lancea are used in traditional Japanese medicine (Kampo) and Chinese medicine to treat numerous diseases and disorders because they contain many pharmacologically active compounds. The major active compounds in A. lancea are essential oil compounds such as β-eudesmol, hinesol, atractylon, and atractylodin. The contents of the compounds in A. lancea exhibit high variability depending on their habitat. We cultivated clonal lines of A. lancea in different years (2016, 2017) and different locations (Hokkaido, Ibaraki) to investigate the influence of genetic and environmental factors on the contents of major compounds, namely, β-eudesmol, hinesol, atractylon, and atractylodin. Broad sense heritability of β-eudesmol, hinesol, atractylon, and atractylodin contents were 0.84, 0.77, 0.86, and 0.87, respectively. The effects of interannual variability on the contents of the compounds were lower than those of genotype. In addition, the cultivated environmental factors were assessed by different locations, and the correlations between Hokkaido and Ibaraki grown plants based on β-eudesmol, hinesol, atractylon, and atractylodin contents were 0.94, 0.94, 1.00, and 0.83, respectively. The results suggest that the contents of β-eudesmol, hinesol, atractylon, and atractylodin in A. lancea are largely influenced by genetic factors, and clonal propagation could be an effective strategy for obtaining populations with high contents of essential oil compounds. Furthermore, the contents of β-eudesmol, hinesol, atractylon, and atractylodin in A. lancea exhibited few correlations with rhizome yields. A. lancea cultivars with not only high contents of essential oil compounds but also high rhizome yield could be developed through selective breeding.
Keywords: Research Article; Biology and life sciences; Biochemistry; Lipids; Oils; Molecular biology; Molecular biology techniques; Cloning; Research and analysis methods; Genetics; Heredity; Agriculture; Agricultural soil science; Ecology and environmental sciences; Soil science; Plant genetics; Crop genetics; Plant science; Agronomy; Plant breeding; Hormones; Plant hormones; Plant biochemistry; Chromatographic techniques; Gas chromatography-mass spectrometry; Physical sciences; Chemistry; Analytical chemistry; Mass spectrometry; Spectrum analysis techniques
Atractylodes lancea De Candolle (Compositae) is a medicinal plant that is distributed in East Asia, mainly in central China [[
Generally, phenotypic variation is a product of both genetic and environmental variation [[
Some studies have reported that the activation of phytohormones such as jasmonic acid and abscisic acid through symbiosis with several endophytes induces the production of the essential oil compounds in A. lancea [[
In the present study, to evaluate effects of genetic factors on the contents of essential oil compounds in A. lancea, we cultivated twenty-five clonal lines of A. lancea in a micro-environment and estimated broad sense heritability for β-eudesmol, hinesol, atractylon, and atractylodin contents. In order to estimate the broad sense heritability, we employed the calculation model for vegetatively propagated crops such as orchardgrass, bromegrass, and tall fescue [[
In the present study, seeds of A. lancea obtained in a previous study were used [[
The 25 clonal lines were cultivated in an experimental field located in Ami-machi, Inashiki-gun, Ibaraki prefecture (35°.99'N, 140°.20'E), Japan, in 2017. Rhizomes of the 25 clonal lines were divided into about 50 g. The rhizomes were planted in November 25, 2016 and harvested on November 23, 2017. The cultivation was performed with 5–20 biological replicates for each clonal line. The replicates for each clonal line were as follows; line1–line17 (n = 20), line18–line24 (n = 10), line25 (n = 5).
To assess the effects of cultivation year on the contents of essential oil compounds in A. lancea, six clones (line1–line6) were also grown in the fields located in Ibaraki prefecture in 2016. The plants were cultivated from November 25, 2015 to November 23, 2016 in Ibaraki prefecture. The cultivation was performed with 20 biological replicates for each clonal line. The six clones were also cultivated in an experimental field located in Kyowa-town, Hokkaido prefecture (43°.01'N, 140°.53'E), Japan, in 2017, to evaluate the effects of cultivation location on the contents of essential oil compounds in A. lancea. Rhizomes of the plants were planted on October 21, 2016 and harvested on October 19, 2017. The plants were cultivated with 8–20 biological replicates for each clonal line. The replicates for each clonal lines were as follows; line1 (n = 20), line2 (n = 12), line3 (n = 8), line4 (n = 20), line5 (n = 18), line6 (n = 20).
The harvested rhizomes of A. lancea were dried in a convection oven at 50°C for 7 days and their dry weights were determined. The dried rhizomes were pulverized using a vibrating mill (Cosmic Mechanical Technology, TI-200). The contents of four essential oil compounds, namely, β-eudesmol, hinesol, atractylon, and atractylodin, were determined by gas chromatography-mass spectrometry (GC-MS) analysis as follows. Powdered samples of A. lancea rhizomes (0.5 g) were extracted with n-hexane (25 mL) for 15 min using a recipro shaker (Taitec, model SR-1) and centrifuged at 1660 × g for 10 min. The supernatant was separated, and the residue was re-extracted with n-hexane (20 mL) in a similar manner as above. The supernatant was combined, and phenanthrene (1.5 mg in 1 mL n-hexane) was added as an internal standard. A solution with a total volume of 50 mL was made by adding of n-hexane. An aliquot (1 μL) of the solution was injected into GC-MS. GC-MS analysis was carried out using an Agilent 7890 gas chromatograph equipped with a 5975 mass spectrum detector (MSD) (Agilent Technologies). Chromatography was performed with a DB-WAX GC column (polyethylene glycol, 30 m×250 μm i.d., 0.25 μm film; Agilent J&W Scientific). The column was used with the following temperature program: column held at 160°C for 2 min after injection, increased by 5°C/min to 200°C, then increased by 8°C/min to 240°C, and held for 5min. Injection temperature was set to 160°C. Helium was used as the carrier gas and the flow rate was 1.0 mL/min. The contents were calculated based on the dry weights of the powdered samples.
Statistical analyses, including one-way ANOVA, two-way ANOVA, and correlation analysis (Pearson's correlation coefficient) were performed for β-eudesmol, hinesol, atractylon, and atractylodin contents using R (version 3.5.0). Broad sense heritability (h
To evaluate the heritability of the contents of essential oil compounds in A. lancea under a microenvironment, 25 clonal lines were grown in Ibaraki prefecture in 2017 and the contents of the compounds in individual plantlets were determined. Fig 1 illustrates the distribution of each compound in different clonal lines. In most clonal lines, the ranges of variation in the contents of β-eudesmol, hinesol, atractylon, and atractylodin were lower than varietal differences, although some lines exhibited relatively high variations among individual plantlets in the same line, for example, line20 and line23 (Fig 1). One-way ANOVA and the estimation of broad sense heritability of those compound contents were performed using the above results. Table 1 shows that the differences in varietal compound contents were significant (P > 0.01) and the heritability for the contents of β-eudesmol, hinesol, atractylon, and atractylodin were high at 0.84, 0.77, 0.86, and 0.87, respectively (Table 1).
Table 1: Broad-sense heritability for contents of the essential oil compounds in A. lancea.
Essential oil compounds Factors Df Mean Sq P-value Effective replication Genotypicvariance Environmentalvariance Broad-sense heritability β-eudesmol Clonal lines 24 539.1 <2e-16** 16.5 32.2 6.3 0.84 Residuals 390 6.3 hinesol Clonal lines 24 698.2 <2e-16** 16.5 41.5 12.7 0.77 Residuals 390 12.7 atractylon Clonal lines 24 23.5 <2e-16** 16.5 1.4 0.2 0.87 Residuals 390 0.2 atractylodin Clonal lines 24 6.1 <2e-16** 16.5 0.4 0.1 0.86 Residuals 390 0.1
- 2 Df; degree of freedom, Mean Sq; Mean square
1 **; Probability value for test of significance < 0.01.
Correlation analyses among various compound contents and rhizome yield were performed, since there is potential linkage among different traits. According to the results, there was no significant correlation between rhizome yields and contents of atractylon or atractylodin, and the correlation coefficient between rhizome yields and contents of β-eudesmol and hinesol were relatively low, 0.20 and 0.28, respectively (Fig 2). In contrast, the correlation between β-eudesmol and hinesol contents was relatively high, although the correlations among the other compounds were comparatively low (Table 2).
Table 2: Correlation matrix of β-eudesmol, hinesol, atractylon, and atractylodin contents.
β-eudesmol Hinesol atractylon atractylodin β-eudesmol - 0.59** 0.29** 0.09 hinesol - - −0.13** 0.39** atractylon - - - −0.04 atractylodin - - - -
3 **; Probability value for test of significance < 0.01.
Climatic datas for each cultivation years and locations were used the data issued by the Japan Meteorological Agency (JMA) [[
To examine the effect of cultivation year on contents of the essential oil compounds, six clones of A. lancea were grown in the same experimental field in 2016 and 2017, and contents of the compounds analyzed using two-way ANOVA. The effects of genotype (G), cultivation year (Y), and G × Y interaction were significant for contents of β-eudesmol, hinesol, and atractylon. However, mean squares of genotypes for each compound's contents were higher than those of cultivation year and G × Y interaction (Table 3). In addition, interaction plots between genotype and cultivation year for β-eudesmol, hinesol, and atractylon showed that there were low qualitative interactions (Fig 3). Atractylodin contents were not significantly influenced by cultivation year, although there were significant differences for genotypes and G × Y interactions (Table 3). Fig 2D illustrates that qualitative interaction between genotype and cultivation year based on atractylodin contents was low, although in line5, the content levels of atractylodin in six clonal lines varied depending on cultivation year. In addition, the correlation coefficients between the two cultivation years for β-eudesmol, hinesol, atractylon, and atractylodin contents were 0.94, 0.94, 1.00, and 0.83, respectively (Fig 4).
Table 3: Two-way ANOVA results for β-eudesmol, hinesol, atractylon, and atractylodin contents in six A. lancea clones grown in 2016 and 2017.
Essential oil compounds Factors Df MeanSq P-value β-eudesmol Genotype (G) 5 12.26 2.0.E-16** Year (Y) 1 0.97 1.4.E-05** G × Y 5 0.16 6.1.E-03** Residuals 228 0.05 hinesol Genotype (G) 5 23.22 2.0E-16** Year (Y) 1 0.27 6.1E-02* G × Y 5 0.22 1.6E-02** Residuals 228 0.08 atractylon Genotype (G) 5 0.64 2.0.E-16** Year (Y) 1 0.07 2.0.E-16** G × Y 5 0.01 1.1.E-14** Residuals 228 0.001 atractylodin Genotype (G) 5 0.20 2.0E-16** Year (Y) 1 0.0.E + 00 0.9 G × Y 5 0.003 2.6E-06** Residuals 228 0.0004
- 5 Df; degree of freedom, Mean Sq; Mean square
- 4 *, **; Probability value for test of significance < 0.05 and < 0.01, respectively.
To determine the effects of cultivation location on contents of the essential oil compounds, six clonal lines of A. lancea were cultivated in different locations. Two-way ANOVA results for β-eudesmol and hinesol contents revealed that there were significant differences in genotypes (G), locations (L), and G × L interactions. In addition, mean squares of genotype were lower than those of location (Table 4). However, low qualitative interactions between genotype and location for contents of β-eudesmol and hinesol were observed (Fig 5). In addition, correlation coefficients for contents of β-eudesmol and hinesol between the two cultivation locations were 1.00 and 0.94, respectively (Fig 6).
Table 4: Two-way ANOVA for β-eudesmol, hinesol, atractylon, and atractylodin contents in six A. lancea clones grown in Hokkaido and Ibaraki prefecture.
Essential oil compounds Factors Df MeanSq P-value β-eudesmol Genotype (G) 5 7.80 2.0E-16** Location (L) 1 16.66 2.0E-16** G × L 5 0.24 2.2E-04** Residuals 206 0.05 hinesol Genotype (G) 5 9.61 2.0E-16** Location (L) 1 52.76 2.0E-16** G × L 5 1.58 2.3E-16** Residuals 206 0.08 atractylon Genotype (G) 5 0.83 2.0.E-16** Location (L) 1 0.23 2.0.E-16** G × L 5 0.01 1.9.E-08** Residuals 206 0.001 atractylodin Genotype (G) 5 0.16 2.0E-16** Location (L) 1 0.01 4.6E-08** G × L 5 0.002 3.9E-05** Residuals 206 0.0004
- 7 Df; degree of freedom, Mean Sq; Mean square
- 6 **; Probability value for test of significance < 0.01.
Two-way ANOVA analyses of atractylon and atractylodin contents revealed that there were significant differences in genotypes, locations, and G × L interaction. However, the mean squares of genotypes were higher than those of locations and G × L interactions (Table 4). Fig 5C and 5D show that there were low qualitative interactions between genotype and location for contents of atractylon and atractylodin. In addition, correlation analyses of the contents of atractylon and atractylodin between the two locations revealed high correlations coefficients, 1.00 and 0.94, respectively (Fig 6).
In the present study, broad sense heritability for β-eudesmol, hinesol, atractylon, and atractylodin contents in A. lancea was high (Table 1), suggesting that the contents of the essential oil compounds in A. lancea are highly influenced by genetic factors. In addition, correlations between each compound contents and rhizome yield were low (Fig 2), indicating that A. lancea strains, which have high contents of essential oil compounds could be selectively bred irrespective of rhizome yields. Therefore, cultivars of A. lancea with not only high contents of essential oil compounds but also high yields could be developed through selective breeding.
Correlation coefficients among different compound contents indicated low correlations, except in the case of the correlation between β-eudesmol and hinesol contents (Table 2). The biosynthetic pathways of β-eudesmol and hinesol are considered potentially closely associate since the compounds have similar chemical structures with the same molecular mass [[
We used the six clonal lines to investigate the influence of cultivation year on the contents of the essential oil compounds. Based on the results of the two-way ANOVA analyses, mean squares of genotype for contents of the essential oil compounds were higher than the interannual variability (Table 3). Additionally, there were low G × Y qualitative interactions (Fig 3), while the correlation coefficients between cultivation years and compound contents were relatively high (Fig 4). The results imply that, regardless of cultivation year, the contents of essential oil compounds in A. lancea clonal lines were stable, and genetic factors had greater influence than cultivation year did.
We cultivated six clones in two locations and evaluated the effects of cultivation location on the contents of essential oil compounds. The effect of cultivation location on the contents of essential oil compounds in A. lancea was larger than that of cultivation year, especially, the mean squares of cultivation location for β-eudesmol and hinesol contents were higher than those of genotype (Table 4). In general, secondary metabolites such as sesquiterpenes are induced by biological and abiotic stress via phytohormone signaling [[
Conversely, low qualitative interactions between genotype and location in the contents of β-eudesmol, hinesol, atractylon and atractylodin were observed (Fig 5), and correlation coefficients between 2 locations for these componds' contents were high (Fig 6). The results suggest that genetic potential of A. lancea regarding the contents of β-eudesmol, hinesol, atractylon and atractylodin are stable regardless of environmental conditions, although the contents of the compounds are influenced by environmental factors as absolute values. It has been reported that artemisinin contents in Artemisia annua clones grown under different cultivation conditions were highly correlated [[
In conclusion, the present study demonstrates that the contents of β-eudesmol, hinesol, atractylon, and atractylodin in A. lancea are influenced mainly by genetic factors, and selective breeding could be an effective strategy for developing A. lancea populations that yield high contents of essential oil compounds. In addition, β-eudesmol, hinesol, atractylon, and atractylodin contents could be selected regardless of rhizome yields in the course of A. lancea cultivar development. Consequently, A. lancea cultivars with high rhizome yields and high contents of each the essential oil compounds could be developed. However, since the contents of the essential oil compounds are influenced by environmental condition, further investigations on the effects of environment factors on the compound contents in A. lancea are required.
S1 Table. The data for Figs 1 and 2 and Tables 1 and 2. The contents of essential oil compounds in A. lancea grown in Ibaraki prefecture on 2017. (PDF)
S2 Table. The data for Figs 3 and 4 and Table 3. The contents of essential oil compounds in A. lancea grown in each cultivation year. (PDF)
S3 Table. The data for Figs 5 and 6 and Table 4. The contents of essential oil compounds in A. lancea grown in each cultivation location. (PDF)
DIAGRAM: Fig 1: Range of variations for contents of the essential oil compounds in A . lancea . The boxes represent the values from the 25th to 75th percentile. The middle lines represent the median. The vertical lines extend from the minimum to the maximum values. Biological replicates within each clonal lines were as follows: line1–line17 (n = 20), line18–line24 (n = 10), line25 (n = 5). A; β-eudesmol contents, B; hinesol contents, C; atractylon contents, D; atractylodin contents.
DIAGRAM: Fig 2: Relationships between contents of the essential oil compounds and rhizome weight in A . lancea . A; β-eudesmol contents, B; hinesol contents, C; atractylon contents, D; atractylodin contents.
DIAGRAM: Fig 3: Interaction plots for interannual variability of the essential oil compound contents in A . lancea . Each point represents the mean of 20 measurements within clonal lines each in 2016 and 2017. A; β-eudesmol contents, B; hinesol contents, C; atractylon contents, D; atractylodin contents.
DIAGRAM: Fig 4: Relationships between contents of the essential oil compounds from six A . lancea clones grown in 2016 and 2017. Each point represents the mean of 20 measurements each in 2016 and 2017. A. β-eudesmol contents, B. hinesol contents, C. atractylon contents, D. atractylodin contents.
DIAGRAM: Fig 5: Interaction plots for contents of the essential oil compounds in six A . lancea clones grown in Hokkaido and Ibaraki prefectures. Data represent mean values of each compounds' contents in A. lancea (n = 20 in line1, n = 12 in line2, n = 8 in line3, n = 20 in line4, n = 18 in line5, n = 20 in line6). A; β-eudesmol contents, B; hinesol contents, C; atractylon contents, D; atractylodin contents.
DIAGRAM: Fig 6: Relation for contents of the essential oil compounds from six A . lancea clones grown in Hokkaido and Ibaraki prefecture. Data represent means ± SD (n = 20 in line1, n = 12 in line2, n = 8 in line3, n = 20 in line4, n = 18 in line5, n = 20 in line6). A. β-eudesmol contents, B. hinesol contents, C. atractylon contents, D. atractylodin contents.
We are grateful to Assistant Prof. Yosuke Yoshioka (University of Tsukuba) for technical support in data analysis. We thank Mikio Sakai, Akiko Uetake and Terue Kurosawa for cultivation assistance. We also thank Kazunori Hashimoto, Kenji Kondo and Yoichiro Nakai for helpful suggestions.
By Takahiro Tsusaka, Writing – original draft; Bunsho Makino, Writing – review & editing; Ryo Ohsawa, Writing – review & editing and Hiroshi Ezura, Writing – review & editing